Background: TTFields induce anti-tumor immunity via simultaneous activation of type-1 interferon (T1IFN) pathways of the STING and AIM2 inflammasomes and immunogenic cell death. Thus, TTFields-treated GBM cells may provide a complete in situ vaccination platform and synergize with immune checkpoint inhibitors to prolong survival in GBM patients. Methods: We enrolled 26 newly diagnosed GBM patients in a pilot phase 2 study combining TTFields, pembrolizumab and maintenance temozolomide (TMZ). To distinguish immune effects of TTFields from those of pembrolizumab, TTFields was started at cycle 1 of TMZ while pembrolizumab (200 mg IV every 3 weeks) at cycle 2 of TMZ. Primary endpoints were progression-free survival (PFS) versus case-matched controls treated with TTFields plus TMZ only in the EF-14 study. Secondary endpoints included overall survival (OS), toxicity, and signature and mechanism of response by multiomics analyses of PBMCs and tumors. Results: The median age was 60.5 years. Fourteen (54%) had biopsy only or partial resection. Nineteen (73%) had unmethylated MGMT and 3 (11.5%) had an IDH mutation. Median PFS was 12.0 months versus 5.8 months in a case-matched control cohort of 26 patients (HR = 0.466; 95% CI: 0.234-0.936; Log Rank P = 0.007). Twelve-month PFS was 50.0% versus 28.2% in controls; P = 0.058. Median OS was 24.8 months versus 14.7 months in controls (HR = 0.388; 95% CI: 0.194-0.775; P = 0.039). Two-year OS was 52.4% versus 12.0% in controls; P = 0.004. Six of 15 (40%) patients with measurable disease achieved partial to complete response. In a Cox regression analysis adjusting for key prognostic factors, P-value reached 0.0317 for PFS and 0.0074 for OS. The most common serious adverse events were thromboses, seizures, and metabolic disturbances in 4 (15%), 3 (11.5%), and 2 (7.7%) patients, respectively. Molecular analyses prior to the addition of pembrolizumab confirmed robust T cell activation by TTFields via the T1IFN trajectory, as evidenced by a high correlation between TCRab clonal expansion and T1IFN responsive plasmacytoid dendritic cells (Spearman coefficient = -0.8; P = 0.014) and defined a T cell-based gene signature of TTFields effects. Subsequently, the ability of the top expanded TCRab clones to adapt to the everchanging tumor microenvironment through successful clonal switching by 2 months after the addition of pembrolizumab strongly predicted response to the triple combination in a Cox HR fit model for OS with a concordance rate of 0.876, Log Rank P = 0.031. Conclusions: The triple combination was well tolerated and demonstrated promising efficacy in newly diagnosed GBM. Multiomics analyses confirmed the robust in situ immunizing property of TTFields with synergy with pembrolizumab and identified potential signatures of response to TTFields and the triple combination. Correlative analysis will be updated. Clinical trial information: NCT03405792.