Session: 711. Cell Collection and Manufacturing of HSPCs, CAR-T Cells, and Other Cellular Therapy Products: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Treatment Considerations, Biological therapies, Immunology, Biological Processes
Previously, we have shown the importance of temporal NOTCH and T-cell receptor signaling in the derivation of CD8αβ+ iT cells (van der Stegen et al. 2022). To further understand the nature of iT cells, we conducted an extensive parallel comparison between CD8αβ+ iT and primary T cells. We demonstrate the functional, phenotypic, and transcriptomic overlap between CD8αβ+ iT and primary T cells, confirming the capacity of iT cells to support CAR-mediated responses. Specifically, to demonstrate the functionality of unedited CD8αβ+ iT cells, we investigated the intrinsic CD3 signaling capacity of iT cells relative to primary T cells by co-culturing with EGFR+ tumor cells in the presence of an anti-CD3, anti-EGFR bi-specific T cell engager (TCE). Similar to primary T cells, iT cells achieved complete clearance of tumors and established an activation profile with early upregulation of CD69 followed by high affinity IL-2Rα (CD25). Furthermore, following CD3 engagement, iT cells exhibited rapid antigen-mediated response by initiating proliferation and achieving equivalent-fold expansion when compared to primary T cells. RNA-seq analyses of two distinct clones of TCRαβ+ iT cells after 12 and 36 hours revealed 2,656 and 2,719 upregulated genes, respectively (FC >1.5, p < 0.05). In comparison, primary T cells from two different donors resulted in the upregulation of 2,048 and 2,167 genes, with 66% of the early-response genes and 54% of the late-response genes overlapping with those upregulated in iT cells. The shared early-activation gene signatures were enriched for pathways indicative of T-cell activation, such as NF-kappa B, JAK-STAT, and TNF signaling. Importantly, TCRαβ+ iT and primary CAR T cells maintained transcriptional similarity following CD3 stimulation, with late-response gene programs revealing sustained cytokine production and proliferation.
CAR-mediated T-cell functionality relies upon the endogenous signaling capability of T cells through redirecting single-chain variable fragment (scFv)-mediated antigen interactions via CD3 and co-stimulatory signal cascades. To this end, TRAC-engineered CD19-CAR iT cells were compared to primary CAR T cells: 75% and 40% of 2-fold upregulated and downregulated genes, respectively, were identical between CAR iT and CAR T cells at 12 hours post antigen exposure relative to non-antigen exposed baselines. The comparability of genetic signatures between the two T-cell groups revealed similar durable functional outcomes for both sets, including complete elimination of target cancer cells in an array of in vivo studies.
Taken together, CD8αβ+ iT cells show significant transcriptional, phenotypic, and functional similarities to primary T cells following CD3 and CAR engagement, confirming the potential use of CAR iT cells as a promising off-the-shelf approach to cell therapy.
Disclosures: Hancock: Fate Therapeutics: Current Employment. Tuncel: Fate Therapeutics: Current Employment. Omilusik: Fate Therapeutics: Current Employment. O'Connor: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yang: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Haynes: Fate Therapeutics: Current Employment. Yzaguirre: Fate Therapeutics: Current Employment. Palomares: Fate Therapeutics: Current Employment. Kothapally Hanok: Fate Therapeutics: Current Employment. Denholtz: Fate Therapeutics: Current Employment. Hosking: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Peralta: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Clarke: Fate Therapeutics: Current Employment. Goulding: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goodridge: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sadelain: Atara Bio: Patents & Royalties: Licensed Patents and Research Funding ; Fate Therapeutics: Patents & Royalties: Licensed Patents and Research Funding ; Mnemo Therapeutics: Current equity holder in publicly-traded company, Patents & Royalties: Licensed Patients and Research Funding ; Takeda Development Center Americas, Inc.: Patents & Royalties: Licensed patents and Research Funding . Valamehr: Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company.