Supplementary Fig1. PGV001 feasibility and recurrence free survival a) Survival plot depicting overall survival (OS). N=12. Median survival depicted by vertical dotted line b) Survival plot depicting recurrence free survival (RFS). Median RFS is depicted by vertical dotted line. N=12. c) Swimmer plot depicting time-line of clinical events for each patient since their curative intent treatment. d) Comparing linear correlation between TMB or neoantigen load vs OS at 60 months from 1st vaccine. Pearson correlation coefficient (r) calculated for fitting of the correlation with 95% confidence interval (95%CI). TMB: tumor mutation burden, NeoAg: neoantigens, OS: overall survival,

Supplementary Fig5. TCR sequencing on neoantigen reactive T cells in patient PBMCs a) (Top) Depiction of methodology utilized to generate ex vivo and in vitro expanded and stimulated (IVS) T cell samples from screen, week 8 (W8) and week 28 (W28) timepoints for TCR sequencing. (Bottom) Gating strategy and representative dot plot for isolation by FAC sorting of IFNγ positive, neoantigen reactive T cells for TCR sequencing. b) (Top) Representative differential abundance plots from PID:012 comparing TCR clones between two conditions listed on X and Y axes. (Bottom) Logic and calculation of “relevant” clones in vivo. First using the IVS T cells, lists of TCR sequences differentially abundant in peptide stimulated (Pepstim) Week 8 (W8) and Week 28 (W28) versus screening were generated, named W8IVS>scr and W28IVS>scr, respectively. Similarly, lists of TCR sequences differentially abundant in peptide expanded W8 and W28 samples versus DMSO expanded cells was obtained, named W8ctrl>IVS and W28ctrl>IVS, respectively. Sequences in W8ctrl>IVS and W28ctrl>IVS were removed from W8IVS>scr and W28IVS>scr to obtain vaccine induced “relevant” TCR sequences in IVS T cells named, W8ΔIVS and W28ΔIVS. Ex vivo T cells were analyzed to identify lists of differentially abundant TCRs in W8 and W28 versus screening, named W8ex>scr and W28ex>scr. Finally, an intersection between relevant clones in IVS (W8ΔIVS and W28ΔIVS) and abundant clones in W8 or W28 ex vivo T cells (W8ex>scr and W28ex>scr) was performed to identify relevant neoantigen reactive TCR sequences in vivo, named W8clones and W28clones. c) Stacked bar plot depicting number and frequency of new (detected only post vaccination) and expanded (existed at baseline but frequency expanded post vaccination) W8clones and W28clones TCR clones in PID:015. Each color represents distinct TCR clones and the height of each stack represents each clone’s frequency. d) For PID:015, Venn diagram showing overlap between ex vivo and IVS clones to obtain W8clones and W28clones and their overlap to identify persisting clones in vivo. For PID:015, no persisting clones were found.

Supplementary Table3: List of mutated peptide IDs and peptide sequence in each patients’ vaccine

Supplementary Fig4. CD4+ and CD8+ T cell activation by PGV001 PBMCs from patients were expanded in vitro in presence of neoantigen peptides (pools of peptides, OLPs+Mins, corresponding to each vaccine SLP) and restimulated with peptide/s followed by intracellular staining and flowcytometry. All data is normalized to MOG. a) Aligned dot plot showing CD8+ and CD4+ T cell responses at baseline induced by 96 evaluated neoantigens across 10 patients. Horizontal dotted line depicts cut-off threshold for immunogenicity. 15 and 32 peptides were found to be immunogenic at baseline against CD8+ and CD4+ T cells, respectively. b) Line plots showing longitudinal CD8+ and CD4+ T cell immune responses induced by neoantigens found to be immunogenic at baseline in (a) and that induced greater than 2-fold increase in response following PGV001 treatment. c) Each pie chart shows proportion of vaccine peptides that induced production of IFNγ, TNFα or IL-2 in CD8+ or CD4+T cells in each patient at any timepoint post vaccine initiation. Refer to Material and Methods for definition of “response” in flowcytometry assay. d) CD8+ and CD4+ T cell immune responses elicited by tetanus peptides at various time points in 10 evaluated patients.

