A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo.

The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.

A rational approach to assess off-target reactivity of a dual-signal integrator for T cell therapy.

Cell therapy is an emerging therapeutic modality with the power to exploit new cancer targets and potentially achieve positive outcomes for patients with few other options. Like all synthetic treatments, cell therapy has the risk of toxicity via unpredicted off-target behavior. We describe an empirical method to model off-tumor, off-target reactivity of receptors used for investigational T cell therapies. This approach utilizes an optimal panel of diverse human cell-lines to capture the large majority of protein-coding gene expression in adult human tissues. We apply this cell-line set to test Jurkat and primary T cells engineered with a dual-signal integrator, called TmodTM, that contains an activating receptor (activator) and a separate inhibitory receptor (blocker). In proof-of-concept experiments, we use CD19 as the activating antigen and HLA-A*02 as the blocker antigen. This specific Tmod system, which employs a blocker targeting a ubiquitously expressed HLA class I antigen to inhibit CAR activation, has an inherent mechanism for selectivity/safety, designed to activate only when a specific HLA class I antigen is lost. Nonetheless, it is important to test off-target reactivity in functional assays, especially given the disconnect between ligand-binding and function among T cell receptors (TCRs) and chimeric antigen receptors (CARs). We show these cell-based assays yield consistent results with high sensitivity and specificity. The general strategy is likely applicable to more traditional single-receptor CAR- and TCR-T therapeutics.