Multispecific Hybrid T Cell Receptors for Sensitive Targeting of Cancer
Authors: Simon Sylvain , Bugos Grace , Prins Rachel , Rajan Anusha , Palani Arulmozhi , Heyer Kersten , Riddell Stanley R. 
Published: December 2023

CAR T cells represent a promising therapy for a growing list of tumors. Despite high initial response rates, 50-60% of patients relapse within 1 year after CAR T cell infusion. Heterogeneity of target antigen expression can lead to outgrowth of tumor cells with low or negative antigen levels. Conventional CARs require high antigen levels for efficient T cell activation and tumor elimination. By contrast, T cell receptors (TCRs) are 10-100 times more sensitive than CARs. To achieve greater antigen sensitivity, we and others designed synthetic hybrid T cell receptors by fusing antibody-based recognition domains to TCRa and TCRb constant chains. Here we extend the initial monospecific design to incorporate two antigen recognition domains.
We evaluated 2 monospecific hybrid receptors, one with VH and VL domains fused to TCRa and TCRb constant chains respectively (Split TCR/CAR) and a second where the entire scFv (VL-linker-VH) is fused only to the TCRa chain (Full TCR/CAR) leaving TCRb available for a second scFv. We used based editing to knock out endogenous TCRa and b chains to avoid mispairing. Using CD19 as a model target we found that both TCR/CAR designs lacked tonic signaling, induced TCR-like changes in Ca+ flux, more efficiently phosphorylated LAT, and exhibited superior recognition of Nalm-6 clones expressing a range of CD19 levels compared to conventional CD19 28z or 4-1BBz CARs. Interestingly, the Full TCR/CAR outperformed the Split format in these assays. Using soluble bilayer and TIRF microscopy, we demonstrated that unlike CARs, TCR/CAR T cells recapitulate a classical TCR-like immunological synapse. I n vivo studies using NSG mice engrafted with CD19+ Raji cells demonstrated potent anti-tumor efficacy, and the Full format outperformed the Split TCR/CAR.
Capitalizing on the superiority of the Full TCR/CAR format, we next designed bispecific hybrid receptors composed of two scFvs of different specificities fused to the TCRa and TCRb chains. Multiple Myeloma is particularly suitable for bispecific targeting as relapse following CAR T cell therapy is common and multiple target antigens have been described. We designed bispecific Full TCR/CARs specific for pairs of myeloma antigens (BCMA/SLAMF7 and BCMA/GPRC5D) and expressed them in primary T cells. We used base editing to knock out SLAMF-7 which can be expressed in T cells and could cause fratricide. Bispecific TCR/CAR receptors conferred sensitive T cell recognition of target cells expressing BCMA and SLAMF-7 as measured by cytokine production, proliferation, and cytotoxicity. T cells equipped with these novel bispecific receptors maintained function when exposed to L363 cells BCMA ko or SLAMF-7 ko demonstrating that both antigen binding domains can trigger T cell functions against target cells. In vivo studies using NSG mice implanted with a mixture composed of 33% of Nalm-6 BCMA+, Nalm-6 SLAMF-7+ and Nalm-6 BCMA+SLAMF-7+ cells to mimic target antigen expression heterogeneity and antigen loss showed superior anti-tumor efficacy for bispecific TCR/CAR cells compared to conventional BCMA and SLAMF-7 BBz CARs. Our hybrid bispecific TCR/CAR also demonstrated superior anti-tumor efficacy compared to a previously described, and systematically optimized bispecific CAR format (Zah et al., 2020). 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 cancer.