Generation and characterization of a CEACAM5 x CDH17 x CD3 tri-specific T-Cell engager for colorectal cancer therapy

Abstract Background CEACAM5 and CDH17 are highly co-expressed in certain solid tumors including colorectal cancer (CRC). Current single-target based immunotherapies, such as antibody-drug conjugates (ADC) and T cell engagers (TCE), have shown promising efficacy in clinical trials. In this study, we generated a novel tri-specific TCE (tsTCE) targeting CEACAM5, CDH17, and CD3, aiming to enhance tumor-targeting selectivity and therapeutic efficacy. Methods Various TCE molecule formats, including 1+1 monovalent (CEACAM5 or CDH17 x CD3), 2+1 bivalent or biparatopic (CEACAM5 x CEACAM5 x CD3, CDH17 x CDH17 x CD3) and 1+1+1 tsTCEs (CEACAM5 x CDH17 x CD3) were generated. A lead tsTCE molecule was selected and investigated by antigen binding affinity, cell binding, T-cell-dependent cellular cytotoxicity, and T-cell activation in vitro. In addition, its’ in vivo antitumor efficacy were evaluated in subcutaneous HT55, SW403, and LS174T tumor xenografts in NCG mice engrafted with human PBMCs. Results The CEACAM5 x CDH17 x CD3 tsTCE (tsTCE3) possesses a moderate binding affinity to human/cynomolgus CEACAM5 (Kd: 51.0 nM / 109 nM), CDH17 (Kd: 42.8 nM / 9.26 nM), and human CD3ε (Kd: 51.9 nM). The tsTCE3 effectively kills tumor cells with variable expression levels of CEACAM5 and/or CDH17 and induces significant tumor growth inhibition in multiple CDX models. In the CEACAM5-high / CDH17-high HT55 tumor model, tsTCE3 was more efficacious than cibisatamab analogue and a 2+1 CEACAM5 x CEACAM5 x CD3 bivalent bispecific TCE, whereas in the LS174T tumor model (CEACAM5-high / CDH17-low) tsTCE3 was more potent than a 2+1 CDH17 x CDH17 x CD3 biparatopic TCE molecule. Notably tsTCE3 elicited minimal cytokine release in HT55 xenograft model. Conclusions Our tsTCE3 exhibits potent in vitro tumor cell killing and superior in vivo efficacy with a favorable safety profile, supporting its potential as a promising therapeutic agent for the treatment of CEACAM5 / CDH17 positive solid tumors.