Supplementary Fig7. Characterizing peripheral cellular immune environment in patients receiving PGV001. Multicolor flowcytometry performed to phenotype circulating immune cells in patients and healthy donors (HD). PID-017 was excluded from analysis. a) Tabulated view of age and sex of the seven healthy donors whose PBMCs are used in the study. b) Frequency of lymphoid immune cell subsets over the course of treatment. Each dot represents a subject. # p value indicates un-paired two-sided Student’s T-test comparing HD with patient cohort # <0.05, ## <0.01. c) Frequency of myeloid immune cell subsets over the course of treatment. Each dot represents a subject. d) Depicting Fold change from “Pre” in listed immune cell subsets over the course of treatment. e) Pie charts depicting CD4+ and CD8+ T cell states in patient blood and healthy donors. f) �4+ T cells expressing TIGIT and CTLA4 shown as a fold change from baseline in patient blood. *p value indicates paired two-sided Student’s T-test comparing post treatment samples with “pre”. * <0.05. Data in pie charts depicts median. Patient samples, N=12. Healthy donor samples, N=7. Data in graphs shown as mean with bar graphs showing +/- SEM.

Supplementary Table2 – Variant count per patient

Supplementary Table4: Number of Subjects who had Treatment Emergent Adverse Events by grade, system (13 subjects)

Supplementary Fig6. Gating strategy for phenotyping of immune cells by flow cytomtery. EM: Effector memory, TEMRA: Effector memory Re-expression RA, CM: Central Memory

Supplementary Fig2. PGV001-induced T cell immunity as measured by ex vivo IFNγ ELISPOT assay PBMC samples were stimulated with neoantigen peptides (pools of peptides: composed of 15mer OLPs+ 9-10mer predicted Mins corresponding to each vaccine SLP) and analyzed by IFNγ ELISPOT. All data is MOG normalized. a) Line diagrams showing longitudinal changes in IFNγ secretion upon stimulation with responder neoantigen peptides in each patient. Each line represents an individual neoantigen and each dot represents an individual time-point. For definition of “Responder” neoantigen: See Materials and Methods. b) Aligned dot plot showing IFNγ secretion at baseline (Pre) induced by all 126 neoantigens used in the study for 13 patients. Horizontal dotted line depicts cut-off threshold for immunogenicity in the assay. Peptides with immunogenicity at baseline, above the dotted line, are labeled. “Immunogenic” neoantigen: See Materials and Methods. c) Line plot showing post-vaccination immune response, as measured by IFNγ release in ex vivo ELISPOT assay, by the neoantigen peptides found to be “immunogenic” at baseline in (b). d) Plot showing number of IFNγ SFCs/million PBMCs elicited by tetanus peptide at various time points in 13 patients. e) Line plots showing immune response, as measured by IFNγ release in ex vivo ELISPOT assay, elicited by mutated neoantigen peptide pools vs their wild type (WT) counterparts at Week 28 over a range of peptide concentrations. Note: For PID:016 Week 31 sample was used. f) Kaplan Meier Curve comparing OS between subjects that responded, in ex vivo ELISPOT assay, to more or less than 40% neoantigens in their vaccines. PID-017 was lost of follow up and excluded from this analysis. g) Comparing number of SFCs/million PBMCs elicited by each neoantigen in patients Alive (N=6) or Deceased (N=4) at 60-month survival follow up. Total 96 neoantigens analyzed. h) Pie chart showing proportion of neoantigens that elicited antigen specific response in patients within Alive (N=6) vs Deceased (N=4) cohorts. SFC: spot forming cells. In f-h patients that expired with no evidence of their disease recurrence are excluded. *p value indicates two-sided Student’s T-test. *** <0.001. # p value indicates Log-rank (Matel-Cox) test ### <0.001. in patients within Alive (N=6) vs Deceased (N=4) cohorts. SFC: spot forming cells. In f-h patients that expired with no evidence of their disease recurrence are excluded. *p value indicates two-sided Student’s T-test. *** <0.001. # p value indicates Log-rank (Matel-Cox) test ### <0.001.

Supplementary Fig3. Neoantigen specific antibody responses induced by PGV001. Patient plasma was subjected to seromics by ELISA using linear SLPs in the vaccines. N=12 patients. PID-017 excluded from analysis due to high background. a) Stacked bar graph depicting number of neoantigens that induced an IgG/A or M response in each patient at Week 8 and Week 28. For seromics by ELISA a “Responder” neoantigen is defined as a peptide that induced an antibody titer of greater than 100 compared to baseline. b) Line diagrams showing changes in antibody isotypes induced by responder peptides in patients from pre-treatment (Pre), through Prime (Week 8) and Post (Week 28, Week 31 or End of treatment (EOT)). c) Line graph showing changes in titers of total IgG-subclasses induced by responder peptides in PID-006 and PID-008. d) Heat map depicting poly-ICLC specific antibody responses in each patient. e) Plot depicting linear correlation between total IgG antibody titer and IFNγ ELISPOT response at Week 8 (Prime) and Week 28/31 (Post). Pearson correlation coefficient and Spearman correlation coefficient calculated for fitting of the correlation with 95% confidence interval (95%CI). Each dot represents a neoantigen. Horizontal line depicts threshold for antibody titer response at 100. The vertical dotted line depicts threshold for ELISPOT immunogenicity at 60 SFC/million PBMCs. For Week 8, 126 neoantigens evaluated while for Week 28/31, 116 neoantigens were evaluated.

