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Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 1 Abbreviated Title: mRNA-Based, Cancer Vaccine CC Protocol Number: 18-C-0074 E IBC Number: RD-18-I-07 OSP Number: 1803-1739 NCT Number: NCT03480152 Version Date: January 23, 2019 PROTOCOL TITLE A Phase I/II Trial to Evaluate the Safety and Immunogenicity of a Messenger RNA (mRNA)- Based, Personalized Cancer Vaccine Against Neoantigens Expressed by the Autologous Cancer NIH Principal Investigator: Steven A. Rosenberg, M.D., Ph.D. Chief of Surgery, Surgery Branch, CCR, NCI Building 10, CRC, Room 3-3940 9000 Rockville Pike, Bethesda, MD 20892 Phone: 240-858-3080; E-ma il: [email protected] Investigational Agent: Drug Name: NCI-4650 (mRNA-based, Personalized Cancer Vaccine) IND Number: 17858 Sponsor: Center for Cancer Research Manufacturer: ModernaTX, Inc. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 2 PRÉCIS Background: x Therapeutic vaccination against cancer has proven very challenging with little clinical benefit.

x Vaccines against non-viral tumors have mainly targeted differentiation antigens, cancer testis antigens, and overexpressed antigens. However, negative selection in the thymus against these normal non-mutated antigens severely limits the ability to generate high avidity anti-cancer T-cells. Such depletion can impair their antitumor activity and limit tumor elimination. x The National Cancer Institute Surgery Branch (NCI-SB) has developed a pipeline for the identification of immunogenic T-cell epitopes derived from neoantigens. x In recent studies, we identified the neoantigens recognized by tumor-infiltrating lymphocytes (TIL) that mediated regression in patients with metast atic melanoma. Using whole exome sequencing of a resected meta static nodule followed by high throughput immunologic screening, we were able to demonstrate that tumor regressions were associated with the recognition by the administered TIL of unique somatic mutations that occurred in the cancer. x We also found that TIL from 29 of 32 patie nts with a wide variety of metastatic gastrointestinal cancers contained lymphoc ytes that recognized unique mutations presented in that patient’s cancer. x We, therefore, aim to use this pipeline to identify immunogenic neoantigens and to predict for neoantigens binding the patient human leukocyte antigen (HLA) molecules from melanoma or epithelial cancer patients and to use these epitopes for a personalized therapeutic messenger RNA (mRNA) vaccine. Objectives: x Primary objectives: o Determine the clinical response rate in patients with meta static melanoma, gastrointestinal, or genitourinary cancers who receive NCI-4650. o Determine the safety of NCI-4650 in patients with meta static melanoma, gastrointestinal, or genitourinary cancers. Eligibility: x Age • 18 years DQG”\HDUV x Evaluable metastatic melanoma, gastrointestin al, or genitourinary cancers refractory to standard of care treatment. x Metastatic cancer lesions suitable for surg ical resection to perform whole exome sequencing and preparation of TIL. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 3 Design: x Patients with metasta tic cancer will undergo surgical re section of tumor followed by exome and RNA sequencing to identify expr essed mutations. This will be conducted under the NCI-SB cell harvest protocol 03- C-0277 (Cell Harvest and Preparation for Surgery Branch Adoptive Cell Therapy Protocols). x Immunogenic neoantigens will be identified from TIL by high throughput immunologic screening using long peptides and tandem minigenes covering all mutated epitopes. x Up to 15 predicted neoantigens will be selected based on exome and RNA sequencing and their binding affinity to the patient HLA molecules. x The mRNA vaccine will be manufacture d and supplied as cGMP product by ModernaTX, Inc. x The patient will be vaccinated with mRNA containing epitopes from immunogenic neoantigens, predicted neoantig ens, and mutations in tumor suppressor or driver genes. x The mRNA vaccine will be administered intramuscularly (IM) for four cycles every two weeks. A patient may receive a second course for a total of eight cycles. x Blood samples will be taken every two weeks (d uring the vaccination period) and at each follow-up visit, and patients will be monitored for the quantity and quality of circulating neoantigen-specific T-cells. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 4TABLE OF CONTENTS PRÉCIS ........................................................................................................................ ..................

2 TABLE OF CO NTENTS ............................................................................................................. 4 1 INTRODUCTION............................................................................................................. 8 Study Objectives ............................................................................................................. 8 1.1.1 Primary Objectives...................................................................................................... 8 1.1.2 Secondary Objective ................................................................................................... 8 Background and Rationale .............................................................................................. 8 1.2.1 Cancer Vaccines.......................................................................................................... 8 1.2.2 Identification of Cancer-Specific Imm unogenic Epitopes Derived from Somatic Mutations ..................................................................................................................... ........... 9 1.2.3 Overview of mRNA-4650, a mRNA-Based, Personalized Cancer Vaccine ............ 11 1.2.4 Preclinical Experiments Evaluating th e Safety of mRNA-Based, Personalized Cancer Vaccines ............................................................................................................... ..... 11 1.2.5 Preclinical Experiments Evaluating the Immunogenicity of a mRNA-Based, Personalized Cancer Vaccine in Mice .................................................................................. 12 1.2.6 Preclinical Experiments Evaluating the Pr esentation of CD4 and CD8 Epitopes by Human Dendritic Cells ......................................................................................................... 16 1.2.7 Rationale for Selecting Defined Epit opes, Predicted Epitopes, and Tumor Suppressor and Driver Neoantigen s to Use in this Trial ....................................................... 17 1.2.8 Structure of NCI-4650 .............................................................................................. 17 1.2.9 Risk for NCI-4650 Monotherapy .............................................................................. 18 2 ELIGIBILITY ASSESSMENT AND ENROLLMENT ..............................................

19 Eligibility Criteria ......................................................................................................... 1 9 2.1.1 Inclusion Criteria ...................................................................................................... 19 2.1.2 Exclusion Criteria ..................................................................................................... 20 Screening Evaluations ................................................................................................... 21 2.2.1 Within 3 Months Prior to Part Two Registration ...................................................... 21 2.2.2 Within 4 Weeks Prior to Part Two Registration ....................................................... 21 2.2.3 Within 7 Days Prior to Part Two Registration .......................................................... 21 Registration and Treatment A ssignment Procedures .................................................... 22 2.3.1 Prior to Registration for this Protocol ....................................................................... 22 2.3.2 Registration Procedure .............................................................................................. 22 2.3.3 Treatment Assignment Procedures ........................................................................... 23 3 STUDY IMPLEMENTATION ...................................................................................... 23 Study Design ................................................................................................................. 2 3 3.1.1 Schema ...................................................................................................................... 23 3.1.2 Pre-Treatment Phase: Pe rformed on 03-C-0277 .......................................................

23 3.1.3 Treatment Phase ........................................................................................................ 24 Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 53.1.4 Dose-Limiting Toxicity ............................................................................................ 24 3.1.5 Phase I – Dose Escalation.........................................................................................

24 3.1.6 Rationale for Dose Le vel Regimen and Route of Administration ............................ 26 3.1.7 Phase II Portion ......................................................................................................... 26 Protocol Stopping Rules ............................................................................................... 27 Drug Administration ..................................................................................................... 27 Baseline Evaluations..................................................................................................... 28 3.4.1 Within 7 Days Prior to the First Vaccination (Day 0) .............................................. 28 3.4.2 Within 2 Days Prior to the First Vaccination (Day 0) .............................................. 28 On-Study Evaluations ................................................................................................... 28 3.5.1 Within 2 Days Prior to Each Subsequent Vaccination (Days 14, 28, & 42 (± 5 days)) 28 3.5.2 After Each Vaccination ............................................................................................. 29 Post-Treatment (Follow-up) Evaluations ...................................................................... 29 3.6.1 First Follow-up Evaluation ....................................................................................... 29 3.6.2 Subsequent Follow-up Evaluations........................................................................... 29 Study Assessment Calendar .......................................................................................... 31 Criteria for Removal from Protocol Therapy and Off-Study Criteria .......................... 33 3.8.1 Criteria for Removal from Protocol Therapy............................................................ 33 3.8.2 Off-Study Criteria ..................................................................................................... 33 3.8.3 Off Protocol Therapy and Off-Study Procedure ....................................................... 33 4 CONCOMITANT MEDICAT IONS/MEASURES ...................................................... 34 5 BIOSPECIMEN COLLECTION .................................................................................. 35 Samples Sent to Figg Lab ............................................................................................. 35 Samples Sent to Surgery Bran ch Cell Production Facility ........................................... 35 Sample Collection Schedule ......................................................................................... 35 Immunological Testing ................................................................................................. 36 Sample Storage, Tracking, and Disposition For Surgery Branch Cell Production Facility ...................................................................................................................... ................ 36 Sample Storage, Tracking, and Disposition for Figg Lab ............................................ 36 5.6.1 Sample Data Collection ............................................................................................ 36 5.6.2 Sample Storage and Destruction ............................................................................... 37 6 DATA COLLECTION AND EVALUATION ............................................................. 37 Data Collection ............................................................................................................. 37 6.1.1 Exclusions to Routine Adverse Event Recording ..................................................... 38 Data Sharing Plans ........................................................................................................ 38 6.2.1 Human Data Sharing Plan ......................................................................................... 38 6.2.2 Genomic Data Sharing Plan ...................................................................................... 38 Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 6 Response Criteria .......................................................................................................... 38 6.3.1 Disease Parameters ...................................................................................................

39 6.3.2 Methods for Evaluation of Measurable Disease ....................................................... 40 6.3.3 Response Criteria Definitions ................................................................................... 41 6.3.4 Duration of Response ................................................................................................ 42 Toxicity Criteria ............................................................................................................ 43 7 SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN 43 Definitions.....................................................................................................................4 3 7.1.1 Adverse Ev ent ........................................................................................................... 43 7.1.2 Suspected Adverse Reaction ..................................................................................... 43 7.1.3 Unexpected Adverse Reaction.................................................................................. 43 7.1.4 Serious....................................................................................................................... 43 7.1.5 Serious Adverse Event .............................................................................................. 43 7.1.6 Disability ................................................................................................................... 4 4 7.1.7 Life-Threatening Adverse Drug Experience ............................................................. 44 7.1.8 Protocol Deviation (N IH Definition) ........................................................................ 44 7.1.9 Non-Compliance (NIH Definition) ........................................................................... 44 7.1.10 Unanticipated Problem.......................................................................................... 44 NIH INTRAMURAL-IRB and Clinical Director (CD) Reporting ............................... 44 7.2.1 NIH Intramural IRB and NCI CD Expedite d Reporting of Unanticipated Problems and Deaths .................................................................................................................... ......... 44 7.2.2 NIH Intramural IRB Requirements for PI Reporting at Continuing Review ........... 45 7.2.3 NIH Intramural IRB Reporting of IND Safety Reports ............................................ 45 IND Sponsor Reporting Criteria ................................................................................... 45 7.3.1 Reporting Pregnancy ................................................................................................. 46 Safety Reporting Criteria to the Pharmaceutical Collaborator ..................................... 46 Institutional Biosafety Committee (IBC) Reporting Criteria ........................................ 47 7.5.1 Serious Adverse Event Reports to IBC ..................................................................... 47 7.5.2 Annual Reports to IBC.............................................................................................. 47 Data and Safety Monitoring Plan .................................................................................. 48 7.6.1 Principal Investigator/Research Team ...................................................................... 48 7.6.2 Sponsor Monitoring Plan .......................................................................................... 48 7.6.3 Safety Monitoring Committee (SMC) ...................................................................... 49 8 STATISTICAL CONS IDERATIONS ..........................................................................

49 Statistical Hypothesis .................................................................................................... 49 8.1.1 Primary Efficacy Endpoints ...................................................................................... 49 8.1.2 Secondary Efficacy Endpoint ................................................................................... 49 Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 7 Sample Size De termination........................................................................................... 49 Populations for Analyses ..............................................................................................

50 8.3.1 Evaluable for Toxicity .............................................................................................. 50 8.3.2 Evaluable for Obj ective Response ............................................................................ 50 8.3.3 Evaluable for Non-Target Disease Response ........................................................... 50 Statistical Analyses ....................................................................................................... 50 8.4.1 General Approach ..................................................................................................... 50 8.4.2 Analysis of the Primary Efficacy Endpoints ............................................................. 50 8.4.3 Analysis of the Secondary Efficacy Endpoint .......................................................... 50 8.4.4 Safety Anal yses ......................................................................................................... 51 8.4.5 Intention-to-Treat Analysis ....................................................................................... 51 8.4.6 Planned Interim Analyses ......................................................................................... 518.4.7 Sub-Group Analyses ................................................................................................. 51 9 COLLABORATIVE AGREEMENTS.......................................................................... 51 ModernaTX, Inc. ........................................................................................................... 51 10 HUMAN SUBJECTS PROTECTIONS ........................................................................ 52 Rationale for Patie nt Selection ...................................................................................... 52 Participation of Children ............................................................................................... 52 Participation of Subjects Unable to Give Consent ........................................................ 52 Evaluation of Benefits an d Risks/Discomforts ............................................................. 52 Risk/Benefit Analysis ................................................................................................... 53 Consent Process and Documentation ............................................................................ 53 10.6.1 Telephone Consent for Part One Registration ...................................................... 53 10.6.2 Informed Consent of Non-English Speaking Subjects ......................................... 53 11 PHARMACEUTICAL INFORMATION ..................................................................... 54 Cell Preparation ............................................................................................................ 54 Intramuscular Injection Preparation .............................................................................. 54 11.2.1 NCI-4650, mRNA-Based Personalized Cancer Vaccine ...................................... 54 12 REFERENCES ................................................................................................................

