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.