Background: Novel T cell-enabling therapies plus checkpoint inhibition may improve OS in GBM. INO-5401 (synthetic DNA plasmid encoding hTERT, WT-1, PSMA) plus INO-9012 (synthetic DNA plasmid encoding IL-12), with cemiplimab (PD-1 inhibitor), was given to patients with newly diagnosed GBM with MRD to evaluate tolerability, efficacy, and immunogenicity. Median OS and immunogenicity at 18 months (OS18) are reported. Methods: This is a phase I/II, single arm, two cohort (A: unmethylated MGMT and B: methylated MGMT) study. Primary endpoint is safety; efficacy and immunogenicity are secondary. Nine mg INO-5401 plus 1 mg INO-9012 (4 doses Q3W, then Q9W) was given IM with EP in combination with cemiplimab (350 mg IV Q3W). Hypofractionated RT (40 Gy over 3 weeks) with TMZ was given to all patients, followed by maintenance (Cohort B only), which was a novel therapeutic approach. Immunogenicity was assessed by quantifying INO-5401-specific peripheral cellular immune responses via IFN-g ELISpot and flow cytometry. Intra-tumoral gene expression was analyzed by RNA-Seq of FFPE GBM tissue. Differences in gene expression were analyzed using the Wilcoxon rank sum test. Results: Fifty-two subjects were enrolled: 32 in Cohort A; 20 in Cohort B (35% women; median age 60 years [range 19-78 years]). The adverse event profile was consistent with known single-agent (INO-5401, INO-9012, EP or cemiplimab) events; most events were ≤Grade 2 and no related events were Grade ≥4. Median OS durations in Cohorts A and B were 17.9 months (95% CI 14.5-19.8) and 32.5 months (95% CI 18.4-not reached), respectively. Flow cytometry revealed activated, antigen specific CD4+CD69+PD1+ and CD8+CD69+PD1+ T cells, the latter with lytic potential as defined by presence of perforin and granzyme A. Both subsets exhibited HR < 1.0 and p < 0.05 when accounting for a 0.1% T cell frequency change, translating to a 23% and 28% reduced risk of death, respectively. Gene expression levels in pre-treatment tissues were similar between alive and deceased groups for INO-5401 antigens and immune cell markers; however, the alive group displayed significantly reduced expression of genes associated with anti-apoptosis, pro-proliferation, and immune response suppression. Post-treatment tumor tissue displayed altered gene expression for immune-related markers versus pre-treatment tissue, including markers of T cell infiltration, activation, and lytic potential. Conclusions: INO-5401 + INO-9012 has an acceptable risk/benefit profile and elicits robust immune responses that correlate with enhanced survival when administered with cemiplimab and RT/TMZ to newly diagnosed GBM patients. Pre-treatment gene expression signatures in MGMT-unmethylated patients were statistically associated with OS18. Overall, INO-5401 elicits antigen-specific T cells that can infiltrate GBM tumors. Clinical trial information: NCT03491683.