P-08.04 Impact of variability of CD4+/CD8+ T cell ratios on the functional properties of CAR T cell products

<h3>Background</h3> Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have shown significant clinical success, particularly against certain leukemias and lymphomas. CAR T cell products generally consist of donor-dependent, highly variable ratios of CD4+ and CD8+ T-cell subsets. However, there is still a lack of comprehensive understanding of how each subset contributes, both collectively and individually, to the different effector functions typically associated to CAR T cell products. While CD8+ T cell responses are classically associated with cytotoxicity, and CD4+ T cells responses with cytokine secretion, their respective roles within CAR T cell products remains controversial across different models. This variability could potentially impact the functional characterization assays and the release criteria of these therapies, highlighting the need for more detailed studies. <h3>Materials and Methods</h3> Automated CAR T cell manufacturing was performed from multiple healthy donor-derived Leukapheresis using the CliniMACS Prodigy® platform. Cell composition, viability and transduction efficiency were determined by flow cytometry. CD4+ and CD8+ T cells were magnetically isolated from the resulting CAR T cell products using REAlease® technology, resulting in label-free cells. Functional characterization of CD4+ and CD8+ CAR T cells was performed after co-culture with antigen-expressing target cells. Cytokine secretion was evaluated using the MACSPlex cytokine bead assay, degranulation by CD107a extracellular exposure, and cytotoxicity by target cell lysis, all measured by flow cytometry. <h3>Results</h3> We analyzed CAR T cell products from multiple donors. The frequency of CD4+ and CD8+ T cells among CAR+ T cells was highly variable, including one donor with &lt;5% CD8+ CAR T cells. However, both CD4+ and CD8+ CAR T cell subsets were able to secrete proinflammatory cytokines (e.g. IFNγ, TNFα and IL-2), degranulate and kill tumor cells. The capacity of each subset to exert each effector function was quantified and compared. <h3>Conclusions</h3> Our study addresses the previously unresolved question of whether the variable frequencies of CD4+ and CD8+ CAR T cells within donor-derived CAR T cell products could influence the functional characteristics of these products. Such variability has the potential to affect the outcomes of functional characterization assays and, consequently, the release criteria for these therapies. However, our findings demonstrate that both CD4+ and CD8+ CAR T cell subsets, regardless of their relative frequencies, are capable of secreting proinflammatory cytokines and exerting cytotoxic effects against tumor cells. This suggests that the functional capabilities of CAR T cell products are maintained across different CD4+/CD8+ ratios, indicating that this variability may not be a critical issue for the efficacy and release of CAR T cell therapies. <b>C. Pitzka:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>N. Hussein:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec. <b>N. Pietz:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>D. Rheindorf:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>N. Tokarew:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG. <b>C. Evaristo:</b> A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. &amp; Co. KG.