Abstract A120: Topological distribution of stimulating factors on artificial antigen-presenting cells defines the therapeutic efficacy of the <i>ex vivo</i> expanded tumor-specific CD8+ T-cells

Abstract Adoptive cellular therapy (ACT) exploits the concept of ex vivo expansion of tumor-specific CD8+ T-cells to restrict the tumor growth and metastasis. Despite remarkable success in the clinical field, further growth of this potential avenue is heavily compromised. The complexities associated with harvesting and reprogramming of autologous professional antigen-presenting cells (such as dendritic cells) from the cancer patient to activate and expand the tumor-specific CD8+ T-cells is one of the major hurdles. Although material-based micro/nano scaffolds have shown promising results in complementing this approach, a precise mechanistic study to understand the importance of the topological distribution of various stimulating factors on micro/nano-scaffolds in regulating the expansion dynamics and therapeutic efficacy of the expanded cells remains unexplored. To have a better insight, we have developed iron-oxide nanoparticle-based artificial antigen-presenting cells (aAPCs) with different valencies of class I peptide-major histocompatibility complex (p-MHC) while with a constant valency of anti-CD28 to activate and expand the tumor-specific CD8+ T-cells following ex vivo culture conditions. Interestingly, we found that only the aAPCs having p-MHC valency (#p-MHCs/nanoparticle) 25 or 40 (high valency aAPCs) can significantly expand (∼15 fold) the antigen-specific T-cells in just 7 days without employing exogenous growth factor (i.e., IL-2) as compared to the aAPCs displaying 10 or 4 p-MHCs/nanoparticle (low valency aAPCs) (only ∼5 fold). In addition, FACS-based immunophenotyping revealed that the high valency aAPCs could differentiate the expanded cells into the non-exhausted effector population with an extraordinary ability to produce cytolytic molecules (such as granzyme B and perforin) as well as effector cytokines (TNF-α and IFN-γ) in significantly higher amounts than low valency aAPC expanded cells. Most importantly, we found that the enhanced therapeutic potential of high valency aAPC-expanded T-cells can exhibit profound anti-tumor immunity by inhibiting the tumor growth and offer enhanced survival, requiring infusion of CD8+ T-cells as low as 1 million in a single dose, compared to low valency aAPC expanded or non-expanded CD8+ T-cells of 10 million. Mechanistically, through confocal microscopy, we found that the p-MHC valency governs the TCR-p-MHC interaction by inducing the magnitude of TCR cluster formation in case of higher valency aAPCs compared to low valency aAPCs at the immune synapse, subsequently facilitating the downstream signaling cascade by modulating the functional activities of critical transcription factor, NFAT (nuclear factor of activated T-cells). Taken together, this study highlights the topological distribution in addition to the types of stimulatory signals as the critical parameters to formulate the nano-scale aAPCs for developing a robust tool for manufacturing the therapeutically potent T-cells to complement the existing methods that would enhance the widespread use of adoptive T-cell therapy for treating cancer. Citation Format: Antara Mondal, Faisal Jamal, Arpita Das, Arnab Kumar. Sahoo, Khushboo Chaudhary, Santiswarup Singha. Topological distribution of stimulating factors on artificial antigen-presenting cells defines the therapeutic efficacy of the ex vivo expanded tumor-specific CD8+ T-cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A120.