Sylvain Simon, Grace Bugos, Anusha Rajan, Rachel Prinsland, Stanley R. Riddell

Fred Hutchinson Cancer Research Center, Seattle, USA; Department of Immunology, University of Washington, Seattle, USA; Department of Medicine, University of Washington, Seattle, WA 98109, USA

Multi-specific hybrid T cell receptors for sensitive targeting of hematologic malignancies

CAR T cells targeting lineage antigens are highly effective therapeutic options for the treatment of B cell malignancies and multiple myeloma. But despite good initial response rates, long term remissions remain infrequent and in the case of multiple myeloma, 50-60% of treated patients will relapse within 1 year after CAR T cell infusion. This is due to heterogeneity of target antigen expression on tumors, which leads to selection and outgrowth of tumor cells with low levels of antigen and/or loss following immune pressure with CAR therapy.  Conventional CARs that encode CD3ζ and a costimulatory domain require high levels of antigen for efficient T cell activation and tumor elimination. By contrast, T cell receptors (TCRs) are 10-100 times more sensitive than CARs as they engage a more complex and diversified signaling machinery following antigen recognition. We and others have designed synthetic hybrid T cell receptors by fusing VH and VL recognition domains to TCR alpha and beta constant chains respectively. When combined with efficient genetic knockout of endogenous TCR chains using base editing, these hybrid molecules provided greater antigen sensitivity than conventional CARs but in current iterations only recognize a single antigen target. We have now addressed the problem of antigen heterogeneity by designing bispecific hybrid receptors composed of two scFvs of different specificities, one fused to the TCRα chain and the second scFv fused to TCRβ chain. Bispecific hybrid receptors for pairs of myeloma antigens (BCMA/SLAMF7; BCMA/GPRC5D; BCMA/CD229) were expressed in primary T cells and bind each of their cognate antigens. Bispecific TCR/CARs conferred T cells with specificity for both targets and were superior for killing heterogeneous tumor cell populations in vitro and in vivo compared to mono-specific CAR- or TCR/CAR T cells. Furthermore, presence of both target antigens on the tumor cells provided increased functional avidity measured by z-movi assay, increased Ca2+ flux and higher amounts of cytokines compared to tumor cell lines expressing a single antigen. Bispecific hybrid T cell receptors embedded with natural TCR signaling machinery represent a promising therapeutic option to address antigen downmodulation/loss and antigen heterogeneity for treatment of MM and other hematologic cancers.