56 13 APPENDICES ................................................................................................................. 5 8 Appendix A: Methods for Selecting Defined Epitopes, Predicted Epitopes, and Tumor Suppressor and Driver Neoantigens ......................................................................................... 58 Appendix B: Performance Status Criteria .....................................................................

62 Appendix C: mRNA-4650 Certificate of Analysis ....................................................... 63 Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 81 INTRODUCTION STUDY OBJECTIVES 1.1.1 Primary Objectives x Determine the clinical response rate in patients with meta static melanoma, gastrointestinal, or genitourinary cancers who receive NCI-4650. x Determine the safety of NCI-4650 in patients w ith metastatic melanoma, gastrointestinal, or genitourinary cancer. 1.1.2 Secondary Objective x Determine whether administration of NCI-4650, a mRNA-based, personalized cancer vaccine (PCV) presenting immunogenic neoa ntigens, can increase the quantity and quality of circulating antigen-specific T-cells in patients with solid tumors. BACKGROUND AND RATIONALE 1.2.1 Cancer Vaccines Protective vaccination against infectious diseases has proven to be one of the most effective health measures. Therapeutic vaccination agains t established diseases such as persistent infections and cancer ha s proven much more challenging. Cancer vaccines are designed to target antigens that can elicit an immune response that selectively attacks cancer cells. Ideally, these antigens should be exclusively presented on ca ncer cells. Until recently, vaccines against non- viral tumors mainly targeted differentiation antigens, cancer testis antigens, and overexpressed antigens. However, central immunological tolera nce has limited the generation of high-avidity reactivity against these normal antigens1, 2. Thus, T-cells prompted using these vaccines underwent depletion of high-avidity clones directed against such antigens. This depletion causes the loss of T-cells bearing high-affinity T-cell re ceptors (TCR) for their cognate antigens, which have the superior cytotoxic capacity, longer persistence in the tumor microenvironment, and decreased susceptibility to immune suppression3. Taken together, such depletion can lead to impaired clinical efficiency following vaccine administration. In recent years, our group and others have extensively studied the importance of neoantigens as targets for immunotherapy. It is now clear that neoantigen-specific T-cells are present in most cancers. Neoantigens derived from somatic muta tions offer a specific and highly immunogenic target for vaccination. Today, with the rapid development of technologies for DNA sequencing, the identification of those mutations becomes widely feasible. Evidence for the power of neoantigen vaccines in pre-clinical and clinical settings was recently published. The first proof of concept of neoantig en vaccines for cancer prevention was shown in the B16 mouse melanoma model4. By prophylactic vaccination with two mutated epitopes, the researchers achieved complete tumor protection in 40% of the mice. In therapeutic settings, they observed significant tumor growth inhibition i nduced by mutation-specific peptide vaccination. Recently, the same group published evidence for the potential of neoantigen vaccines as tested in three independent murine models5. In this paper, the researchers showed that a considerable fraction of the non-synonymous mutations in the cancer were immunogenic, and the majority of them were recognized by CD4+ T-cells. Vaccination with these CD4+ epitopes induced potent tumor control and rejection of established tumors. Similar results were published in another independent mutation-based cancer model in mice. In this work, the authors developed a pipeline Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 9 to identify immunogenic mutant peptides usin g whole-exome and transcriptome sequencing together with mass spectrometry. Again, the researchers were able to show that vaccination with mutation-specific peptides induced therapeutically active T-cell re sponses. Results of the current pre-clinical data support the eval uation of neoantigen vaccines as an effective cancer treatment. The first human trial involving a personalized cancer vaccine was conducted by a group from Washington University in St. Louis which report ed on a first-in-man clinical trial evaluating neoantigen vaccines in patients with metastatic melanoma6. These researchers vaccinated three patients with mature de ndritic cells presenting seven pred icted mutation-specific HLA-A*02:01 restricted epitopes. They showed that vaccina tion with high-affinity, patient-specific mutated epitopes augmented T-cell immunity directed against naturally occurring dominant neoantigens and broadened the response by revealing subdominant neoantigen s; but no clinical responses were observed. However, this did show that va ccination against mutated epitopes was safe as none of the three patients experienced any autoimmune adverse events (AEs). Recently, two trials involving personalized cance r vaccines in patients with advanced melanoma were published7, 8. The first7 used synthesized, clinical-grade long peptides targeting up to 20 neoantigens per patient, admixed with the Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA-5). This trial demonstrated the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumor neoantigens where the vaccine-induced polyfunctional CD4+ and CD8+ T-cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T-cells discriminated mutated from wild-type antigens, and in some cases directly recognized autologous tumor. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, while two with recurrent disease were subsequently treated with anti-programmed cell death-1 (anti-PD-1) therapy and experienced complete tumor regression, with expansion of the repertoire of neoantigen-specific T-cells. The authors concluded that these data provide a strong rationale for further development of this approach, alone a nd in combination with checkpoint blockade or other immunotherapies. The second trial8 implemented an RNA-based poly-neoepitope approach to mobilize immunity against a spectrum of cancer mutations also in patients with melanoma. All patients in this trial developed T-cell responses against multiple vacci ne neoepitopes at up to high single-digit percentages. Vaccine-induced T-cell infiltration and neoepitope-specific killing of autologous tumor cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression- free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses . One of these patients had a late relapse owing WRRXWJURZWKRIȕ -microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient devel oped a complete response to vaccination in combination with PD-1 blockade therapy. These authors concluded that individual mutations can be exploited by way of personalized immunotherapy for patients with cancer. 1.2.2 Identification of Cancer-Specific Imm unogenic Epitopes Derived from Somatic Mutations The NCI-SB has developed a process to iden tify immunogenic T-cell epitopes derived from neoantigens ( Figure 1 ). The identification of these antigens is done in four main stages: (1) Whole exome sequencing and RNA-seq analysis of the tumor and a ma tched normal apheresis Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 10sample to identify highly expressed somatic mutations; (2) Construction of tandem minigenes and synthesis of long peptides covering these mutations; (3) Expression of the constructs in autologous antigen-presenting cells; and, (4) In vitro co-culture assay to identify T-cells recognizing mutated epitopes from TIL9. In earlier studies done by the NCI-SB (published and unpublished data), 25 patients with melanoma were screened in the above manner, and 64 antigenic somatic mutations were identified with no overlapping neoantigens between patient tumors10, 11. Recently, a study to identify antigenic mutations fr om patients with epithelial cancers, including those of the gastrointestinal tract, genitourinary tract, and breast, identified 57 non-overlapping somatic mutations. In this study, two patient s were identified who developed a T-cell response against the mutated KRAS oncogene12. Thus far, more than 121 immunogenic epitopes have been identified from multiple cancer types including melanoma, ovarian, colorectal, lung, and breast cancers9, 10, 12-15. This process, which can be applie d to any cancer type and can be completed in several weeks, ensures that the selected antigens are not just expressed in the tumor but can also prompt a significant T-cell-mediated immune response. Figure 1. A pipleline for the identification of immunogenic somatic mutations. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 11 1.2.3 Overview of mRNA-4650, a mRNA-Based, Personalized Cancer Vaccine ModernaTX, Inc. (Moderna) is developing a new mRNA technology that allows for induced production of a broad array of se creted, membrane-bound and intra cellular proteins both in cell culture and in many animal models and hum ans. The mRNA vaccines are produced by a well-controlled, characterized, s calable process that is agnostic to the antigen being produced. Additionally, host cell production a nd presentation of the antigen more closely resemble viral antigen expression and presentation compared to an exogenously produced, purified, and formulated protein antigen. This offers advant ages in speed, precision, and adaptability of antigen design and production that cannot be replicated by conventional platforms, which may be especially valuable for personalized cancer vaccines (PCV). This mRNA vaccine production process allows for rapid mRNA production and fo rmulation (within approx imately eight weeks) at sufficient quantities to suppor t individualized manufacture and dosing in a clinical study. Moreover, this mRNA-based vaccine technology overcomes the challenges other nucleotide approaches pose such as preexisting immunity or antivector immunity for viral vectors, the risk of genome integration, or the high doses and devices needed (e.g., electroporation) for DNA- based vaccines. Using a lipid nanoparticle (LNP), Moderna has gene rated very high levels of transient expression of several different types of pr oteins (including those for proph ylactic vaccines for infectious diseases), in several animal species as wells as humans. The approach also avoids the use of conventional immunostimulatory compounds for e nhancement of immunogenicity. Specifically, Moderna has demonstrated that LNP-based, modified-mRNA vaccine technology can generate robust and protective immune responses in mice, ferrets, and cynomolgus monkey, and initial data from early trials in huma ns have shown such mRNA-based vaccines to be safe and well- tolerated. As an example, Moderna has shown that a range of doses of formulated mRNA encoding the hemagglutinin (HA) protein of either H7N9 or H1 0N8, can stimulate rapid, robust, long-lasting, and dose-related immune responses as measured by a hemagglutination inhibition (HAI) test, microneutralization assay, and protection from viral challenge16. Importantly, mRNA is a potent and flexible component for vaccina tion, inducing rapid, durable, and high levels of antigen-specific B- and T-cell responses to protein antigens encoded by mRNA. Moreover, mRNA vaccines can also induce potent T-cell resp onses to murine and human neoantigens, while not inducing strong responses to epitope s derived from the wild-type sequences5. For this clinical trial, Moderna will manufact ure NCI-4650 encoding immunogenic and predicted neoantigens expressed by the patie nt autologous tumor identified by the NCI-SB. The goal of the approach is to prime, activate, and expand a di verse population of neoantigen-specific T-cells against these immunogenic and predicted neoantigens for the treatment of solid tumors. As part of this approach, next-gener ation sequencing (NGS), HLA epitope prediction, and high throughput epitope screens will be employed to enable robust and consistent vaccine design within this clinical study. 1.2.4 Preclinical Experiments Evaluating the Safe ty of mRNA-Based, Personalized Cancer Vaccines To support the proposed Phase I clinical study, the safety and tolerability of a similar, mRNA- based PCV , mRNA-4157 (IND 17390), was evaluate d in a Good Laboratory Practices (GLP)- compliant one-month toxicity stud y, in outbred Sprague Dawley® rats at 0.012, 0.035, and 0.117 mg per dose via IM injection administered weekly for a total of 5 doses, and followed by a Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 12 14-day post-study recovery period. These doses ap proximate to human e quivalent doses (HED) of 0.29, 0.97, and 3.51 mg, respectively (based on group mean body weights of 0.46, 0.40, and 0.37 kg in rats, respectively, and using an average human body weight of 70 kg), and thus approximate margins of 7-, 24- and 88-fold over the human starting dose of 0.04 mg.

The study involved a more dose-dense regimen than the proposed human dosing frequency of every 2 weeks which is being used herein. Although more frequent administration of antigens can impact immunogenic responses, it is unlikely that more frequent dosing would interfere with immunogenicity of this drug product as the antig ens expressed are not intended for the rodent and are selected to cross a broad spectrum of major histocompatibility complex (MHC) specificities. A more aggressive regimen than the intended clinical dosing frequency was selected to thoroughly characterize drug product toxicity. Although the vaccine design of NCI-4650 is 100% personalized (i.e., sequences coding for tumor-associated neoantigens unique to the subject and predicted to be most immunogenic will be administered for each subject), the sequence tested in this toxicology study represented a human-based sequence of comparable size and composition to the NCI-4650 that will be generate d for each subject in the clinical study, and was manufactured in a similar ma nner to how NCI-4650 will be manuf actured in this clinical study, with the exception that the immunogenic neoantigens coded by NCI-4650 will be determined by laboratory techniques at the NC I-SB and bioinformatics prediction algorithms described in Section 3.2.R in IND 17390. The primary driver of safety that can be adequately assessed non-clinically is that of the LNP and mRNA chemistry, and any immunological effects of antigen expression is likely to be subject-specific and more adequately assessed clinically. The results of the repeat-dose toxicity study in rats indicated that mR NA-4157 was tolerated at all dose levels (no mRNA-4157 related mortality was observed). In addition, no changes in body temperature (measured indirectly by clinical observation), serum chemistry, urinalysis, or ocular findings were noted. Weekly administration of mRNA-4157 to Sprague Dawley rats (both male and female) at dose levels of 0.012, 0.035, and 0.117 mg was associated with a dose-dependent VOLJKWWRPLOGGHFUHDVHLQERG\ZHLJKWJDLQDWGRVHV• 0.012 mg and this correlated with reduced food consumption. Clinical observations of inject ion-site reaction (ISR) were observed at all doses and included erythema (slight to mild) and edema (slight to moderate) that increased in incidence and severity with increasing dose. These clinical observations were associated with gross and microscopic changes at the injection site and associated enlargement of lymph nodes. Hematology changes consistent with inflammation were also observed and included mild to moderate increased white blood cells (WBCs) (dri ven primarily by neutrophils), and a decrease in lymphocytes. Systemic effects were primarily considered responses to local inflammation at the injection site.

