Background
Cadherin-17 (CDH17) is specifically overexpressed in colorectal cancer (CRC), making it a promising tumor-associated antigen for targeted immunotherapy. While conventional CD3-based bispecific T-cell engagers (TCEs) show potential in solid tumors, their effect on T-cell activation is often limited in the immunosuppressive tumor microenvironment, in part due to a lack of T-cell co-stimulation. To overcome this limitation, we developed a novel trispecific TCE targeting CDH17, CD3, and the co-stimulatory receptor CD28 to drive potent and sustained tumor-localized T-cell activation and tumor cell killing while minimizing systemic toxicity.
Methods
The binding affinity and specificity of the CD3xCD28xCDH17 trispecific TCE were evaluated using BLI and flow cytometry. In vitro T-cell activation, proliferation, and cytokine release were assessed by co-culturing human PBMCs with CDH17-positive CRC cell lines. In vitro cytotoxicity was evaluated in cells of varying CDH17 expression levels. In vivo anti-tumor efficacy was investigated in PBMC-reconstituted B-NDG mice bearing CDH17 tumors. Safety profiles were examined in humanized mouse models.
Results
The trispecific TCE demonstrated robust and selective binding to CDH17 and mediated potent, target-dependent T-cell cytotoxicity and cytokine release against CDH17-positive cancer cells. The addition of the CD28 co-stimulatory arm significantly enhanced T-cell proliferation and sustained cytotoxicity compared with its bispecific counterpart in vitro and in vivo. In vivo, the trispecific TCE showed potent and dose-dependent tumor inhibition and demonstrated superior in vivo efficacy to that of a bispecific CDH17-targeted TCE. In non-tumor-bearing B-hCD3E/h- CD28 humanized mice, the trispecific TCE induced no significant body weight loss or cytokine release.
Conclusion
This novel CD3xCD28xCDH17 trispecific TCE exhibits superior anti-tumor efficacy with a wide safety window in preclinical models. The data establish a strong rationale for clinical translation as a promising therapeutic for colorectal cancer.
