................................................ 54 Appendix 2: Certificate of Analysis for Immature Dendritic Cell s.............................. 57 Appendix 3: Certificate of Analy sis for Mature Peptide Pulsed D endritic Cells ......... 58 Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 81 INTRODUCTION STUDY OBJECTIVES 1.1.1 Primary Objective xTo determine the clinical respons e rate in patients with metast atic melanoma or epithelial cancer who receive this DC vaccine. 1.1.2 Secondary Objectives xTo determine whether administration of a DC vaccine presenting defined mutated neoantigens can increase the qu antity and quality of circulatin g antigen-specific T cells. xTo determine the safety of the vaccine. BACKGROUND AND RATIONALE 1.2.1 Cancer Vaccines Protective vaccination against infectious diseases has proven t o be one of the most effective health measures. Therapeutic v accination against established dis eases such as persistent infections and cancer has proven much more challenging. Cancer vaccines are designed to target antigens that can elicit an immun e response that selectively at tacks cancer cells. Ideally these antigens should be exclusively presented on cancer cells. Until recently, vaccines against non- viral tumors mainly targeted diffe rentiation antigens, cancer te stis antigens, and overexpressed antigens. However, central immunological tolerance has limited t he 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-a ffinity TCRs for t heir cognate a ntigens which have the superior cytotoxic capacity, longer persis tence in the tumor microenviro nment, and decreased susceptibility to immune suppression3. Taken together, such deple tion can lead to impaired clinical efficiency following vaccine administration. In recent years our group and others have extensively studied the importan ce of neoantigens as targets fo r immunotherapy. It is now clear that neoantigen specific T cells are present in most cancers. Ne oantigens derived from somatic mutations offer a specific and hi ghly immunogenic target for vac cination. Today, with the rapid development of technologies for DNA sequencing, the identificati on of those mutations becomes widely feasible. Evidence for the power of neoantigen vaccines i n pre-clinical and clinical settings was recently published. T he first proof of concept of neoantigen vaccines was shown in the B16 mice melanoma model4. By prophylactic vaccination wit h two mutated epitopes, the researchers achieved complete tumor protection in 40% of the mi ce. In therapeutic settings, they observed significant tumor grow th inhibition induced by mutatio n-specific peptide vaccination. Recently, the same group publishe d 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 mut ations in the cancer were immu nogenic, and the majority of them were recognized by CD4+T cells. Vaccination with these CD4+epitopes induced potent tumor control and rejection of e stablished tumors. Similar results were published in another independent mutation based cancer model. In this work the autho rs developed a pipeline to identify immunogenic mutant peptides using whole-exome and trans criptome sequencing together with mass spectrometry. Again, the researchers were abl e to show that vaccination with mutation specific peptides induce d therapeutically active T cell responses. Results of the current Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 9pre-clinical data support the evaluation of neoantigen vaccines as an effective cancer treatment. Although studied extensively i n pre-clinical models, only one limited human trial has been published. In this trial, a group from Washington University in St. Louis reported on a first-in- man clinical trial evaluating ne oantigen vaccine in patients wi th metastatic melanoma6. The researchers vaccinated three patients with mature dendritic cel ls presenting seven predicted mutation-specific HLA-A*02:01 restr icted epitopes. They showed that vaccination with high affinity, patient specific mutated epitopes augmented T cell im munity directed against naturally occurring dominant neoantigens and broadened the response by re vealing subdominant neoantigens; but no clinical res ponses were observed. However, t his did show that vaccination against mutated epitopes was sa fe as none of the three patients experienced any autoimmune adverse events. 