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.