At the end of the recovery period, all changes were fully or partially resolved (absolute body weights remained lower than controls). 1.2.5 Preclinical Experiments Evaluating the I mmunogenicity of a mRNA-Based, Personalized Cancer Vaccine in Mice To determine the ability of a poly-epitope concatemer mRNA vaccine to generate T-cell responses to multiple epitopes in C57Bl/6 mic e, we produced a con catemer mRNA vaccine containing 15 known mouse epitopes (sourced from Immune Epitope Database [IEDB]) that bind to alleles H-2-Kb, H-2-Db, a nd H-2-IAb in C57Bl/6 mice ( Table 1 ). Mice were vaccinated on Days 0 and 14, and spleens were harvested on Day 21. Splenocytes were restimulated with peptides corresponding to the vaccine epitopes and positive responses were detected via IFN- Ȗ ELISpot assay. Positive T-cell responses were measured for 4 of 5 Class II epitopes targeting Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 13IAb, 3 of 5 Class I epitopes targeting Kb, a nd 5 of 5 Class I epitopes targeting Db ( Figure 2 ). Restimulation with vehicle produced no T-cell res ponses and robust responses were detected to phorbol myristate acetate/ionomycin in both the empty SM-102 and concatemer vaccine groups. As expected, the negative control SM-102 vacc ine containing no mRNA did not induce T-cell responses upon peptide restimulation. Positive T-ce ll responses were measured for 4 of 5 (CAP2- CAP5) Class II epitopes targeting IAb, 3 of 5 (CAP6, CAP7, and CAP9) Class I epitopes targeting Kb, and 5 of 5 (CAP11-CAP 16) Class I epitopes targeting Db ( Figure 2 ) when splenocytes from mice vaccinated were restimulate d with peptides corresponding with epitopes in the vaccine. These data demonstrate the ability of a poly-epitope concatemer vaccine to elicit robust T-cell responses to multiple epitopes targeting distinct Class I and Class II alleles in a single mRNA construct. The vaccine design used in this study is similar to the proposed design for the clinical trial; these data provide evidence that concatemerizing multiple epitopes together in a single mRNA sequence will lead to eff ective protein translation, processing, and presentation of the epitopes on MHC molecules. Although capable of eliciting T-cell responses against known T-cell epitopes, we decided to fu rther determine if neoantigen-encoding mRNA vaccines can elicit antigen-specific T-cell responses in C57Bl/6 mice. Table 1. Concatemer mRNA vaccine epitopes. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 14 Figure 2. Concatemer mRNA vaccine epitopes. To test the immunogenicity of neoantigen speci fic mRNA vaccine, mice were vaccinated either subcutaneously (SQ) or intravenously (IV) tw ice (prime day 0, boost day 14) with three mRNA vaccines (given in a single injection), with each vaccine containing an mRNA sequence encoding 1 of 3 mutant epitopes identif ied by Yadav and colleagues17 (Figure 3 ) in the MC38 tumor cell line. Three days after, the boost spleens were harv ested and cells were rest imulated with peptides corresponding to the mutant or wild-type peptid e sequences for the 3 neoantigens. To compare the mRNA vaccine to a common peptide vaccine, mice were also vaccinated intraperitoneally with long peptides corresponding to the neoan tigens sequence together with PolyI:C and anti- CD40 antibody. To evaluate the phenotype of the neoantigen-specific T-cells elicited by the vaccine, T-cells were also tested for CD62L and CD44 expression. Figure 3. MC38 neoantigen-specific T-cell responses in C57Bl/6 mice. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 15Strong CD8+IFN-Ȗ+ T-cell responses were detected from sp lenocytes of mice vaccinated SQ or IV with the combination mRNA vaccine containing the three mutant neoantigens when restimulated with the mutant peptides ( Figure 3 ); the mutant-specific responses were significantly higher than the responses measure to wild-type peptides or when there was no peptide restimulation (vehicle). Re sponses were comparable for IV and SQ administration of the mRNA vaccine, and vaccination with peptide+ad juvant+anti CD40 against the neoantigens resulted in minimal/low specific T-cell responses upon restimulation with mutant peptides (Figure 3 and Table 2 ) that were significantly lower than the responses measured with mRNA vaccines. The neoantigen-specific T-cells respondi ng to restimulation with mutant peptide were demonstrated to be primarily of the CD62L lo and CD44hi phenotype, resembling an effector memory cell type (representative flow cytometry plots are included in ( Figure 3 ). Table 2. MC38 neoantigen-specific T-cell responses in C57B1/6 mice. To summarize, robust neoantigen-specific CD8+IFN-Ȗ+ T-cell responses were induced in mice vaccinated, either SQ or IV, with the combination vaccine containing three mutant sequences; the T-cell responses were comparable betw een the SQ and IV vaccinated groups. The neoepitope-specific T-cells generated throug h mRNA vaccination had an effector memory phenotype which is thought to be necessary for a successful anti-tumor response 18. The antigen- specific responses to the mutant peptides were significantly higher than responses to restimulation with either the wild-type antigen sequences or vehicle, demonstrating the ability of the neoepitope mRNA vaccine to produce highly -specific responses. In addition to being specific, the response to mRNA vacci nation were also significantly higher than responses to the adjuvanted peptide vaccine, validating the use of the mRNA platform in delivering neoepitopes for the generation of antigen-specific T-cell responses in the context of a PCV. These data demonstrate that mRNA vaccines can induce potent and specific T-cell responses to multiple murine neoantigens. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 161.2.6 Preclinical Experiments Evaluating the Presentation of CD4 and CD8 Epitopes by Human Dendritic Cells To test if human CD4 and CD8 epitopes ca n be processed and presented from mRNA concatemer, a screening system to evaluate the presentation of two CD8 and two CD4 epitopes in one assay was developed. A concatemeric mR NA encoding 25mer long antigens either on the 3’ (CA-48) or the 5’ (CA-49) of the mRNA back bone was produced. As a control, we used a single 25mer mRNA corresponding to each epito pe and a peptide pool containing all four epitopes. To test antigen presentation, dendritic cells (D C) were transfected with the concatemer constructs or loaded with a peptide pool containi ng all four epitopes. DCs were co-cultured with peripheral blood mononuclear cells (PBMC) tr ansduced with TCRs recognizing the corresponding antigens, and antigen recognition was evaluated by flow cytometry for 41BB expression and ELISA for IFN- Ȗ(Figure 4 ). The results clearly show that both CD8 and CD4 epitopes can be processed and presented efficiently after transfection with concatemers harboring several CD8 and CD4 epitopes. While known that human DC are the major population of antigen-presenting cells to uptake and process mRNA molecules in vivo19, these results support the assumption a concatemer mRNA vaccine can gi ve rise to widespread T-cell activation upon mRNA delivery. Figure 4. Presentation of mRNA-derived CD4 and CD8 epitopes by human dendritic cells. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 171.2.7 Rationale for Selecting Defi ned Epitopes, Predicted Epitope s, and Tumor Suppressor and Driver Neoantigens to Use in this Trial Historically, preclinical and clinical ne oantigen vaccines used epitopes predicted in silico . Although in silico prediction can select for proper antigens, in many cases there is no evidence that those antigens are processed and presented by the tumor or antigen presenting cells in the microenvironment. As described above, in addition to up to 15 predicted neoantigens, patient TIL will be screened to identify defined mutated antigens ( Table 3 ) recognized by T-cells guaranteeing that those antigens are processed and presented for T-cells in the tumor or its draining lymph nodes. Table 3. Defined mutated antigen s recognized by TIL. There will be one exception for using predicted or laboratory-derived, immunogenic neoantigens in this trial. There is growing evidence to su ggest that targeting tumor driver and suppressor genes can elicit superior clinical response. Muta tions in these genes are highly common and have been found in many tumors. When mutations are identified in KRAS, NRAS, p53, EGFR, IDH1, PIK3CA or any other potential driver mutations, nuc leotides coding for peptides covering those epitopes will be synthesized and used in the vaccine composition. Augmenting T-cell responses against defined and driver neoantigens should increase the likelihood of a significant clinical response. Appendix A describes the three different classes of immunogenic antigens that will be included in the vaccine backbone, and provides the met hods for identifying the defined epitopes and selecting the predicted epitopes. 1.2.8 Structure of NCI-4650 A schematic of NCI-465 0 is provided in ( Figure 5 ). NCI-4650 is chemically identical to naturally occurring mammalian mRNA with the exception that the uridine nucleoside normally Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 18present in mammalian mRNA is fully replaced with N1-methyl-pseudouridine, a naturally occurring pyrimidine base present in mammalian tRNAs20, 21. The cap structure used in NCI-4650 is identical to the natural mammalian Cap 1 structure22, 23. Each mRNA construct will be between 500 and 3000 nucleotides in length codi ng for neoantigens between 15 and 41 amino acids in length. Figure 5.

Schematic of mRNA-4650 design. 1.2.9 Risk for NCI-4650 Monotherapy The risk to subjects receiving NCI-4650 is expect ed to be low and primarily involving mild to moderate toxicities which have been observed in animal studies and generally observed and expected for other IM administered vaccines. Thes e local reactions may consist of transient and dose-dependent pain, swelling, and erythema. Possible mild to moderate systemic reactions, which are also transient, may include fever, fatigue, chills, headache, myalgias, and arthralgias. Such AEs may, in part, be due to the poor biodegradability of LNP formulations used in other vaccine studies; the LNP that comprises NCI-4650 is designed to have superior biodegradability and thus may have a further improved safety prof ile. Also, other AEs that have generally been associated with approved IM administered vaccines have included mild hematological and clinical chemistry abnormalities, which are usually reversible. Subjects receiving NCI-4650 could experience signs and symptoms compatib le with a C activati on-related pseudoallergy (CARPA) response, which has been observed in the administration of approved liposomal products, contrast agents, pegy lated proteins, and antibodies 24, as well as for small interfering RNA products formulated in LNPs25, 26. The signs and symptoms of CARPA resemble those of an acute hypersensitivity reaction. However, complement activation is far less likely to be associated with clinical signs for LNP products such as NCI-4650, which are administered IM at a much lower dose on a mg/kg basis than other clinical entities that cause CARPA in humans and nonclinical species. The design of this study allows an escalation of dose with intensive safety monitoring to ensure the safety of subjects, the study will use a traditional “3+3” design.

Any potential safety risks, such as ISRs, will be monitored closely throughout the study. Dosing will be staggered by three weeks between the first dose administered to the first patient and the first dose administered to the second patient in Dose Level 2 (starting dos e level) of the Phase I Cohort for safety monitoring purposes. Subsequent patients may begi n treatment at an interval of one week. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 192 ELIGIBILITY ASSESSMENT AND ENROLLMENT ELIGIBILITY CRITERIA 2.1.1 Inclusion Criteria Measurable (per RECIST v1.1 criteria), meta static melanoma, ga strointestinal, or genitourinary cancer with at least one lesi on that is resectable. Only patients with metastatic gastrointestinal cancer will be eligible for enrollment on the Phase I portion of the study. Patients with metastatic melanoma, gastrointestinal, or genitourinary cancer will be eligible for enrollment on the Phase II portion of the study. Confirmation of diagnosis of metastatic cancer by the NCI Laboratory of Pathology. Patients with 3 or fewer brain metastases that are < 1 cm in diamet er and asymptomatic are eligible. Lesions that have been treated with stereotactic ra diosurgery must be clinically stable for one month after treatment for the patient to be eligible. Patients with surgically resected brain metastases are eligible. Prior therapy with at least one first-line standard of care treatment or second-line treatment of proven effectiv eness. Patients must have pr ogressive disease after prior treatment. Prior first- or second-line treatments would include the following: x Patients with metasta tic melanoma: Receipt of a chec kpoint inhibitor as first-line therapy. x Patients with metastatic melanoma with an activating mutation of KIT: Receipt of imatinib. x Patients with a BRAF V600 activating mutation: Receipt of appropriate targeted therapy. x Patients with metastatic gastrointestinal cancer: Receipt of up to two forms of approved first- and/or second-line chemotherapy regimens. x Patients with metastatic genitourinary cancers: Receipt of a first- or second-line therapy appropriate for their histologic subtype. $JH•\HDUVDQG”\HDUV Clinical performance status of ECOG 0 or 1 (see Appendix B ). Women of child-bearing potential must have a negative pregnancy test because of the potentially dangerous effects of the treatment on the fetus. Serology x Seronegative for HIV antibody. (The experimental treatment being evaluated in this protocol depends on an intact immune system. Patients who are HIV seropositive may have decreased immune-com petence and thus may be less responsive to the experimental treatment and more susceptible to its toxicities.) x Seronegative for hepatitis B antigen, and seronegative for hepatitis C antibody. If hepatitis C antibody test is positive, then the patient must be tested for the presence of antigen by RT-PCR and be HCV RNA negative. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 20 Hematology x ANC > 1000/mm3 without the support of growth factors. x :%&•PP3 x 3ODWHOHWFRXQW•PP3 x Hemoglobin > 8.0 g/dL.