Supplementary Table5: Cellular Immunophenotyping antibody panels

Supplementary Table1. Staging at time of enrollment, adjuvant treatment following curative intent treatment until the end of vaccination and vaccination timing

SIGNIFICANCE:The PGV001 platform is feasible, safe, and immunogenic. The OpenVax pipeline predicted immunogenic neoantigens in tumors with wide-ranging mutational burdens. Data from this study prompted three additional PGV001 trials, one in newly diagnosed glioblastoma, one in urothelial cancer in combination with an ICI, and another in prostate cancer.

Abstract The tumor microenvironment (TME) of glioblastoma (GBM) is populated by cells that foster immunosuppression. Reversing immunosuppression and promoting tumor surveillance by T cells that recognize the antigens generated by tumor-specific mutations (neoantigens) is critical to eliminating the tumor cells. In a phase 1 clinical trial of newly diagnosed GBM (NCT03223103), patients were treated with personalized neoantigen vaccines (PNV) combined with standard of care (resection, radiotherapy, temozolomide chemotherapy, and TTFields). The neoantigens were identified using the OpenVax computational pipeline. Primary endpoints included safety and feasibility, and the secondary endpoints included PFS, OS, TME analysis, and immunogenicity assays. The study enrolled 12 patients, ages 32-84, between December 2017 - July 2020. With a feasibility endpoint of one successful PNV administration, all patients got at least 6. All developed PNV-related injection site reactions, and flu-like symptoms grade 1-2. Concomitant use with TTFields did not increase toxicity. None of the patients exhibited dose-limiting toxicity within 30 days of receiving the PNV. However, one developed grade 3 SAE, consisting of seizures, neck pain, and progressive neurological deficits, after vaccine 10, culminating in a grade 5 SAE and brain demyelination 10 months later. Assays performed on this subject showed induction of neoantigen-specific responses with PNV. PNV induced CD4+ and CD8+ T cells reactive against multiple vaccine peptide sequences with CD8+ T cell cross-reactivity against one wild-type epitope. Other patients had immunogenicity against the vaccinated peptides. Overall, the 6 months PFS was 100%, with 67% 2-year and 58% 3-year survival. Although the mutation burden is low in GBM, neoantigens were detected in all tumors, and PNV were manufactured for all patients. PNV with synthetic long peptides combined with standard care treatment may help improve outcomes in GBM. Cross-reactivity may potentially be responsible for post-vaccination T-cell demyelination. Further evidence is needed to substantiate a direct effect. Funding: CRI - V Foundation CLIP (#3680)

Overlapping peptides covering the entire NY-ESO-1 protein

Supplemental Figure 3. Selected BTM geneset enrichments (FDR < 0,25) of SD (patient 002) and PD patients (004 and 008) at tumor site. Gene names of enriched genesets are publicly available online in supplemental data for original paper23.

Supplemental Table 1. Quantitative Immunohistochemistry (IHC). In the patient 002 with clinical benefit (stable disease, right two columns), IHC analysis of tumor showed increased CD4 (60x), CD8 (10x), PD1 (20x) and PDL1 (3x). In patients with disease (001, 004, 005, 008), quantitative IHC showed unchanged or decreased levels of CD4, CD8, PD-1, and PDL-1 over treatment periods. Individual antigens were quantified using the CRI Inform software (Perkin Elmer) which applies user-directed antigen thresholds to generate percentages normalized to the total tumor area.

One-way ANOVA analysis comparing expression of cytokines between Avelumab treated and Avelumab combined with Bevacizumab

Supplemental Figure 2. Hierarchical clustering of total gene expression of PBMC and tumor RNA samples. Only genes commonly expressed in PBMC and tumor samples are shown. Of note, up-regulation of specific genes at tumor site may come from tumor rather immune cells. On x-axis, each column represents patient at specific treatment time point, as numbered below. For example, 002- C1W1 is patient 002 at time point Cycle 1 week1. 1 - 002_Screen 12 - 004_Screen 2 - 002_C1W1 13 - 004_C1W2 3 - 002_C1W3 14 - 008_Screen 4 - 002_C1W7 15 - 008_C1W1 5 - 002_C1W10 16 - 008_C1W3 6 - 002_C2W11 17 - 008_C1W7 7 - 002_C2W12 18 - 008_C1W10 8 - 002_C2W13 9 - 002_C2W17 10 - 002_C2W20 11 - 002_C2W26