Abstract 640: Augmenting T-cell bispecific antibody therapy in AML through co-stimulatory agonists

The classical two-signal model states that T-cell activation requires TCR/CD3 engagement (signal 1) and co-stimulation (signal 2) via e.g. CD28 or 4-1BB. T cell bispecific antibodies (TCBs) activate T cells solely through CD3 engagement, bypassing co-stimulatory pathways. However, the efficacy of TCB-mediated cytotoxicity against acute myeloid leukemia (AML) cells is determined by co-stimulatory and co-inhibitory ligands expressed on AML cells (Marcinek et al., 2023). Thus, we hypothesized that TCB-mediated T-cell responses could be enhanced by combination with targeted co-stimulation, particularly in AML cases lacking co-stimulatory ligands. Accordingly, we evaluated the combination of the AML-targeting Wilms tumor 1 (WT1)-TCB with co-stimulatory agonistic bispecific antibodies, targeting 4-1BBL or OX40 and fibroblast activation protein (FAP) expressed on bone marrow stromal cells.Efficacy of WT1-TCB + FAP-4-1BBL or FAP-OX40 was evaluated in co-culture assays with human AML cell lines or murine Ba/F3 cell lines expressing hu4-1BBL or huCD86, healthy donor T cells and an NIH-3T3 fibroblast cell line expressing FAP. In addition to short-term assays (4 days), we co-cultured cells over 28 days to address T-cell exhaustion in the context of continuous target antigen exposure. Lastly, we investigated the impact of co-stimulation in an ex vivo setting with primary AML cells (pAML). T-cell activation, phenotype, cytotoxicity, and metabolic fitness were assessed by MPFC, bulk RNA sequencing, and Seahorse assays. Addition of FAP-4-1BBL strongly enhanced WT1-TCB-mediated cytotoxicity against the AML cell line Set-2, which shows a low expression of co-stimulatory ligands. Although FAP-OX40 did not enhance cytotoxicity, it increased T-cell proliferation. T-cell responses against AML cell lines with high expression of co-stimulatory ligands (SKM-1, OCI-AML3, OCI-AML2) did not show any improvement by the combinatorial approaches. The results were confirmed in the murine Ba/F3 system, showing that especially the presence of hu4-1BBL on the cell surface cancels out the positive effect of the combinatorial approaches. Results could be validated using pAML samples. Importantly, considering the continuous application of TCBs in the clinics, long-term cultures over 28 days showed that adding FAP-4-1BBL or FAP-OX40 preserved T-cell function (proliferation, cytokine secretion, cytotoxicity, and metabolic fitness) over time compared to WT1-TCB alone. Improvements were again stronger and more durable for the combination with FAP-4-1BBL than with FAP-OX40. We conclude, that combining a WT1-targeting TCB with a 4-1BB agonist significantly enhanced T-cell activity against AML cell lines and primary AML samples. Notably, this positive immune modulation persisted in long-term cultures, counteracting T-cell exhaustion caused by continuous TCB stimulation. Citation Format: Anne-Sophie Neumann, Gerulf Hänel, Vesna Kramar, Christina Claus, Kieron White, Amelie Muth, Lis Winter, Monika Sponheimer, Giulia Rappa, Sebastian Theurich, Veit L. Bücklein, Pablo Umaña, Christian Klein, Marion Subklewe. Augmenting T-cell bispecific antibody therapy in AML through co-stimulatory agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 640.