Subjects may be transfused to reach this cut-off. Chemistry x Serum ALT/AST ” 5.0 x ULN x Serum creatinine < 1.5 x ULN or measured creatinine clearance (calculated using Cockcroft-Gault formula) > 40 mL/min x 7RWDOELOLUXELQ” 2.0 mg/dL, except in patients with G ilbert’s Syndrome, who must have a total bilirubin < 3.0 mg/dL. More than four weeks must have elapsed since any prior systemic therapy at the time the patient receives the immunization regimen, and patients’ toxicities must have recovered to a grade 1 or less (except for toxicities such as alopecia or vitiligo).

Ability of subject to understand and the willingness to sign a written informed consent document. Subjects must be co-enro lled on protocol 03-C-0277. 2.1.2 Exclusion Criteria Pregnant or breastfeeding women who do not consent to stop breastfeeding while on study treatment and for 30 days after the use of the investigational vaccine, where pregnancy is confirmed by a positive, rising hCG laboratory test. Women of child-bearing potential, defined as all women capable of becoming pregnant, unless they agree to use an appropriate me thod of contraception during dosing and for 120 days after the last dose (i.e., final vacci ne). Effective contraception methods include a combination of any two of the following (unless method is abstinence or sterilization, in which only one method is required): x Use of oral, injected, or implanted hormona l methods of contraception, or other forms of hormonal contraception that ha ve comparable efficacy (failure rate < 1%). In case of use of oral contraception, women should have been stable on the same pill for a minimum of 6 months before taking study treatment. xPlacement of an intrauterine device or intrauterine system. x Barrier methods of contraception: condom or occlusive cap (diaphragm or cervical/vault caps) with spermicidal fo am/gel/film/cream/vaginal suppository. x Total abstinence x Female sterilization at least eight weeks before taking study treatment. x Male sterilization (at least six months prior to screening). Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 21 Sexually active males must use a condom dur ing intercourse during dosing and for 120 days after the last dose (i.e., final vaccine), a nd should not father a child in this period. Any systemic steroid therapy or other fo rm of immunosuppressive therapy within 7 days of the first dose of the vaccine. A ph ysiologic dose of systemic corticosteroids may be approve G,QKDOHGRUWRSLFDOVWHURLGVDQG DGUHQDOUHSODFHPHQWVWHURLG GRVHV” 10 mg daily prednisone equivalent are permitted in the absence of active autoimmune disease. Active systemic infections requiring anti-infective treatment, coagulation disorders, or any other active or uncompensated major medical illnesses of the cardiovascular, respiratory, or immune system.

Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency Disease). Concurrent opportunistic infections (The experimental treatment bein g evaluated in this protocol depends on an intact immune sy stem. Patients who ha ve decreased immune- competence may be less responsive to the ex perimental treatment and more susceptible to its toxicities). Any vaccinations four weeks prior to the first vaccination cycle or live vaccines at any time during the study. SCREENING EVALUATIONS Note: Testing for screening evaluation is c onducted under the NCI-SB companion protocol, 99- C-0128 (Evaluation for NCI Surgery Bran ch Clinical Research Protocols). 2.2.1 Within 3 Months Prior to Part Two Registration x HIV antibody titer, HBsAg determination, and anti-HCV x Confirmation of diagnosis of metastatic melanoma, gastrointestinal, or genitourinary cancer by the NCI Laboratory of Pathology. (Note: Testing is pe rmitted to be conducted at any time prior to Part Two registration.) 2.2.2 Within 4 Weeks Prior to Part Two Registration x Complete history and physical examination. (Note: Patient history may be obtained within 8 weeks prior to Part Two registration.) x Imaging evaluation to determine baseline di sease status. This may include CT, MRI, PET, and/or photography. 2.2.3 Within 7 Days Prior to Part Two Registration x Screening blood tests: o CBC w/differential o Chemistries: Creatinine, ALT/GPT, AST/GOT, Total bilirubin o Lymphocyte phenotyping (TBNK) x Urinalysis, with culture if indicated Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 22x Physical examination, including ECOG of 0 or 1 (see Appendix B ) x Weight and vital signs x ȕ-HCG pregnancy test (serum or urine) on all women of child-bearing potential REGISTRATION AND TREATMENT ASSIGNMENT PROCEDURES 2.3.1 Prior to Registration for this Protocol Patients will initially sign the consent for a nd enroll on protocol 03-C-0277 prior to tumor harvest and leukapheresis for ge neration of the cell product. Patie nts will then sign the consent for and register for Part One of this trea tment protocol. If the mRNA vaccine meets the requirements specified in the Certificate of Analysis (CoA) ( Appendix C ) to include USP <71> sterility testing, the vaccine will be released by Moderna and shipped to the NIH Clinical Center Pharmacy, where it will be disp ensed and administered to the patient after receipt by the pharmacy of a physician’s order. The patient must meet the eligibility criteria and be registered to Part Two of this treatment protocol before receiving the vaccine. If the mRNA vaccine does not meet the requirements specified in the CoA, the vaccine will not be released by Moderna and the patient will be ta ken off-study (Section 3.8.2). 2.3.2 Registration Procedure Registration will be a two-part process. Authorized staff must register an eligible candidate with the NCI Central Registration Office (NCI-CRO) w ithin 24 hours of signing consent. To initially register a subject after the participant has signed the consent, complete the top portion of the registration Eligibility Checklist from the website (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) indicating that the patient is being registered for Part One and send via encrypted email to the NCI-CRO at [email protected]. Once the mRNA vaccine meets the requirements specified in the CoA and the vaccine is released by Moderna, and eligibility is confirmed after completion of scr eening studies, authorized staff must complete the remainder of the eligibility checklist, indicating that the patient is being registered for Part Two (treatment on study). Email the completed registration Eligibility Checklist to the NCI-CRO at [email protected]. After confirmation of eligibility at the NCI-CRO, CRO staff will call pharmacy to advise them of the acceptance of the patient on the protocol prio r to the release of any investigational agents. Verification of registration will be forwarded electronically via email to the research team. A recorder is available during non-working hours. Subjects that do not meet scr eening criteria should be removed from the study following the procedure in Section 3.8.3.

Note: Patients are not considered to be fully enrolled on this treatment prot ocol unless they meet the eligibility criteria and are registered to Part Two. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 23 2.3.3 Treatment Assignment Procedures Cohorts Number Name Description 1 Phase I Gastrointestinal Cancer First 12-18 patients enrolled with a diagnosis of gastrointestinal cancer 2a Phase II Melanoma Patients with a diagnosis of melanoma 2b Phase II Gastrointestinal or Genitourinary Cancer Patients with a diagnosis of gastrointestinal or genitourinary cancer Arms Number Name Description 1 Phase I Escalating doses of mRNA vaccine 2 Phase II MTD of mRNA vaccine established in Phase I Randomization and Arm Assignment This is a non-randomized study. All patients will be directly assigned based on cohort as follows: x Subjects in Cohort 1 will be directly assigned to Arm 1.

x Subjects in Cohort 2a will be directly assigned to Arm 2. x Subjects in Cohort 2b will be directly assigned to Arm 2. 3 STUDY IMPLEMENTATION STUDY DESIGN 3.1.1 Schema 3.1.2 Pre-Treatment Phase: Performed on 03-C-0277 Patients with metasta tic melanoma, gastrointestinal, or genitourinary cancers will undergo resection of the tumor and apheresis under pr otocol 03-C-0277. Whole exome sequencing will be performed under protocol 03-C-0277 to identif y all cancer mutations using our standard procedures. TIL will be grown and expanded for this trial according to Standard Operating Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 24 Procedures (SOPs) which can be found in BB -IND 17858. After a sufficient yield of TIL (5x107 cells) is expanded, immunogenic neoantigens will be identified by high throughput immunologic screening using long peptides and tandem minigenes covering all mutated epitopes to identify the exact mutations recognized by autologous T-cells. Up to 15 predicted neoantigens will be selected based on exome and RNA sequencing and their binding affinity to the patient HLA molecules (see Appendix A ). The final nucleotide sequences of the mutated 15-41mer epitopes and/or driver genes will then be transmitted electronically via Globus, a secure research data management system, to Moderna for pr oduction of the NCI-4650 drug product. 3.1.3 Treatment Phase Patients will receive a mRNA-based vaccine IM at two-week intervals for four cycles. All patients may receive one course of treatment (four vaccination cycles). Immunologic tests will be performed as per Section 5.4 to quantitate circulating T-cells reactive with the immunizing antigens. If a 3-fold increase in the precursor level of pre-existing neoantigen-specific T-cells or new reactivity appears in the blood at detectable levels, patients may be vaccinated with a second and final course of treatmen t using the same vaccine dose. The second course may start approximately four weeks (± 2 weeks) from the last vaccine dose of the first course. If the patient receives only one course of treatment (four vaccination cycles), the start date of the course will be the date of the first vaccination; the end date will be the date of the first post- treatment evaluation. If the patient receives a second course (four additional vaccination cycles), the start date of the first course will be the date of the first va ccination. The date of the fifth vaccination will be both the end date of the first course and the start date of the second course. The end date of the second cour se will be the date of the fi rst post-treatment evaluation. 3.1.4 Dose-Limiting Toxicity The dose-limiting toxicity (DLT) evaluation period for determination of the maximum tolerated vaccine dose (MTD) will occur during the 21 days af ter the first vaccination for each patient. A DLT is defined as follows: All grade 3 or gr eater toxicities related to the mRNA vaccine, with the exception of : x Grade 3 fever x Grade 3 pruritus/itching x Grade 3 fatigue x Grade 3 metabolic laboratory abnormalities without signifi cant clinical sequela that resolve to grade 2 or less within 7 days. x Grade 3 autoimmune toxicity that resolves to grade 2 or less within 7 days.

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.

x The NCI Pharmacy Manual contains details regarding sterility release of NCI-4650. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 28 BASELINE EVALUATIONS 3.4.1 Within 7 Days Prior to the First Vaccination (Day 0) x Physical examination, including ECOG (see Appendix B ) x Weight and vital signs x Baseline blood tests: o CBC w/differential o Acute Care Panel (sodium, potassium, chlo ride, bicarbonate, creatinine, glucose, BUN), Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), Mineral Panel (a lbumin, calcium, magnesium, phosphorus), Uric acid, Creatinine kinase, Lactate dehydrogenase, Total protein o PT-INR/PTT o Anti-CMV antibody titer, HSV and VZV serology, and EBV panel (Note: Patients who are known to be positive do not need to be retested. May be performed within 3 months prior to Part Two registration.) x ȕ-HCG pregnancy test (serum or urine) on all women of child-bearing potential x Urinalysis, with culture if indicated x Chest x-ray x EKG x Repeat imaging if not performed within 4 weeks of Day 0. This may include CT, MRI, PET, and/or photography. 3.4.2 Within 2 Days Prior to the First Vaccination (Day 0) x Lymphocyte phenotyping (TBNK) x Review of baseline symptoms x Research samples, as described in Section 5.3 ON-STUDY EVALUATIONS 3.5.1 Within 2 Days Prior to Each Subsequent Vaccination (Days 14, 28, & 42 (± 5 days)) x Physical examination, including ECOG (see Appendix B ) x Weight and vital signs x Toxicity assessment, including a review of systems x CBC w/differential x Acute Care Panel (sodium, potassium, chlori de, bicarbonate, creatinine, glucose, BUN), Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), Mineral Panel (albumin, calcium, magnesium, phosphorus ), Uric acid, Creatinine kinase, Lactate dehydrogenase, Total protein Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 29x ȕ-HCG pregnancy test (serum or urine) on all women of child-bearing potential x Research samples, as described in Section 5.3 3.5.2 After Each Vaccination x Vital signs will be mon itored hourly (± 15 minutes) for two hours. The post- administration monitoring period may be adjusted by the PI. POST-TREATMENT (FOLLOW -UP) EVALUATIONS 3.6.1 First Follow-up Evaluation All patients (unless the patient receives a second course of treatment) will return to the NIH Clinical Center for their first follow-up evaluati on on day 56 (± 5 days), which is approximately two weeks after the fourth vaccination. If a patient receives a second course of four vaccines, the first follow-up evaluation will be two weeks (± 5 days) after the eighth vaccination. At the first follow-up evaluation, patients will undergo: x Physical examination, including ECOG (see Appendix B ) x Weight and vital signs x Toxicity assessment, including a review of systems x CBC w/differential x Acute Care Panel (sodium, potassium, chlori de, bicarbonate, creatinine, glucose, BUN), Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), Mineral Panel (albumin, calcium, magnesium, phosphorus ), Uric acid, Creatinine kinase, Lactate dehydrogenase, Total protein x Lymphocyte phenotyping (TBNK) x ȕ-HCG pregnancy test (serum or urine) on all women of child-bearing potential x Research samples, as described in Section 5.3. Approximately 72 mL of blood will be obtained. PBMC will be cryopreserved so th at immunologic testing may be performed. This will be performed on protocol 03-C-0277. x Imaging studies as performed at baseline to determine tumor response. If clinically indicated, other scans or x-rays may be performed, e.g.

brain MRI, bone scan. x A 5-liter apheresis may be performed. If th e patient is unable to undergo apheresis, approximately 96 mL of blood may be obtained. This will be performed on protocol 03- C-0277. 3.6.2 Subsequent Follow-up Evaluations All patients will return to the NIH Clinical Ce nter for their second follow-up evaluation one month (± 5 days) after the first follow-up evalua tion. Subsequent follow-up evaluations will take place every 1-2 months for the first year, and then annually for up to five years or until clinically proven progressive disease, whichever comes first. Patients will be co-enrolled and followed on the NCI-SB long-term follow-up protocol 09-C-0161 (Follow-up Protocol for Subjects Previously Enrolled on NCI Surgery Branch Studies). As noted in Section 3.6.1, additional Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 30 apheresis may be performed only at the first follo w-up evaluation. It will not be performed at any other post-treatment evaluations.

