Rationally Designed Transgene-Encoded Cell-Surface Polypeptide Tag for Multiplexed Programming of CAR T-cell Synthetic Outputs
Adam Johnson, Jia Wei, James M. Rosser, Annette Künkele, Cindy A. Chang, Aquene N. Reid, Michael C. Jensen
Abstract
Abstract Synthetic immunology, as exemplified by chimeric antigen receptor (CAR) T-cell immunotherapy, has transformed the treatment of relapsed/refractory B cell–lineage malignancies. However, there are substantial barriers—including limited tumor homing, lack of retention of function within a suppressive tumor microenvironment, and antigen heterogeneity/escape—to using this technology to effectively treat solid tumors. A multiplexed engineering approach is needed to equip effector T cells with synthetic countermeasures to overcome these barriers. This, in turn, necessitates combinatorial use of lentiviruses because of the limited payload size of current lentiviral vectors. Accordingly, there is a need for cell-surface human molecular constructs that mark multi-vector cotransduced T cells, to enable their purification ex vivo and their tracking in vivo. To this end, we engineered a cell surface–localizing polypeptide tag based on human HER2, designated HER2t, that was truncated in its extracellular and intracellular domains to eliminate ligand binding and signaling, respectively, and retained the membrane-proximal binding epitope of the HER2-specific mAb trastuzumab. We linked HER2t to CAR coexpression in lentivirally transduced T cells and showed that co-transduction with a second lentivirus expressing our previously described EGFRt tag linked to a second CAR efficiently generated bispecific dual-CAR T cells. Using the same approach, we generated T cells expressing a CAR and a second module, a chimeric cytokine receptor. The HER2txEGFRt multiplexing strategy is now being deployed for the manufacture of CD19xCD22 bispecific CAR T-cell products for the treatment of acute lymphoblastic leukemia (NCT03330691).