Combination of Three Unique Anti-Tumor Modalities Engineered into iPSC-Derived T Cells Demonstrate a Synergistic Effect in Overcoming Tumor Heterogeneity and Cancer Escape
Authors: Yang Bi-Huei , Lin Yu-Sheng Eason , Shirinbak Soheila , Yeh Wen-I , Pribadi Mochtar , Chu Hui-Yi , Gutierrez Alma , Mehta Amit , Avramis Earl , Petrovic Roseanna , Whitlock Benjamin , ORourke Jason , van der Stegen Sjoukje , Lee Tom , Witty Alec , Peralta Eigen , Hosking Martin , Chang Chia-Wei , Valamehr Bahram 
Published: November 2021

Abstract
Chimeric antigen receptor (CAR) is known to trigger an effective immune response through surface antigen recognition enhanced by T-cell activation signal one (ex. CD3) and signal two (ex. CD28); however, targeting neoantigens and intracellular antigens remains a challenge. On the other hand, the T-cell receptor (TCR) can target neo/intracellular antigens presented by MHC molecules, but often the response is not as potent. The CD16 Fc receptor, which is naturally expressed on NK cells, mediates antibody-dependent cellular cytotoxicity (ADCC), but its application in T cells is yet not fully appreciated.
Utilizing our proprietary induced pluripotent stem cell (iPSC) platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-T cells (CAR-iT cells), we aimed to study the combination of these three targeting modalities, CAR, TCR, and CD16 (tri-modal), to determine whether challenges associated with the treatment of heterogeneous tumors may be overcome.
To evaluate the benefit of the three distinct targeting modalities, we tested the functional activity of individual and various combinations of i) anti-CD19 CAR, anti-MICA/B CAR, and anti-BCMA CAR, ii) high-affinity, non-cleavable CD16 (hnCD16), and iii) MR1 and NYESO1 TCR modalities in iT cells. All tested combinations successfully expressed the designated edits and differentiated into iT cells (T-lymphocytes &gt; 95%). Initially, we tested CD19 CAR and MR1 TCR in a 9-day serial killing assay of Nalm6 leukemia cells (CD19 high, MR1 +), where we observed CD19 CAR-iT cells induce prompt CAR-mediated tumor growth inhibition (TGI), saw similar effective killing by MR1 TCR-iT cells but with a 24-hr delay, and observed the most effective response of tumor cell elimination when both where combined in the same iT cell population (relative tumor counts; Day 1, no stim: 2.6, CAR: 0.28, TCR: 1.02, CAR+TCR: 0.02; Day 2, no stim: 5.36, CAR: 0.25, TCR: 0.05, CAR+TCR: 0.02). All conditions (CAR, TCR, and CAR+TCR) reached and maintained complete TGI by Day 9 of the assay (relative tumor count, Day 9, no stim: 50.97, all other stim conditions: &lt;0.01). In line with killing kinetics, the time for the activation marker CD25 upregulation differed between CAR and TCR (peak time and percentage of CD25 +, CAR: Day 1, 49.9%; TCR: Day 5, 74.6%). Co-triggering of CAR and TCR in combination revealed quickest, highest, and sustained CD25 upregulation levels (CD25 +, CAR+TCR Day 1: 62.5%, Days 3 to 9: &gt;90%), indicating a synergistic effect and compatibility between CAR and TCR.
Assessing anti-MICA/B CAR and hnCD16, we confirmed the hnCD16-mediated response in iT cells in the presence of anti-MICA/B CAR when crosslinking hnCD16 via biotinylated anti-CD16 antibody with streptavidin (phosphorylated CD3zeta peaked at 10 min upon ADCC triggering), indicating the compatibility between a CAR-iT cell and the hnCD16 motif.
Lastly, combining iT cells expressing anti-BCMA CAR + MR1 TCR + hnCD16 with daratumumab (anti-CD38 mAb) in a 9-day serial killing assay demonstrated the best TGI among the groups with a near-elimination of transgenic Nalm6 cells (area under curve, no stim: 30.29, TCR: 1.564, CAR: 0.7087, hnCD16+mAb: 1.452, trimodal+mAb: 0.5824). To assess the function of the tri-modal iT cells in vivo, we used a disseminated xenograft model of B-cell leukemia where a heterogenous mixture of transgenic Nalm6 leukemia cells was used to mimic tumor heterogeneity. Assessment of the bone marrow revealed the unique capacity of each target modality to eliminate its target designated Nalm6 leukemia group, with tri-modal iT cells effectively clearing all populations (Figure 1).
In summary, using the unique approach to engineer iPSCs at the clonal level to create a distinct population of engineered iT cells, we successfully demonstrated the compatibility between CAR, TCR, and hnCD16 to mitigate tumor heterogeneity. This approach is an ideal strategy to create off-the-shelf cellular immunotherapy for a promising therapeutic approach to combat heterogeneous and difficult to treat solid tumors, including those that are resistant due to antigen escape.
Figure 1 Figure 1.

Disclosures
Yang: Fate Therapeutics, Inc.: Current Employment. Lin: Fate Therapeutics, Inc.: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Pribadi: Fate Therapeutics, Inc.: Current Employment. Chu: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Mehta: Fate Therapeutics, Inc.: Current Employment. Avramis: Fate Therapeutics, Inc.: Current Employment. Whitlock: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Witty: Fate Therapeutics, Inc.: Current Employment. Peralta: Fate Therapeutics, Inc.: Current Employment. Hosking: Fate Therapeutics: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.