Note: Patients may be seen more frequently as clinically indica ted. Patients who are unable or unwilling to return for follow-up evaluati ons may be followed via phone or email contact. Patients may be asked to send laboratory, imaging, and physician exam reports performed by their treating physician. At each scheduled follow-up evaluation, patients will undergo: x Physical examination, including ECOG (see Appendix B ) x Weight and vital signs x Toxicity assessment, including a review of systems x CBC w/differential x Acute Care Panel (sodium, potassium, chlori de, bicarbonate, creatinine, glucose, BUN), Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), Mineral Panel (albumin, calcium, magnesium, phosphorus ), Uric acid, Creatinine kinase, Lactate dehydrogenase, Total protein x Lymphocyte phenotyping (TBNK) x ȕ-HCG pregnancy test (serum or urine) on all women of child-bearing potential (complete at all follow-up evaluations within 120 days after the last vaccine) x Research samples, as described in Section 5.3. Approximately 72 mL of blood will be obtained at each follow-up visit. PBMC will be cryopreserved so that immunologic testing may be performed. This will be performed on protocol 03-C-0277. x Imaging studies as performed at baseline to determine tumor response. If clinically indicated, other scans or x-rays may be performed, e.g.

brain MRI, bone scan. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 31 STUDY ASSESSMENT CALENDAR AssessmentsScreening (Prior to Part Two registration) Baseline (Prior to first vaccine) On-Study/Course of Treatment (1 vaccine = 1 cycle)1 Post-Treatment Follow-up Within 3 MonthsWithin 4 WeeksWithin 7 DaysWithin 7 DaysWithin 2 DaysDay 0Day 14 (±5 d)Day 28 (±5 d)Day 42 (±5 d)Day 562 (±5 d)Subsequent Follow-up Visits3 Confirmation of diagnosis by NCI Lab of Pathology4 X Medical history5 X Physical exam X X X X X X X X Performance status (ECOG)6 X X X X X X X Weight X X X X X X X Vital signs7 X X X X X X X X ȕ-HCG pregnancy test8 X X X X X X X9 Urinalysis10 X X EKG X Toxicity assessment11 X12 X X X X X Serology 1 Assessments must be performed within 2 days prior to each vaccination on Days 14, 28, & 42. Patients may receive a 2nd course of treatment for a total of 8 cycles. 2 If a patient receives a second course of four vaccines, the first follow-up evaluation will be two weeks (± 5 days) after the eighth vaccination. 3 All patients will return to the NIH Clinical Center for their second follow-up evaluation one month (± 5 days) after the firs t follow-up evaluation. Subsequent follow- up evaluations will take place every 1-2 months for the first year, and then annually for up to five years or until clinically proven progressive disease, whichever comes first. Patients may be seen more frequently as clinically indicated. Patients who are unable or unwilling to return for follow- up evaluations may be followed via phone or email contact. Patients may be asked to send laboratory, imaging, and physician exam reports performed by their treating phy sician. 4 Confirmation of diagnosis of metastatic melanoma, gastrointestinal, or genitourinary cancer. Testing is permitted to be condu cted at any time prior to Part Two registration. 5 Patient history may be obtained within 8 weeks prior to Part Two registration. 6 ECOG of 0 or 1 (see Appendix B ). 7 After each vaccination, vital signs will be monitored hourly (± 15 minutes) for two hours. The post-administration monitoring period may be adjusted by the PI. 8 Serum or urine; on all women of child-bearing potential. 9 Complete at all follow-up evaluations within 120 days after the last vaccine. 10 With culture if indicated. 11 Including a review of systems.

12 Review of baseline symptoms. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 32 Assessments Screening (Prior to Part Two registration) Baseline (Prior to first vaccine) On-Study/Course of Treatment (1 vaccine = 1 cycle)1 Post-Treatment Follow-up Within 3 Months Within 4 Weeks Within 7 Days Within 7 Days Within 2 Days Day 0 Day 14 (±5 d) Day 28 (±5 d) Day 42 (±5 d) Day 562 (±5 d) Subsequent Follow-up Visits3 HIV antibody titer, HBsAg, anti- HCV X Anti-CMV antibody titer, HSV and VZV, EBV panel 13 X Laboratory Procedures CBC w/differential X X X X X X X Blood chemistries14 X X X X X X X PT-INR/PTT X Lymphocyte phenotyping (TBNK) X X X X Additional apheresis15 X Correlatives16 CPT tubes (SB-CPF) X X X X X X SST tubes (Figg Lab) X X X X X X Imaging CT, MRI, PET, and/or photography X17 X18 X19 X19 Chest x-ray X Treatment/Intervention NCI-4650 mRNA vaccine20 X X X X 13 Patients who are known to be positive do not need to be retested. May be performed within 3 months prior to Part Two registr ation.

14 Screening: Creatinine, ALT/GPT, AST/GOT, Total bilirubin. All other times: Acute Care Panel (sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN), Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), Mineral Panel (albumin, calcium, magne sium, phosphorus), Uric acid, Creatinine kinase, Lactate dehydrogenase, Total protein. 15 A 5-liter apheresis may be performed. If the patient is unable to undergo apheresis, approximately 96 mL of blood may be obt ained. This will be performed on protocol 03-C-0277, and will only be performed at the first follow-up evaluation.

16 Research samples, as described in Section 5.3. 17 Imaging evaluation to determine baseline disease status. 18 Repeat imaging if not performed within 4 weeks of Day 0. 19 As performed at baseline to determine tumor response. If clinically indicated, other scans or x-rays may be performed, e.g., brain MRI, bone scan. 20 Administered intramuscularly. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 33 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF-STUDY CRITERIA Prior to removal from study, effort must be made to have all subjects complete an evaluation safety visit approximate ly 30 days following the first follow-up evaluation (at the second follow- up evaluation). 3.8.1 Criteria for Removal from Protocol Therapy Patients will be taken off treatment for the following: x Completion of second follow-up evaluation x Grade 3 or 4 toxicity due to the vaccine, excluding those known events such as local injection site reactions, skin rash, pruritus, fatigue, fever and local adenopathy. x Progression of disease x Patient requests to be withdrawn from protocol therapy x Investigator discretion x Positive pregnancy test 3.8.2 Off-Study Criteria Patients will be taken off-study for the following: x Vaccine does not meet CoA requirements x Completion of study follow-up period x Progression of disease x Patients begins a new therapy for their cancer x Patient requests to be w ithdrawn from the study x Significant noncompliance x Investigator discretion x Patient lost to follow-up x Patients who require the use of an y medications listed in Section 4 for clinical management. x Death All patients will be co -enrolled on protocol 09-C-0161. Patients who are taken off-study for progressive disease or study closure on the trea tment protocol will be followed on protocol 09-C- 0161.Once a subject is taken off-study, no further data can be collected on this treatment protocol. 3.8.3 Off Protocol Therapy and Off-Study Procedure Authorized staff must notify the NCI-CRO when a subject is taken off protocol therapy and when a subject is taken off-study. A Partic ipant Status Update Form from the website Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 34 (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to the NCI-CRO at ncicentralregist [email protected]. 4 CONCOMITANT MEDICATIONS/MEASURES Use of all medications taken at any time from consent to the last safety follow-up visit must be recorded in the subject’s electronic case report form (eCRF).

This will include all prescription drugs, herbal products, vitamins, minerals, and over-the-counter (OTC) medications. Any changes in concomitant medications also will be recorded in the subject’s eCRF. Any concomitant medication deemed necessary for the welfare of the subject during the study may be given at the discretion of the inve stigator, including palliative radiation. Blood transfusions are allowed at any time during the study, except to meet eligibility criteria. x Antineoplastic systemic chemotherapy or biological therapy is prohibited. x Systemic corticosteroids are prohibited for any purpose other than to treat symptoms from an event of clinical interest of su spected immunologic etiology. The use of physiologic doses of corticosteroids may be approved after consultation with the investigator. If in the interest of safety, the investigator must treat the subject immediately. x ,QKDOHGRUWRSLFDOVWHURLGVDQGD GUHQDOUHSODFHPHQWVWHURLGGR VHV” 10 mg daily prednisone equivalent, are permitted in the absence of active autoimmune disease. x Subjects already taking erythropoietin at the time of screening for the study may continue, provided they have be en taking it for more than one month at the time study treatment is started. Prophylac tic erythropoietin should not be started during screening or the first two weeks of study treatment but may be started thereafter. x Granulocyte colony stimulating factors should not be used prophylactically during the first two weeks of study treatment. Use of prophylactic colony stimulating factors may be considered thereafter following discussion with the investigator. x Live vaccines within 30 days prior to the first dose of NCI-4650 and while participating in the study are prohibited. Examples of liv e vaccines include but are not limited to the following: measles, mumps, rubella, chic ken pox, yellow fever, rabies, Bacillus Calmette-Guérin, and typhoid (o ral) vaccine. Seasonal influe nza vaccines for injection are generally killed virus vaccines and are allowed. However, intranasal influenza vaccines (e.g., FluMist ®) are live attenuated vaccines, and are prohibited. x Radiation therapy to a symptomatic solitary lesion or to the brain may be considered on an exceptional case-by-case basis after docum ented consultation with the investigator. The subject must have clear measurable disease outside the radiated field. Administration of palliative radiation therapy will be considered clinical progression. Subjects who, in the assessment by the investigator , require the use of any of the aforementioned treatments for clinical management should be removed from the trial.

Subjects may receive other medications that the investigator deems to be medically necessary. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 355 BIOSPECIMEN COLLECTION Blood and tissue are tracked at the patient level and can be linked to all protocols on which the patient has been enrolled. Sample s will be used to support the sp ecific objectives listed in the treatment protocol(s), e.g., immunologic monitoring, cytokine levels, persistence, as well as to support long-term research efforts within the NC I-SB and with collaborators as specified in protocol 03-C-0277. The amount of blood that may be drawn from a dult patients for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any eight-week period. Samples will be ordered in CRIS and tracked through the Clinical Trial Data Management system.

