Development of Induced Pluripotent Stem Cell-Derived T Cells Exhibiting Phenotypic and Functional Attributes of Primary CAR T Cells
Authors: Hancock Bryan , Tuncel Jonatan , Omilusik Kyla , O'Connor Alison , Yang Bi-Huei , Haynes Matthew , Yzaguirre Amanda , Palomares Karina , Kothapally Hanok Sandeep , Denholtz Matthew , Hosking Martin , Peralta Eigen , Lee Tom , Clarke Raedun , Van Der Stegen Sjoukje , Goulding John , Goodridge Jode , Sadelain Michel , Valamehr Bahram 
Published: December 2024

Autologous chimeric antigen receptor (CAR) T-cell therapy has produced remarkable outcomes for patients with B cell malignancies that have failed multiple lines of previous treatments. The success of CAR T-cell therapies, however, is met with significant logistical challenges, including limitations in generating uniform drug product after genetic engineering and the inability to manufacture substantial quantities of drug product for broad patient access. Consequently, there is a requirement for alternative approaches that can address the challenges of autologous CAR T-cell therapy while achieving or exceeding their therapeutic profile. The use of induced pluripotent stem cell (iPSC) technology to derive iPSC-derived T (iT) cells represents a promising approach, with the potential for large-scale manufacture of high-quality drug product having true off-the-shelf availability.

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 &gt;1.5, p &lt; 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.