Background: The development and clinical application of immune checkpoint inhibitors has transformed the therapeutic landscape for cancer treatment in recent years. Balstilimab (AGEN2034) is a fully human, monoclonal IgG4 antibody that binds with high affinity to programmed death 1 (PD-1), thus preventing the interaction between this receptor and its ligands programmed death ligand 1 and 2 (PD-L1, PD-L2). Emerging evidence suggests that balstilimab exhibits a differentiated activity profile compared to currently approved anti-PD-1 agents, including pembrolizumab and nivolumab. Methods: Balstilimab as monotherapy was evaluated in a large phase 2 study in patients (pts) with recurrent/metastatic cervical cancer who had relapsed after a platinum-based treatment regimen for advanced disease. Pts were dosed at 3 mg/kg once every 2 weeks for up to 24 months and antitumor activity was assessed using RECIST v1.1. The tumor cell killing activity of balstilimab was evaluated preclinically in a human co-culture system of (1) primary T cells engineered to recognize NY-ESO-1 and (2) NY-ESO-1⁺ cancer cell lines, including PD-L1 and/or PD-L2-deficient engineered lines. The co-culture system was maintained for ̃ two weeks to drive partial T cell exhaustion; a state where cytotoxicity is compromised but recoverable with PD-1 blockade. Cytotoxicity of these partially exhausted T cells was quantified against PD-L1/L2 double positive, single positive, or double negative cancer cells in the presence or absence of PD-(L)1 antibodies. Results: In the second-line treatment setting for pts with advanced cervical cancer, balstilimab showed a numerically higher objective response rate (ORR) in subjects with PD-L1⁺, squamous cell carcinoma (SCC) tumors (21%, 95% CI, 12.7-32.6%) than those reported for pembrolizumab. Unlike pembrolizumab, balstilimab showed activity in PD-L1(-) pts, and irrespective of tumor histology (ORR 7.9%, 95% CI, 2.7-20.8%). Despite lower overall PD-L1 positivity compared to SCC (41.7 v 72.9%), an ORR of 12.5% (95% CI, 5.9-24.7%) was observed in the subset of pts with a poorer prognosis, those with cervical adenocarcinoma. Concordant with clinical observations, balstilimab demonstrated superior rescue of antigen-specific T cell cytotoxicity in vitro relative to pembrolizumab, nivolumab, or atezolizumab. Balstilimab also induced cytotoxicity against PD-L1 and/or PD-L2 deficient target cancer cells. Conclusions: Taken together, these data suggest functional differentiation of balstilimab from other PD-1 inhibitors with potentially important implications for extending the therapeutic reach of anti-PD-1 therapy. Investigation of the underlying mechanistic basis for these findings is ongoing. Clinical trial information: NCT03104699