Background Colorectal cancer (CRC) remains of high unmet medical need. T cell engagers as a modality have gained attraction in solid tumors, but despite the early data that T cells can infiltrate cold CRC tumors with therapies such as Cibisatamab, treatment remains challenging due to poor target specificity and related on-target off-tumor toxicity. Through our ATLAS platform that analyzes healthy and tumor tissues at the single-cell level, we identified CB21 (target undisclosed), a highly tumor-specific antigen, with minimal expression in healthy cells, from primary CRC tumors. Based on both the expression profile and target cleanliness, we built a bispecific T cell engager as a therapeutic targeting CB21. Here, we describe the expression profile of CB21 in healthy and patient tissues and relevant in vitro and in vivo models of CRC to interrogate the biology and efficacy of targeting CB21. Based on target expression profile and target cleanliness, a bispecific T cell engager targeting CB21 was tested for therapeutic potential and safety in a non-human primate study.

Methods CB21 target expression from CRC patient samples was determined using IHC and quantitative flow cytometry. Tumor cell lines with varying levels of CB21 were used to determine in vitro activation, cytotoxicity and cytokine production of T cells in the presence of CB21 TCE. Efficiency of the TCE was further tested in spheroid assays and killing kinetics over time using a Cellcyte imaging platform. Xenograft mouse models of CRC were established to evaluate the efficacy of CB21 TCE treatment in vivo.

Results Analysis of primary patient samples revealed CB21 expression at varying levels primarily in microsatellite stable (MSS) and instability-low (MSI-L) CRC. CB21 TCE showed strong binding to the target antigen and demonstrated significant cytotoxicity across multiple cell lines with varying CB21 levels, which were concordant with T cell activation and cytokine production. Similarly, in xenograft models with a CB21-positive endogenously expressing CRC line, the CB21 TCE showed robust tumor control in a dose dependent manner. The CB21 TCE, which is fully cynomolgus cross-reactive, was administered to non-human primates at doses that achieved concentrations exceeding those predicted for clinical efficacy. No significant cytokine release or meaningful changes in clinical blood chemistry were observed.

Conclusions CB21 is a highly specific and promising target for treatment of MSS CRC patients. We have demonstrated efficacy and safety of Cartography’s CB21 TCE in a preclinical setting. Data generated strongly supports clinical development of this CB21 TCE.