Characterizing the immune effects of enfortumab vedotin and pembrolizumab (EV+P) on peripheral blood mononuclear cells (PBMCs) in metastatic urothelial cancer patients (mUC).

4571 Background: Enfortumab vedotin improves response and outcomes over chemotherapy in mUC, with further benefit when combined with pembrolizumab. Although EV alone has limited immune-activating effects, we hypothesize that the combination of EV+P modulates peripheral T-cell activation states and correlates with clinical response. Methods: We conducted a retrospective analysis of mUC patients treated with EV+P at Memorial Sloan Kettering Cancer Center. PBMCs were isolated from patients on C1D1 and C2D1 of EV+P (n=33) and analyzed with a 32-color spectral flow cytometry panel. Immune subsets were compared between responders (complete [CR] or partial response [PR]) and non-responders (stable [SD] or progressive disease [PD]) using Wilcoxon rank sum test. Response was determined by a radiologist’s assessment of the best-overall response on EV+P according to RECIST v1.1. Results: Among 33 patients, median age was 71 years and 48% were male. 12.1% (4/33) received prior platinum chemotherapy and 18.1% (6/33) received prior PD-1/PD-L1 immunotherapy. The objective response rate was 67% (CR=10; PR=12); eight patients had SD and three had PD. At C1D1, CD8+ T cell frequencies did not differ by response. At C2D1 however, circulating CD8+ T cells were significantly higher in non-responders (p < 0.05). From baseline to C2D1, CD8+ T cells showed increased expression of CTLA4+ (p < 0.0001), Ki67+ (p < 0.01) and HLA-DR+ (p < 0.01), with higher expression of each marker at C2D1 significantly correlated with response (all p < 0.05, Table 1). At C2D1, CD4+ effector T cells showed increased expression of Ki67+ and HLA-DR+ (both p < 0.05), both significantly higher in responders (both p < 0.05). CD4+ regulatory T cells showed increased CTLA4+ (p < 0.001), Ki67+ (p < 0.01) and HLA-DR+ (p < 0.01) expression from C1D1 to C2D1, with significantly higher expression in responders (CTLA4+ p < 0.05, Ki67+ p < 0.0001, HLA-DR+ p < 0.001). Across all T cell subtypes, PD1+ expression significantly decreased (p < 0.05) from C1D1 to C2D1, but this decrease did not correlate with response. The frequencies of circulating memory CD8+ and CD4+ T cells did not change on treatment or correlate with response at either timepoint. Conclusions: EV+P induces early peripheral T cell activation in mUC, with increased expression of activation and proliferative markers in responders at C2D1. Ongoing studies are investigating correlation between TCR specificity, immune activation and responses to EV+P. Peripheral CD8+ T cell frequencies (median) at C1D1 and C2D1 from mUC patients on EV+P. CD8+ T Cell C1D1 C2D1 p-value C2D1 in Responders C2D1 in Non-Responders p-value CD8+ (%CD45+) 8.54 7.8 0.6 7.27 13.8 0.03 CTLA4+ (%CD8+) 1.15 3.94 0.0001 4.57 2.85 0.02 Ki67+ (%CD8+) 3.18 6.27 0.005 7.26 5.4 0.04 HLA-DR+ (%CD8+) 4.66 9.37 0.004 10.22 4.13 0.04 PD-1+ (%CD8+) 45.6 29.8 0.0005 28.7 31.5 0.48