Abstract 2756: Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape
Authors: Chang Chia-Wei , Yang Bi-Huei , Lin Eason , Shirinbak Soheila , Yeh Wen-I , Pribadi Mochtar , Chu Helen , Gutierrez Alma , Avramis Earl , ORourke Jason , Lee Tom , Witty Alec , Peralta Eigen , Hosking Martin , Valamehr Bahram 
Published: June 2022

Adoptive T-cell therapy with chimeric antigen receptor (CAR) has shown promising results in cancer treatment, however, antigen escape and tumor heterogeneity are major causes for disease relapse. While CARs are known to trigger an effective immune response through surface antigen recognition many solid tumor cancer antigens are intracellular and presented by MHC molecules recognized by T cell receptors (TCRs). In addition, many therapeutic antibodies have shown clinical efficacy in solid tumor settings. However, antibody-dependent cellular cytotoxicity (ADCC) is mediated by the CD16 Fc receptor naturally expressed on NK cells although 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, to determine whether challenges associated with the treatment of solid tumors, which are heterogeneous and challenging to treat, may be overcome.To test the base line activity of CAR-iT cells in the solid tumor setting, we selected our anti-MICA/B CAR, previously shown to effectively target stress ligands found on transformed cells, to demonstrate effective anti-tumor activity against multiple solid tumor cell lines (72 hrs cytotoxicity: A2058 = 99%; 786-O = 98%; versus non-specific CAR-iT cells: A258 = 13%; 786-O = 17%). To test compatibility of TCR in our iT cell platform, we engineered MR1-TCR in iT cells to show increased cytokine release and degranulation upon stimulated with MR1 positive lung carcinoma epithelial cells line A549 (fold change compared to un-stimulated: IFNg = 210, p = 0.0032; TNFa = 76.9, p = 0.0005; CD107ab = 115.0, p=0.0013). Notably, with the engineering of tumor antigen specific TCR in TCR-less CAR-iT cells, CD3 complex can be re-established to provide an opportunity to combine with bispecific T cell engagers. Lastly, combining CAR-iT cells with MR1-TCR and hnCD16 uniquely demonstrated synergistic tumor growth inhibition and validated our approach to target multiple antigens at once for an effective anti-tumor response (A549 cytotoxicity: tumor only = 3.68±2.04%; effector+TCR = 41.31±2.27%; effector+TCR+ADCC = 90.28±1.87%). 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.

Citation Format: Chia-Wei Chang, Bi-Huei Yang, Eason Lin, Soheila Shirinbak, Wen-I Yeh, Mochtar Pribadi, Helen Chu, Alma Gutierrez, Earl Avramis, Jason ORourke, Tom Lee, Alec Witty, Eigen Peralta, Martin Hosking, Bahram Valamehr. Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2756.