A Chimeric Antigen Receptor That Binds to a Conserved Site on MICA.

Abstract

The NKG2D ligand MHC class I chain-related protein A (MICA) is expressed on many varieties of malignant cells but is absent from most normal tissues, and thus represents a potential target for chimeric Ag receptor (CAR) T cell-based therapeutics. However, there are more than 100 alleles of MICA, so the ability to target a conserved site is needed for a therapy to be used in most patients. In this study, we describe a fully human anti-MICA CAR created by fusing the single-chain fragment variable B2 to the full length DAP10 protein and the traditional CD3ζ signaling domain. Human T cells expressing the B2 CAR killed MICA-positive tumor cells, produced IFN-γ when in contact with MICA-positive tumor cells or plate-bound MICA protein, and inhibited PANC-1 growth in a mouse xenograft model. To localize B2's epitope on MICA, we used novel computational methods to model potential binding modes and to design mutational variants of MICA testing these hypotheses. Flow cytometry using a commercial anti-MICA/MICB Ab indicated that the variant proteins were expressed at high levels on transduced P815 cell lines. One variant protein (R38S/K40T/K57E) showed reduced staining with a B2-IgG1 fusion protein compared with controls and did not induce IFN-γ production by human T cells expressing the B2 CAR. These results show antitumor activity of MICA-specific CAR T cells and indicate an essential role for a conserved site in the exposed loop involving aa 38-57 of MICA. This study describes a novel MICA-specific CAR and discusses its potential use as a cancer therapeutic.