Should a CRIS screen not be available, the CRIS downtime procedures will be followed. Samples will not be sent outside NIH without NIH Intramural IRB notification and an executed MTA. SAMPLES SENT TO FIGG LAB x Venous blood samples will be colle cted in either a 4-mL or an 8-mL SST tube to be processed for serum and stored for future research. o Record the date and exact time of draw on the tube. Blood tubes may be kept in the refrigerator until pick-up. o For sample pick-up, page 102-11964. o For immediate help, call 240-760-6180 (main Blood Processing Core number) or, if no answer, 240-760-6190 (main clin ical pharmacology lab number). o For questions regarding sample processing, contact the Blood Processing Core (BPC) at [email protected] or 240-760-6180. o The samples will be processed, barcoded, and stored in the Figg lab until requested by the investigator. SAMPLES SENT TO SURGERY BRANCH CELL PRODUCTION FACILITY x Venous blood samples will be colle cted in 8-mL CPT tubes to be processed and stored for future research. o Record the date and exact time of draw on the tube. Blood tubes are kept at room temperature until pick-up. o Samples will be picked up by the research nurse or designee and transported to the Surgery Branch Cell Production Facility (S B-CPF) within 24 hours of blood draw. o The samples will be processed, barcoded, and stored in SB-CPF. SAMPLE COLLECTION SCHEDULE Collection Point Test/Assay Volume Blood (approx.) Type of Tube Location of Specimen Analysis Prior to Each Vaccination Immune- Monitoring 64 mL 8 mL CPT SST SB-CPF Figg Lab Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 36Each Follow-up Evaluation Immune- Monitoring 64 mL 8 mLCPT SSTSB-CPF Figg Lab IMMUNOLOGICAL TESTING x Apheresis may be performed prior to a nd approximately 2 weeks following the administration of the final vaccine product. At other time points, patient peripheral blood lymphocytes (PBL) will be obtained fro m whole blood by purification using centrifugation on a Ficoll cushion. Aliquots of these PBMC will be cryopreserved for immunological monitoring of cell function. x A variety of tests including evaluation of spec ific lysis and cytokine release, metabolomic and bioenergetic studies (using Seahorse), intracellular FACS of cytokine production, ELISA-spot assays, and lymphocyte subset analysis may be used to evaluate the immunological correlates of trea tment. In general, differences of 2- to 3-fold in these assays over the baseline measurement are indicative of true biologic differences. SAMPLE STORAGE ,TRACKING , AND DISPOSITION FOR SURGERY BRANCH CELL PRODUCTION FACILITY Blood and tissue collected during the course of this study will follow the Cell Tracking and Labeling System established by the SB-CPF. The Cell Tracking and Labeling System is designed to unambiguously ensure that patient/data verifi cation is consistent. The patients’ cell samples (blood or tissue) are tracked by distinct iden tification labels that include a unique patient identifier and date of specimen collection. Cryopreserved blood and tissue samples also bear the date the sample was frozen. All cryopreserved samples are tracked for freezer location and storage criteria. All samples are stored in monitored freezers/refrigerators in 3NW NCI-SB laboratories at specified temperatures with alarm systems in place. Serum samples will be sent to the BPC for storage. Samples will be barcoded and stored onsite or offsite at NCI Frederick Central Repository Services in Frederick, MD. All collected samples (blood or tissue) are entered into a central computer database with identification and stor age location, and this database is backed up every night. If, at any time, a patient withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed and reported as such to the NIH Intramural IRB. Any samples lost (in transit or by a researcher) or destroyed due to unknown sample integrit y (i.e. broken freezer allows for extensive sample thawing, etc.) will be reported as such to the NIH Intramural IRB. Blood and tissue collected during the course of this study will be stored, tr acked, and disposed of as specified in protocol 03-C-0277. SAMPLE STORAGE ,TRACKING , AND DISPOSITION FOR FIGG LAB 5.6.1 Sample Data Collection All samples sent to the BPC will be barcoded, with data entered and stored in the LABrador (aka LabSamples) utilized by the BPC, and data w ill be updated to the NCI-SB central computer database weekly. This is a secure program, with access to LABrador limited to defined Figg lab personnel, who are issued individual user acc ounts.

Installation of LABrador is limited to computers specified by Dr. Figg.

These computer s all have a password-r estricted login screen. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 37All Figg lab personnel with access to patient information annually complete the NIH online Protection of Human Subjects course. LABrador creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without LABrador access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Patient demographics associated with the Clinical Center patient number are provided in the system. For eac h sample, there are notes associated with the processing method (delay in sample proce ssing, storage conditions on the ward, etc.). 5.6.2 Sample Storage and Destruction Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80°C according to stability requirements. These freezers are located onsite in the BPC and offsite at NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times.

Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protoc ol. All requests are monitored and tracked in LABrador. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the NIH Intramural IRB-approved protocol) and that any unused samples must be returned to the BPC. It is the responsibility of the NCI Principal Investigator (PI) to ensure that the samples requested are being used in a manner consistent with NIH Intramural IRB approval. Following completion of this study, samples will remain in storage as de tailed above. Access to these samples will only be granted following NIH Intramural IRB approval of an additional protocol, granting the rights to use the material. If, at any time, a patient withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed and reported as such to the NIH-IRB. Any samples lost (in transit or by a researcher) or destroyed due to unknown sample integrity (i.e. broken freezer allows for extensive sample thawing, etc.) will be reported as such to the NIH Intramural IRB. Sample barcodes are linked to patient demographics and limited clinical information. This information will only be provided to investigators listed on this protocol, via registered use of the LABrador. It is critical that the sample remains linked to patient information such as race, age, dates of diagnosis and death, and histological info rmation about the tumor, in order to correlate genotype with these variables. Blood and tissue collected during the course of this study will be stored, tr acked, and disposed of as specified in protocol 03-C-0277. 6 DATA COLLECTION AND EVALUATION DATA COLLECTION The PI will be responsible for overseeing entry of data into an in-house password-protected electronic system (C3D) and ensuring data accuracy, consistency and timeliness. The PI, associate investigators (AI), research nurses, and/or a contracted data manager will assist with the data management effo rts. All data obtained during the conduc t of the protocol will be kept in secure network drives or in appr oved alternative sites that comply with NIH security standards. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 38Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. Data will be entered into the NCI CCR C3D database. All AEs, including clinically significant abnor mal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Patients will be followed for AEs from the time the patient receive s the first mRNA vaccine dose to the time of the second follow-up evaluation, or until off-study, whichever comes first. An abnormal laboratory value will be re corded in the database as an AE only if the laboratory abnormality is characterized by any of the following: x Results in discontinuation from the study x Is associated with clinical signs or symptoms x Requires treatment or any other therapeutic intervention x Is associated with death or another serious adverse event, including hospitalization x Is judged by the investigator to be of significant clinical impact x If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient’s outcome. All AEs must be recorded on the AE case report form unless otherwise noted below in Section 6.1.1. End of study procedures: Data will be stored according to HHS and FDA regulations, and NIH Intramural Records Retenti on Schedule as applicable. Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, the NIH Intramural IRB will be notified. 6.1.1 Exclusions to Routine Adverse Event Recording There are no exclusions to routine AE reporting/recording for this protocol. DATA SHARING PLANS 6.2.1 Human Data Sharing Plan Coded, linked human data generated for use in future and ongoing research will be shared through a NIH-funded or approved repository (Clin icalTrials.gov) and BTRIS. At the completion of data analysis, data will be submitted to ClinicalTrials.gov either before publication or at the time of publication or shortly ther eafter. Data may also be used to support long-term research efforts within the NCI-SB, and coded, linked data may also be shared with collaborators as specified in protocol 03-C-0277. 6.2.2 Genomic Data Sharing Plan The NIH Genomic Data Sharing Policy does not apply to this study. RESPONSE CRITERIA For the purposes of this study, patients should be re-evaluated for resp onse approximately two weeks following the last vaccination, then one month later x1, then every 1-2 months for the first Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 39 year, and then annually for up to five years or until clinically proven progressive disease, whichever comes first. In addition to a baseline scan, confirmatory scans should also be obtained at least 4 weeks (but not less than 4 weeks) following initial documentation of objective response. Response and progression will be ev aluated in this study using the new international criteria proposed by the revised Response Evaluation Cr iteria in Solid Tumors (RECIST) guideline (version 1.1) 28 Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST v1.1 criteria. 6.3.1 Disease Parameters Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as: x By chest x-ray: • 20 mm x By CT scan: o Scan slice thickness 5 mm or under: • 10 mm o Scan slice thickness > 5 mm: double the slice thickness x With calipers on clinical exam: • 10 mm All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Malignant lymph nodes: To be c onsidered pathologically enlarged and measurable, a lymph node must be > 15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At base line and in follow-up, only the short axis will be measured and followed. Non-measurable disease: All othe r lesions (or sites of disease), including small lesions (longest diameter < PPRUSDWKRORJLFDOO\PSKQRGHVZLWK• 10 to < 15 mm short axis), are considered non-measurable disease. Bone le sions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflamma tory breast disease, and abdominal masses (not followed by CT or MRI) are considered as non-measurable. Target lesions: All measurable lesions up to a ma ximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, shoul d be identified as target lesions and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repe ated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesion s, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum di ameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 40 Non-target lesions: All ot her lesions (or sites of disease) in cluding any measurable lesions over and above the 5 target lesions should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up. 6.3.2 Methods for Evaluation of Measurable Disease All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment a nd the same technique should be used to characterize each identified and reported lesion at baseline a nd during follow-up. Imaging-based evaluation is preferred to evaluation by clin ical examination unless the lesi on(s) being followed cannot be imaged but are assessable by clinical exam. Clinical lesions: Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes) and • 10 mm diameter as assessed using calipers (e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended. Conventional CT and MRI: This guideline has de fined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be pe rformed with breath-hold scanning techniques, if possible. FDG-PET: While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-P ET scanning to complement CT scanning in assessment of progression (partic ularly possible ‘new’ disease) . New lesions on the basis of FDG-PET imaging can be identified according to the following algorithm: x Negative FDG-PET at baseline, with a positiv e FDG-PET at follow-up is a sign of PD based on a new lesion. x No FDG-PET at baseline and a positive FDG-PET at follow-up: If the positive FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD. If the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, additional follow-up CT scans are needed to determine if there is truly progression occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-PET scan). If the positive FDG-PET at follow-up corresponds to a pre-existing site of disease on CT that is not progressing on the ba sis of the anatomic images, this is not PD. x FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in cases where a residual radiographic abnormalit y is thought to represent fibrosis or scarring. The use of FDG-PET in this circum stance should be prospectively described in the protocol and supported by disease-specif ic medical literature for the indication. However, it must be acknowledged that both approaches may lead to false positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 41Note: A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of the surrounding tissue on the attenuation corrected image. 6.3.3 Response Criteria Definitions Evaluation of Target Lesions Complete Response (CR): Disappearance of a ll target lesions. Any pathological lymph nodes (whether target or non-target) must ha ve reduction in short axis to < 10 mm. Partial Response (PR): At least a 30% decrease in the sum of th e diameters of target lesions, taking as reference the baseline sum of diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative incr ease of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progressions.) Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study. Evaluation of Non-Target Lesions Complete Response (CR): Disappearance of all non-target le sions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (< 10 mm short axis). (Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response.) Non-CR/Non-PD: Persistence of on e or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression of existing non-targ et lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease st atus change, not a single lesion increase. Although a clear progression of “non-target” le sions only is exceptional, the opinion of the treating physician should prevail in such circum stances, and the progression status should be confirmed at a later time by the review panel (or PI). Evaluation of Best Overall Response The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both meas urement and confirmation criteria. For Patients with Measurable Disease (i.e., Target Disease) Target Lesions Non-Target Lesions New Lesions Overall Response Best Overall Response when Confirmation is Required* CR CR No CR •4 weeks confirmation** CR Non-CR/Non-PD No PR •4 weeks confirmation** Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 42 CR Not evaluated No PR PR Non-CR/Non- PD/Not evaluated No PR SD Non-CR/Non- PD/Not evaluated No SD Documented at least once •4 weeks from baseline** PD Any Yes or No PD No prior SD, PR, or CR Any PD*** Yes or No PD Any Any Yes PD * See RECIST v1.1 manuscript for further details on what is evidence of a new lesion. ** Only for non-randomized trials with response as primary endpoint.

*** In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as disease progression. Note: Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be reported as “symptomatic deterioration .” Every effort should be made to document the objective progression even after discontinuation of treatment. For Patients with Non-Measurable Disease (i.e., Non-Target Disease) Non-Target Lesions New Lesions Overall Response CR No CR Non-CR/Non-PD No Non-CR/Non-PD* Not all evaluated No Not evaluated Unequivocal PD Yes or No PD Any Yes PD * ‘Non-CR/non-PD’ is preferred over ‘stable di sease’ for non-target disease since SD is increasingly used as an endpoint for assessment of efficacy in some trials; so, to assign this category when no lesions can be measured is not advised. 6.3.4 Duration of Response Duration of overall response: Th e duration of overall respons e is measured from the time measurement criteria are met for CR or PR (whichev er is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 43 TOXICITY CRITERIA The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP website (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm). 7 SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN DEFINITIONS 7.1.1 Adverse Event Any untoward medical occurrence in a human subject, including any abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the subject’s participation in re search, whether or not considered related to the subject’s participation in the research. 7.1.2 Suspected Adverse Reaction Suspected adverse reaction means any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, ‘reasonable possibility’ means there is evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug. 7.1.3 Unexpected Adverse Reaction An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in the Investigator’s Brochure or is not listed at th e specificity or severity that has been observed; or, if an Investigator’s Brochure is not required or available, is not consistent with the risk information described in the gene ral investigational plan or else where in the current application. "Unexpected” also refers to adverse events or suspected adverse reactions that are mentioned in the Investigator’s Brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are no t specifically mentioned as occurring with the particular drug und er investigation. 7.1.4 Serious An unanticipated problem or prot ocol deviation is serious if it meets the definition of a serious adverse event or if it compromises the safety, welfare, or rights of subjects or others. 7.1.5 Serious Adverse Event An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the Sponsor, it results in any of the following: x Death x A life-threatening adverse drug experience x Inpatient hospitalization or prolongation of existing hospitalization Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 44x Persistent or significant disability/incapacity or substantial disruption of the ability to conduct normal life functions x A congenital anomaly/birth defect x Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. 7.1.6 Disability A substantial disruption of a person’s ability to conduct normal life functions. 7.1.7 Life-Threatening Adverse Drug Experience Any adverse event or suspected adverse reaction that places the patient or subject, in the view of the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it does not include a reaction that had it occurred in a more severe form, might have caused death. 7.1.8 Protocol Deviation (NIH Definition) Any change, divergence, or departure from the NIH Intramural IRB-approved research protocol. 7.1.9 Non-Compliance (NIH Definition) The failure to comply with applicable NIH Human Research Protections Program (HRPP) policies, NIH Intramural IRB requirements, or regulatory requirements for the protection of human research subjects. 7.1.10 Unanticipated Problem Any incident, experience, or outcome that: 1. Is unexpected in terms of nature, severity, or frequency in relation to: a. the research risks that are described in the NIH Intramural IRB-approved research protocol and informed consent document; Investigator’s Brochure or other study documents, and b. the characteristics of the s ubject population being studied; AND 2. Is related or possibly related to participation in the research; AND 3.