1.2.2 Identification of Cancer-Speci fic Immunogenic Epitopes Deri ved from Somatic Mutations The NCI SB has developed a proce ss to identify immunogenic T cel l epitopes derived from neoantigens ( Figure 1 ).The identification of these antigens is done in four main stages: 1) Whole exome sequencing and RNA-s eq analysis of the tumor and a matched normal apheresis sample to identify highly expre ssed somatic mutations; 2) Constr uction of tandem minigenes (TMGs) and synthesis of long peptides covering these mutations; 3) expression of t he constructs in autologous antigen presenting cells; 4) In vitro co-culture as say to identify T cells recognizing mutated epitopes from TIL and PBMC7. In earlier studies done by the NCI SB (published and unpublished data), 25 patients with melanoma were screened in th e above manner, 64 antigenic somatic mutations were identified with no overlapping between pa tient tumors8,9. Recently, a study to identify antigenic mutations from patients with epithel ial cancers including those of the GI tract, genitourinary tract and breast, identified 57 non-ove rlapping somatic mutations. In this study, two patients were i dentified who developed T-cell response against the mutated KRAS oncogene10. Work published by the NCI SB in Nature Medicine proved for the first time that neoantigen specific T cells can also be isolated from patie nts’ PBMC7. By using this process, neoantigen specific lym phocytes were identified in the peripheral blood of three of four patients with melanoma. Despite their low fre quency in the circulation, these CD8+PD-1+ cell populations had lymphocytes that targeted unique pa tient-specific neoantigens. This method provides a novel noninvasive approach to identif y antigenic neoantigens. Thus far, more than 190 immunogenic epitopes have been identifi ed (Table 1 ) from multiple cancer types including melanoma, ovarian, colorectal, lung and breast cancers7,8,10-13. This process, which can be applied t o any cancer type and can be com pleted 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. 1.2.3 Preclinical Studies for the G eneration of a Dendritic Cell Vaccine Development of a personalized n eoantigen vaccine puts forward s everal hurdles. The vaccination must address those issues that are unique to the cancer model. The number of epitopes is variable. xThe vaccine should optimally in clude both CD4 and CD8 epitopes. Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 10xThe vaccine may induce a cell-mediated Th1 response. xThe vaccine manufacturing shoul d avoid expensive and time-consuming processes. To address these issues, ex vivo g enerated DC will be used. Manu facturing of DC from patient PBMC is a well-established and s traightforward method. DC vacci nes are proven to induce T cell responses against multiple m alignancies an d infectious dis eases. Numerous clinical trials incorporating and using differe nt administration routes have demonstrated the safety and tolerance of DC vaccines14. In addition, the use of DCs both as antigen presenting cells i n the initial high throughput screenin g for recognition of neoantigen s and in the delivery of these antigens in the vaccine product, should ensure that all antigen s will be properly processed and presented to T cells upon vaccination. DCs Matured with PolyI:C, R848, and IFN JUpregulate Co-Stimulatory Molecules and Secrete Pro-Inflammatory Cytokines DCs are one of the most effective antigen-presenting cells to in duce T cell immunity. Even though immature DCs (iDCs) can upt ake, process, and present ant igens, they fail to secrete proinflammatory cytokines, and t herefore have been shown to be tolerogenic, or at best weakly immunogenic15. Multiple maturation cocktails have been used in the clinic to produce fully mature DCs. Until recently, researchers used mainly immature DC s or cells activated by a cytokine cocktail (Il-1 E, IL-6, TNF Dand PGE2). Lately, several reports citing the use of TLR ligands such as PolyI:C and R848 a s DCs stimulators have been p ublished6. To evaluate which maturation method genera ted the highest number of DCs secreting IL-12p70/TNF Dday six monocyte-derived DCs were incuba ted with LPS, CD40L expressing 3T3 cells, TLR mix (PolyI:C, R848, and IFN J) and cytokine cocktail (Il-1 E, IL-6, TNF Dand PGE2). Sixteen hours post incubation the level of cos timulatory molecules was measur ed by flow cytometry and the secretion of multiple cytokines a nd chemokines was measured by multiplex assay ( Figure 2 ). From Figure 2 it can be clearly seen that the TLR mix induced the secretion o f IL-12p70, TNF D, IP-12, MCP-1 and MIP-1 E. The TLR cocktail is capable o f maturing DCs to secrete TH1 polarizing cytokines and multiple chemokines. Recently, a group from Washington University in St. Louis (WUSTL) used a combi nation of K562-CD40L cells with P olyI:C, R848, and IFN Jto mature monocyte-derived DCs. To evaluate if the addition of K56 2-CD40L cells to the TLR mix could induce superior DCs maturation, clinical grade K562-CD40L cells (K602C14) were produced and their contribution to D C stimulationwas evaluated. The original cells used by the WUSTL group (K463H) ( Figure 3 ) were used as a positive contro l. This indicates that TLR mix alone is enough to produce full y mature DCs, and thus no additi onal stimulation is needed. DCs Loaded with Long Peptides a nd Minimal Epitopes Stimulate both CD4 and CD8 T Cells To evaluate the best method of loa ding DCs with antigenic deter minants, mRNA electroporation of TMGs was compared to loading w ith long peptides and minimal epitopes. A screening system to evaluate the presentation of three CD8 and three CD4 epitopes in one assay was developed. A series of TMG constructs harboring diffe rent signal peptides and targeting signals and encoding both long peptides and minimal epitopes covering all antigenic determinants ( Figure 4 ) were produced. Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 11To test antigen presentation, DCs were transfected with the TMG constructs or, loaded with long peptide or minimal epitopes. DCs were co-cultured with PBMC tran sduced with TCRs recognizing the corresponding antigens and antigen recognition was evaluated by flow cytometry for 41BB expression ( Figure 5 ). The results clearly showed that while long peptides and minimal epitopes were recognize d by CD4 and CD8 cells, TMGs eli cited only CD8 responses likely indicating that CD4 epit opes cannot be processed through cytosolic proteins thus abrogating the use of TMGs as a n efficient platform for antigen presentation. To test if mature DCs could efficiently present both CD4 and CD8 epitopes DCs wer e loaded with long peptides and minimal epitopes and stimula ted for 16 hours using the TLR mix ( Figure 6 ). As shown the mature DCs efficiently presented both minimal and long epitopes for CD4 and CD8 epitopes. Thus, mature DCs loaded with long peptides or minimal epitopes c overing both CD8 and CD4 epitopes will be used in this trial. Mature DCs can Stimulate Both Mem ory, Effector, and Naïve Neoant igen Specific T Cells An efficient vaccine should be abl e to augment neoantigen specif ic T cells and to expand the breadth of the anti-tumor immune r esponse. To test if DCs mature d with TLR mix are capable of stimulating naïve, antigen-exper ienced and terminally differentiated effector cells, an in vitro stimulation experiment was conducte d. Apheresis samples from a pa tient with colorectal cancer were thawed and then incubated in a n appropriately sized tissue cu lture flask. Adherent monocytes were differentiated i nto DCs, loaded with neoantigen peptide pools and matured using a TLR mix. Non-adherent cells were collected, and memory, n aïve and terminally differentiated effector memory cells (T EMRA ) were sorted and in vitr o stimulated with mature peptide-pulsed autologous DCs ( Figure 7 ). Ten days after the first stimulation T cells were restimulated with DCs loaded with all pep tide pools, and sorted based on 41BB and OX40 expression to enrich for neoantigen-specific cells. Cells were then tested for neoantigen specific recognition using single peptides derived from the most reactiv e peptide pools. By applying this method, neoantigen specific T cells from naïve, antigen-experien ced and T EMRA cells ( Figure 8 ) could be identified. These results show that peptide-loaded, TL R mix stimulated DCs are capable of in vitro activation of T cells de rived from memory or naïve p recursors. 1.2.4 Rationale for Selecting Defin ed, Tumor Suppressor and Driver Neoantigens to use in this Trial Historically, preclinical and c linical neoantigen 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 processe d and presented by the tumor or antigen presenting cells in the microenvironment. As described above, patient TIL and PBMC will be screened to identify defined mutated antigens recogniz ed by T cells guaranteeing tha t those antigens are processed and presented for T cells in the tumor or its draining lymph nodes. There will be one exception for using defined neoantigens in th is trial. There is growing evidence to suggest that targeting tumor driver and suppressor genes can elicit superior clinical response. Mutations in these genes are highly common and have been found in many tumors. When mutations are iden tified in KRAS, NRAS, p53, EGFR, IDH1, PIK3CA or any other potential driver mutations, peptides coveri ng those epitopes will be synthesized and used in the vaccine composition. The method/process for epitope selection and synth esis is shown in Figure 9 . Abbreviated Title: Personalized DC Vaccine Version Date: March 26, 2018 12Augmenting T cell responses against defined neoantigens, should increase the likelihood of a significant clinical response. 1.2.5 Relevant Preclini cal and Clinical Experience to Support th e Proposed Route and Dose of the DC Vaccine As shown by the NCI SB, neoantig en specific cells can be found both in the tumor and in the blood of cancer patients7,10. To boost responses against define d neoantigens, we are using a systemic vaccine dose and sche dule covering both the blood and peripheral tissues. For that purpose, we decided to use two rout es of administration: Intrav enous (IV) to stimulate T cells that reside in the lungs, spleen and the bone marrow of the pat ients and subcutaneously (SQ) to stimulate T cells residing in pe ripheral lymph nodes. This concept was also shown before in a mouse model16.