Abstract The EVOLVE platform utilizes a CD3 T-cell engager (TCE) with integrated CD2 costimulation to enhance the efficacy and durability of T cell responses. CD2 is a costimulatory receptor expressed on the majority of T cells in the tumor microenvironment (TME). We hypothesize that T cell co-engagement via CD3 and CD2 will result in superior T cell activation in the TME, conferring a greater anti-tumor effect. EVOLVE104, which recently entered the clinic, is a tri-specific molecule targeting CD3, CD2 and tumor antigen ULBP2/5/6. We showed previously that ULBP2/5/6, while minimally expressed on normal human tissues, is upregulated in solid tumors of squamous origin as well as in bladder cancer (1). In xenograft mouse models, EVOLVE104 showed significant tumor growth inhibition compared to bispecific TCE (2). In this study, we use a series of in vitro tests to compare T cell phenotypic and functional profiles after stimulation with EVOLVE104 or with other first generation bispecific TCE formats which lack integrated costimulation. We find that chronic stimulation of human peripheral blood-derived T cells with EVOLVE104 led to greater than a 30-fold increase in CD8+ T cell effector-to-naïve ratios compared to CD3 stimulation alone, demonstrating greater differentiation of T cells after exposure to EVOLVE. These EVOLVE104-stimulated effector cells showed two-fold or greater upregulation of activation markers such as CD25, CD69 and CD137 in a concentration-dependent manner after 24 hours when compared to bispecific TCE-treated T cells, with expression of these markers persisting through multiple rounds of stimulation. EVOLVE104-treated T cells also showed faster and sustained proliferation kinetics in chronic stimulation assays and TDCC assays. In this model, approximately 50% of effector CD8 T cells were proliferating by day 3 with EVOLVE104, compared to 20% with a bispecific TCE, which increased to 80% after multiple rounds of stimulation. Additionally, T cell function was maintained in vitro following chronic stimulation as measured by tumor cell killing of ULBP2/5/6+ tumor cells and by cytokine release in a mixed lymphocyte reaction assay. In conclusion, T cells stimulated with EVOLVE TCE platform with CD2 integrated costimulation shows differentiated and superior T cell phenotype with sustained tumor cell killing and cytokine production in vitro compared to T cells stimulated with TCE bispecific formats lacking CD2 costimulation. This study supports the hypothesis that the EVOLVE platform may provide deeper and more durable anti-tumor responses than first generation bispecific T cell engager formats. A first-in-human clinical dose escalation trial with EVOLVE104 is currently underway (NCT07217171). Citation Format: Emma Stewart, Jennifer Zeiger, Donal Ryan, Roukiah Khalil, William DeMaria, Colleen Brown, Kerry Ralph, Abliz Abduqadir, Jeremy S. Myers, Jay S. Fine, Oksana A. Sergeeva, Sergio Trombetta, Stella Martomo, . Integrated CD2-costimulation on CD3 T cell engager EVOLVE platform yields differentiated and superior T cell effector profile abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4055.
Stewart et al. (Fri,) studied this question.