Suggests that the research places subjects or others at a greater risk of harm (including physical, psychological, economic, or social harm) than was previously known or recognized. NIH INTRAMURAL-IRB AND CLINICAL DIRECTOR (CD) REPORTING 7.2.1 NIH Intramural IRB and NCI CD Expedite d Reporting of Unanticipated Problems and Deaths The protocol PI will report in the NIH Problem Form to the NIH Intramural IRB and NCI Clinical Director: x All deaths, except deaths due to progressive disease Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 45x All protocol deviations x All unanticipated problems x All non-compliance Reports must be received within 7 days of PI awareness via iRIS. 7.2.2 NIH Intramural IRB Requirements for PI Reporting at Continuing Review The protocol PI will report to the NIH Intramural IRB: 1. A summary of all protocol deviations in a ta bular format to include the date the deviation occurred, a brief description of th e deviation, and any corrective action.

2. A summary of any instances of non-compliance. 3. A tabular summary of the following adverse events: x All grade 2 unexpected events that are possibly, probably, or definitely related to the research x All grade 3 and 4 events that are possibly, probably, or definitely related to the research x All grade 5 events regardless of attribution x All serious events regard less of attribution. Note: Grade 1 events are not required to be reported. 7.2.3 NIH Intramural IRB Reporting of IND Safety Reports Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported to the NIH Intramural IRB. IND SPONSOR REPORTING CRITERIA From the time the subject receives the investiga tional agent/intervention to the time of the second follow-up evaluation, the investigator must immediately report to the sponsor, using the mandatory MedWatch Form FDA 3500A or equivale nt, any serious adverse event, whether or not considered drug-related, including those listed in the protocol or Investigator’s Brochure and must include an assessment of whether there is a reasonable possibility that the drug caused the event. For serious adverse events that occur after the second follow-up evaluation, only those events that have an attribution of at least possibly related to the agent/intervention will be reported. Required timing for reporting per the above guidelines: x Deaths (except death due to progressive dis ease) must be reported via email within 24 hours. A complete report must be s ubmitted within one business day. x Other serious adverse events as well as de aths due to progressive disease must be reported within one business day. Events will be submitted to the Center for Cancer Research (CCR) at [email protected] and to the CCR PI and study coordinator. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 46All serious adverse events will be reported by the Sponsor (Center for Cancer Research) as per FDA 21 CFR 312.32. 7.3.1 Reporting Pregnancy Maternal Exposure If a patient becomes pregnant during study treatment, the treatment should be discontinued immediately. Pregnancies occurring during study treatment and for the first 120 days after the last vaccination should be reported to the Sponsor. The potential risk of exposure of the fetus to the investigational agent should be documen ted using the MedWatch Form FDA 3500A or equivalent. Pregnancy itself is not regarded as an SAE. Ho wever, as patients who become pregnant on study risk intrauterine exposure of the fetus to agents which may be teratogenic, the CCR is requesting that pregnancy should be reported in an expe dited manner as grade 3 “Pregnancy, puerperium and perinatal conditions - Other (pregnancy)” under the Pregnancy, puerperium and perinatal conditions SOC. Congenital abnormalities or birth defects and spontaneous miscarriages should be reported and handled as SAEs. Elective abortions without comp lications should not be handled as AEs. The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) should be followed up and documented. If any pregnancy occurs in the course of the study, then the investigator should inform the Sponsor within one day, i.e., immediately, but no later than 24 hours of when he or she becomes aware of it. The designated Sponsor representative will work with the investigator to en sure that all relevant information is provided to the Sponsor within 1- 5 calendar days for SAEs and within 30 days for all other pregnancies. The same timelines apply when outcome information is available. Paternal Exposure Male patients should refrain from fathering a ch ild or donating sperm during the study and for 120 days after the last vaccination. Pregnancy of the patient’s partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective ter mination, ectopic pregnancy, normal birth, or congenital abnormality) occurring fr om the date of the first dose until 120 days after the last dose should, if possible, be followed up and documented. SAFETY REPORTING CRITERIA TO THE PHARMACEUTICAL COLLABORATOR The Sponsor (Center for Cancer Research) will pr ovide Moderna with: (1) copies of all safety reports concurrently with their submission to the FDA; (2) a cumulative listing of adverse events (including suspected adverse reactions) on a quart erly basis; (3) notice within 48 hours of case completion with respect to a serious adverse event; and, (4) any other information affecting the safety of human subjects. This information will be submitted to Moderna at [email protected]. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 47Note: The quarterly cumulative listing of adverse events (including susp ected adverse reactions) will be submitted by the PI/research team to the Sponsor for reporting to Moderna. INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA 7.5.1 Serious Adverse Event Reports to IBC The PI (or delegate) will notify IBC of any unexpected fatal or life-threatening experience associated with the use of the gene transfer produ ct as soon as possible but in no event later than 7 calendar days of initial receipt of the informa tion. Serious adverse events that are unexpected and associated with the use of the gene transfer product, but are not fatal or life-threatening, must be reported to the NIH IBC as soon as possible, but not later than 15 calendar days after the investigator’s initial receipt of the information. Adverse events may be reported by using the MedWatch Form FDA 3500A or equivalent. 7.5.2 Annual Reports to IBCWithin 60 days after the one-year anniversary of the date on which the IBC approved the initial protocol, and after each subsequent anniversary until the trial is completed, the PI (or delegate) shall submit the information described below. Alternatively, the NIH Intramural IRB continuing review report can be sent to the IBC in lieu of a separa te report. Please include the IBC protocol number on the report. Clinical Trial Information A brief summary of the status of the trial in progress or completed during the previous year. The summary is required to include the following information: x Title and purpose of the trial x Clinical site x Principal Investigator x Clinical protocol identifiers x Participant population (such as disease indi cation and general age group, e.g., adult or pediatric) x Total number of participants planned for inclusion in the trial; the number entered into the trial to date whose participation in the trial was completed; and the number who dropped out of the trial with a br ief description of the reasons x Status of the trial, e.g., open to accrual of subjects, closed but data collection ongoing, or fully completed x If the trial has been completed, a brief description of any study results. Progress Report and Data Analysis Information obtained during the previous year's clinical and non-clin ical investigations, including: x Narrative or tabular summary showing the most frequent and most serious adverse experiences by body system Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 48x Summary of all serious adverse events submitted during the past year x Summary of serious adverse events that were expected or considered to have causes not associated with the use of the gene transf er product such as disease progression or concurrent medications x If any deaths have occurred, the number of pa rticipants who died during participation in the investigation and causes of death x Brief description of any information obtained that is pertinent to an understanding of the gene transfer product’s actions, including, fo r example, informati on about dose-response, information from controlled trials, a nd information about bioavailability. DATA AND SAFETY MONITORING PLAN 7.6.1 Principal Investigator/Research Team The clinical research team will meet on a weekly basis when patients are being actively treated on the trial to discuss each patient. Decisions about enrollment and dose escalation, if applicable, will be made based on the toxicity data from prior patients. All data will be collected in a timely manner and reviewed by the PI or AI. Adverse events will be reported as required above. Any safety concerns , new information that might affect either the ethical and/or scientific conduct of the trial, or protocol devia tions will be reported to the NIH Intramural IRB using iRIS. The PI will review AE and response data on each pa tient to ensure safety and data accuracy. The PI will personally conduct or supervise the inve stigation and provide ap propriate delegation of responsibilities to other members of the research staff. 7.6.2 Sponsor Monitoring Plan As a Sponsor for clinical trials, FDA regulati ons require the CCR to maintain a monitoring program. The CCR’s program allows for confirmation of: study data, specifically data that could affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and SOPs; and human subject’s protection. This is done through independent verification of study data with source documentation focusing on: x Informed consent process x Eligibility confirmation x Drug administration and accountability x Adverse events monitoring x Response assessment The monitoring program also extends to multi-site research when the CCR is the coordinating center. This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified by training and experience to monitor the progres s of clinical trials. Personnel monitoring this study will not be affiliated in any way with the trial conduct. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 497.6.3 Safety Monitoring Committee (SMC) This protocol will require oversight from the SM C. Initial review will occur as soon as possible after the annual NIH Intramural IRB continuing re view date. Subsequently, each protocol will be reviewed as close to annually as the quarterly meeting schedule permits or more frequently as may be required by the SMC. For initial and subsequent reviews, protocols will not be reviewed if there is no accrual within the review peri od. Written outcome letters will be generated in response to the monitoring activities and submitted to the PI and Clinical Director or Deputy Clinical Director, CCR, NCI. 8 STATISTICAL CONSIDERATIONS STATISTICAL HYPOTHESIS 8.1.1 Primary Efficacy Endpoints x Determine the clinical response rate in patients with meta static melanoma, gastrointestinal, or genitourinary cancers who receive NCI-4650. x Determine the safety of NCI-4650 in patients w ith metastatic melanoma, gastrointestinal, or genitourinary cancer. 8.1.2 Secondary Efficacy Endpoint x Determine whether administration of NCI-4650, a mRNA-based, PCV presenting immunogenic neoantigens, can increase the quantity and quality of circulating antigen-specific T-cells in patients with solid tumors. SAMPLE SIZE DETERMINATION The study will be conducted using a phase I dos e escalation followed by a phase II Simon minimax design in two cohorts: Cohort 2a, patients w ith melanoma and Cohor t 2b, patients with gastrointestinal or genitourinary cancer. Following dose escalation, the objective will be to determine if NCI-4650, given at the MTD, is associated with a clinical respons e rate that can rule out 5% ( p0=0.05) in favor of a modest 20% PR+CR rate (p1=0.20) in each of the two cohorts in Phase II. For each cohort in the Phase II portion of th is study, using a two-stage Simon minimax design, with alpha=0.10 (10% probability of accepting a p oor therapy) and beta =0.20 (20% probability of rejecting a good therapy), initially 12 evaluable patients will be enrolled. If 0 of the first 12 evaluable patients experience a clin ical response, then no further patients will be enrolled in that cohort. 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 that cohort. As it may take several weeks to determine if a patient has experienced a clinical response, a temporary pause of up to 6 months in the accrual to the trial may be necessary to ensure that enrollment to the second stage is warranted. If 1-2 of the 21 ev aluable patients have a cl inical response, then this will be considered inadequa te for further investigation. If 3 or more of the 21 evaluable patients have a clinical response, then this w ill indicate that this strategy provides a new approach that may be worthy of further consid eration in that cohort. Under the null hypothesis (10% response rate), the probability of early termination per cohort is 54.0%. The dose escalation portion of the study may requ ire 6 patients per dose level. For purposes of sample size estimation, we will a ssume that as few as 12 and no more than 18 patients will be Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 50required to perform the initial safety evaluation. In order to complete th e dose escalation phase and Phase II, a total of up to 18+46 =64 patients may be required (allowing up to 2 inevaluable patients per Phase II cohort). 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. Provided that about 2-3 patients per month will be able to be enrolled onto this trial, approximately 2-3 years may be needed to accrue the maximum number of requi red patients. However, as ade quate responses to proceed to the second stage of accrual may not occur, the trial may end up accruing many fewer patients. POPULATIONS FOR ANALYSES 8.3.1 Evaluable for Toxicity All patients will be evaluable for toxicity fro m the time of their first mRNA vaccine dose. 8.3.2 Evaluable for Objective ResponseOnly those patients who have measurable disease present at baseline, have received at least one course of therapy, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified accordin g to the definitions stated in Section 6.3.3. 8.3.3 Evaluable for Non-Target Disease Response Patients who have lesi ons present at baseline that are evalua ble but do not meet the definitions of measurable disease, have received at least one course of therapy, and have had their disease re- evaluated will be considered evaluable for non-tar get disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions. STATISTICAL ANALYSES 8.4.1 General Approach In the Phase I cohort, the toxicities experienced by patients at each dos e level will be reported per dose level. In the two Phase II cohorts, clinical responses will be determined and the fraction with a clinical response will be repor ted, along with confidence intervals. 8.4.2 Analysis of the Primary Efficacy Endpoints In the Phase I cohort, the toxicities experienced by patients at each dos e level will be reported per dose level. The grade of the toxicity as well as the type of toxicity will be tabulated per dose level. In the Phase II cohorts, the fraction of patients who experience a clinical response in each cohort will be reported along with 80% and 95% two-sided confidence intervals. 8.4.3 Analysis of the Secondary Efficacy Endpoint The study will aim to determine whether treatment administration can increase the quantity and quality of circulating antigen-specific T-cells, by measuring the reactivity of circulating cells against the immunizing peptides in Elispot and 41BB upregulation assays. The comparisons will be done using descriptive statistics, without formal statistical testing required. