Increasing the odds: antibody-mediated delivery of two distinct immunogenic T-cell epitopes with one antibody

Wulp, W. van der; Remst, D.F.G.; Koster, C.S.; Wouters, A.K.; Ressing, M.E.; Schuurman, J.; Kasteren, S.I. van; Bleijlevens, B.; Hoeben, R.C.; Guelen, L.; Heemskerk, M.H.M.

doi:doi:10.1080/2162402X.2025.2508050

Antibody-epitope conjugates (AECs) proved to be a promising new therapeutic strategy to redirect virus-specific CD8+ T cells toward cancer cells by delivering T-cell epitopes. To be able to redirect a larger fraction of the virus-specific T-cell population, it is beneficial to deliver a broader selection of T-cell epitopes. We investigated two different methods to generate AECs with two distinct virus-specific T-cell epitopes fused to one antibody. Epitopes were either placed in a tandem-like fashion at the C-terminus of the AEC (t-AEC) or bispecific-AECs (bs-AECs) were generated via controlled Fab-arm exchange to generate bs-AECs with two identical antigen binding domains, but two distinct epitopes on each Fab-arm. Our study revealed that maintaining a free epitope terminus was required for efficient delivery of the virus-specific T-cell epitopes. Consequently, viral-epitope delivery using t-AECs was suboptimal as the concatenated epitopes were less effectively delivered to the...Show moreAntibody-epitope conjugates (AECs) proved to be a promising new therapeutic strategy to redirect virus-specific CD8+ T cells toward cancer cells by delivering T-cell epitopes. To be able to redirect a larger fraction of the virus-specific T-cell population, it is beneficial to deliver a broader selection of T-cell epitopes. We investigated two different methods to generate AECs with two distinct virus-specific T-cell epitopes fused to one antibody. Epitopes were either placed in a tandem-like fashion at the C-terminus of the AEC (t-AEC) or bispecific-AECs (bs-AECs) were generated via controlled Fab-arm exchange to generate bs-AECs with two identical antigen binding domains, but two distinct epitopes on each Fab-arm. Our study revealed that maintaining a free epitope terminus was required for efficient delivery of the virus-specific T-cell epitopes. Consequently, viral-epitope delivery using t-AECs was suboptimal as the concatenated epitopes were less effectively delivered to the target cells. However, well-defined bs-AECs containing both CMV and EBV epitopes were successfully generated and both in vitro and in vivo efficacy was evaluated. Our results demonstrate that bispecific-AECs can efficiently deliver EBV and CMV epitopes simultaneously to multiple cancer cell lines from different origins, thereby redirecting and activating two distinct populations of virus-specific T cells. Furthermore, our in vivo findings indicate that when both virus-specific T-cell populations are present and tumor cells express the proteases required for efficient epitope delivery, bs-AECs exhibit similar efficacy in reducing tumor burden compared to AECs. To conclude, our study demonstrates the feasibility of redirecting two groups of virus-specific T cells using a single antibody and highlights the potential of bs-AECs both in vitro and in vivo.Show less

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Increasing the odds: antibody-mediated delivery of two distinct immunogenic T-cell epitopes with one antibody