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 518.4.4 Safety Analyses Safety will be monitored by identifying the type and severity of any adverse events. The fraction of patients who experience a DLT will be identified at a given dose level, with information reported about the number and grade of each type of DLT identified. 8.4.5 Intention-to-Treat Analysis An intention-to-treat (ITT) analysis will be condu cted of all patients enrolled to the study. A full CONSORT flow diagram will be included in all ITT analyses. 8.4.6 Planned Interim Analyses As noted in Section 8.2, results after the first stage of a Simon minimax two-stage design in each Phase II cohort will be examin ed to determine if the minimum number of responses has been obtained. 8.4.7 Sub-Group AnalysesThere are no planned analyses based on demographic characteristics since the power to do subgroup analyses would be limited a nd make interpretation difficult. 9 COLLABORATIVE AGREEMENTS MODERNA TX, INC. CTA #01051-17 Tumor specific antigens (TSAs) containing be tween 320 and 639 amino acids will be sent to Moderna (formerly Moderna Therapeutics, Inc.) for the manufacture of a single concatemeric mRNA construct to be formulated into a vaccin e for the same cancer patient. Sequencing data will be transferred electronically via the secu re, encrypted Globus file transfer system (Globus.org) under the current NCI license for this transfer software. The vaccine product and associated data will be returned to the NCI-SB. CDA #14358-18 The following data will be transferred to Moderna: (1) raw sequencing data, including tumor DNA whole exome, tumor RNA whole transc riptome, and normal DNA whole exome sequencing data in FASTQ and/or BAM format; (2) somatic variant calling results in VCF format; (3) gene expression quan tification results in plain text format; (4) patient HLA types (major alleles: HLA-A, -B, -C, -DRB1, and any ot her HLA class II alleles that are typed, in 4 digits resolution (e.g., HLA-A:02:01)) in plain text format; (5) a list of all neoantigens that had been tested; and, (6) a list of a ll neoantigens tested to be imm unogenic (i.e., true positives). Moderna will compare the neoantigen ranks predicted by Moderna’s algorithm with the true positive and negative data from the NCI-SB. Moderna will also perform an investigation on features that are important for ne oantigens to be immunogenic. Data will be transferred electronically via a secure encrypted Amazon Web Service S3 bucket or via the secure, encrypted Globus file transfer system (Globus.org) under the current NCI license for this transfer software. No physical specimens will be shipped under this agreement. Results from Moderna’s neoantigen prediction algorithm (a prioritized list of neoantigens for each patient), and a comparison with NCI-SB’s results, will be returned to the NCI-SB. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 5210 HUMAN SUBJECTS PROTECTIONS RATIONALE FOR PATIENT SELECTION Subjects from both genders and all r acial/ethnic groups are eligible fo r this study if they meet the eligibility criteria. To date, there is no information that suggests that differences in drug metabolism or disease response would be expected in one group compared to another. Efforts will be made to extend accrual to a representative population, but in this preliminary study, a balance must be struck between patient safety considerations and limitations on the number of individuals exposed to potentially toxic and/or ineffective treatments on the one hand, and the need to explore gender and ethnic aspects of clinical research on the other hand. If differences in outcome that correlate to gender or to ethnic identity are noted, accrual may be expanded or a follow-up study may be written to investigate those differences more fully. PARTICIPATION OF CHILDREN Since the efficacy of this experimental pro cedure is unknown, it does not seem reasonable to expose children to this risk without further evidence of benefit. Should results of this study indicate efficacy in treating metast atic cancer, which is not responsiv e to other standard forms of therapy, future research can be conducted in th e pediatric population to evaluate potential benefit in that patient population. PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (see Section 10.5 DOOVXEMHFWV• DJHZLOOEHRIIHUH GWKHRSSRUWXQLW\WR fill in their wishes for research and care, and assign a substitute decision maker on the “NIH Advance Directive for Health Care and Medical Research Participation” form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for ev aluation as needed for the following: an independent assessment of whethe r an individual has the capacity to provide consent; assistance in identifying and assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint a surrogate. For those subjects that become incapacitated and do not have a pre-determined substitute decision maker, the procedures described in MAS Policy 87-4 and OHSRP SOP 14E for appointing a surrogate de cision maker for adult subjects who are (a) decisionally impaired, and (b) who do not have a le gal guardian or durable power of attorney, will be followed. EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS The experimental treatment has a chance to provi de clinical benefit though this is unknown. The NCI-SB has extensive experience with adoptive cell therapy (ACT) following treatment with high-dose aldesleukin, however this experimental treatment does not include the standard NCI-SB preparative regimen or high-dose aldesleuki n. Although we have seen responses to prior NCI-SB ACT treatments, we do not know if this change in our proces s will improve patient outcome. The risks associated with ACT are substa ntial, including, a delay in treatment due to the need to harvest and grow the cells, a surgical procedure (possible major) to obtain tumor for Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 53the cell product, the possibility that a cell produc t cannot be generated, infection due to the surgical procedure, and death. The risks in this treatment are detailed in Section 11. RISK/BENEFIT ANALYSIS Because all patients in this prot ocol have metastatic or recurr ent/refractory locally advanced cancer and limited life expectancies the potential benefit is thought to outweigh the potential risks. The risk/benefit analysis for adults with the capacity to consent, as well as for adults who may become unable to provide consent, is greater than minimal risk because the study treatment may be associated with severe side effects, with the prospect of direct benefit because the treatment may reduce the cancer burden. CONSENT PROCESS AND DOCUMENTATION Patients are initially consente d on protocols 99-C-0128 and 03-C- 0277. If the patient meets the thorough screening for eligibility, the patient, with family members or friends at the request of the patient, will be presented with the informed consent document for this protocol, who is requested to review it and to ask questions pr ior to agreeing to participate. The specific requirements, objectives, and potential advantages and disadvantages will be presented to the patient. The patient is reassured that participation on trial is entirely voluntary and that he/she can withdraw or decide against treatment at an y time without adverse consequences. The PI, AI, or clinical fellow is responsible for obtaining written consent from the patient. 10.6.1 Telephone Consent for Part One Registration Because many patients will have previously been treated on another NCI-SB protocol prior to beginning treatment on this protoc ol, these patients may not be onsite at the NIH Clinical Center in order to sign the informed consent document for this protocol. In these circumstances, in order to complete Part One of the re gistration procedure (see Section 2.3.2), the informed consent process and a detailed description of the protocol treatment will be discussed with the potential participant via phone. The informed consent document will be sent to the subject. An explanation of the study will be provided over the telephone after the subject has had the opportunity to read the consent form. The subject will sign and date the informed consent. A witness to the subject’s signature will sign and date the consent. The original informed consent document will be sent back to the consenting investigator who will sign and date the consent form with th e date the consent wa s obtained via telephone. A fully executed copy of the informed consent will be returned via mail to the subject. The informed consent process will be documented on a progress note by the consenting investigator. 10.6.2 Informed Consent of Non-English Speaking Subjects If there is an unexpected enrollment of a research participant for whom there is no translated extant NIH Intramural IRB-approved consent documen t, the PI and/or those authorized to obtain informed consent will use the Short Form Oral Consent Process as described in MAS Policy M77-2, OHSRP SOP 12, 45 CFR 46.117 (b) (2), and 21 CFR 50.27 (b) (2). The summary that will be used is the English version of the extant NIH Intramural IRB-approved consent document. Signed copies of both the English version of the consent and the translated short form Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 54will be given to the subject or their legally authorized representative and the signed original will be filed in the medical record. Unless the PI is fluent in the prospective subject’s language, an interpreter will be present to facilitate the conversation (using either the long-translated form or the short form). Preferably someone who is independent of th e subject (i.e., not a family member) will assist in presenting information and obtaining consent. Whenever possible, interpreters will be provided copies of the relevant consent documents well before the consent convers ation with the subject (24-48 hours if possible). We request prospective NIH Intramural IRB appr oval of the use of the short form process for non-English speaking subjects and will notify the NI H Intramural IRB at the time of continuing review of the frequency of the use of the Short Form. 11 PHARMACEUTICAL INFORMATION CELL PREPARATION The procedure for growing and expanding the autologous young TIL is similar to what is approved by the Food and Drug Administration and used in other NCI-SB TIL clinical studies. TIL will be grown and expanded fo r this trial according to SOPs which can be found in BB-IND 17858. INTRAMUSCULAR INJECTION PREPARATION 11.2.1 NCI-4650, mRNA-Based Persona lized Cancer Vaccine Description: The CoA for the NCI-4650 mRNA vaccine is in Appendix C , and the SOPs for the preparation of the mRNA vaccine are found in BB-IND 17858. This product will be provided for investigational use under BB-IND 17858, which is sponsored by the CCR. The vaccine is composed of cGMP grade mRNA. Neoantigens will be identified from a sequence comparison of a patient’s tumor and normal DNA sample. An al gorithm will predict muta ted sequences with a high probability of binding to a patient’s specific HLA repertoire. Neoantigens will be prepared as peptides, either as 25mers, with the mutate d amino acid residue situated at position 13, or as minimal epitopes. The patient’s TIL, cultured from a biopsy sample, will be screened for their ability to recognize these neoantigens pulsed onto dendritic cells. Neoantigens recognized by the patient’s TIL will be used to formulate the vaccine. The vaccine will be formulated for each administration as indicated below. The identif ied immunogenic neoantig en sequences will be sent electronically to Moderna for vaccine manufacturing. Upon receipt of the final NCI-4650 product, vaccine will be administered via intramuscular injection. Mode of Action: NCI-4650 is taken by antigen-p resenting cells that process and present the neoantigens on HLA molecules to tumor-specific cytotoxic T-lymphocytes, leading to a major histocompatibility complex-restricted cytotoxi c response against tumor cells bearing these neoantigens. Formulation: NCI-4650 is a novel mRNA-based, PCV directed against s ubject-specific tumor neoepitopes. The individual mRNA sequences are designed for each subject based on the pipeline for the identification of immunogenic T-cell epitopes derived from neoantigens recognized by TIL developed by the NCI-SB . Each NCI-4650 vaccine consists of mRNA encoding neoantigens designed specifically to each individual subject’s tumor mutanome, and HLA type. Each subject-specific NCI-4650 vaccine is formulated in LNPs comprised of four Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 55 lipid excipients: SM-102; 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. SM- 102 is a novel, ionizable lipid that complexes with the mRNA component of NCI-4650 (mRNA product intermediate [MPI]) to promote the formation of the LNPs. The commercially-available lipids, DMG-PEG 2000, DSPC, and cholesterol, al so contribute to the overall pharmaceutical properties of the LNPs. NCI-4650 is provided as a sterile liquid for injection in a 93 mM Tris, 7% propylene glycol, 1 mM DTPA buffer, at a concentration of 1.0 mg RNA/mL. Stability: Please refer to the NCI Pharmacy Manual. Storage: NCI-4650 must be stored in a restri cted access area under the storage conditions indicated on the product label and in the NCI Phar macy Manual. Vials should be stored frozen at -20°C (± 5°C) (-13°F to 5°F). How Supplied: NCI-4650 is supplied as solution for injection in 2-mL glass vials (with a septum stopper). Each vial contains 0.6 mL. Preparation: 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. The NCI Pharmacy Manual contains specific instructions for the preparation and administration of each dose level of NCI-4650. The NCI Pharmacy Manual also contains details regarding sterility release of NCI-4650. Administration: For all subjects, NCI-4650 will be administered by IM injection into the deltoid muscle (alternating deltoid musc le sites should be used for each subsequent injection, and injection sites may also be rotated to thigh muscles). Toxicities: Risk to subjects receiving NCI-4650 is expected to be low and primarily involving mild to moderate injection site reactions, which have been observed in animal studies and generally observed and expected for other IM administered vaccines. These local reactions may consist of transient and dose-dependent pain, swelling, and erythema. Possible mild to moderate systemic reactions, which are also transient, may include fever, fatigue, chills, headache, myalgias, and arthralgias. Such AEs may, in part, be due to the poor biodegradability of LNP formulations used in other vaccine studies; the LNP that comprises NCI-4650 is designed to incorporate biodegradable properties that may have an improved safety pr ofile. In addition, other AEs that have been generally associated with approved IM administere d vaccines have included mild hematological and clinical chemistry abnormalities, which are usually reversible. Subjects receiving NCI-4650 could experience signs and symptoms compatible with a C activation- related pseudoallergy (CARPA) response, which has been obser ved in the administration of approved liposomal products, contrast agents, pegylated proteins, and antibodies, as well as for small interfering RNA products formulated in LNPs. The signs and symptoms of CARPA resemble those of an acute hypersensitivity reac tion. However, complement activation is far less likely to be associated with clinical sign s for LNP products such as NCI-4650, that are administered IM at a much lower dose on a mg/kg basis than other clinical entities that cause CARPA in humans and nonclinical species. Special Handling: Please refer to the NCI Pharmacy Manual. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 56 12 REFERENCES 1. Klein L, Hinterberger M, Wirnsberger G, Kyewski B. Antigen presentation in the thymus for positive selection and central toleran ce induction. 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