An engineered Trop2/CD3 bispecific antibody for treatment of gastric, pancreatic, and colorectal cancers.

Abstract

e14521 Background: Given its over-expression and poor prognosis in a variety of solid tumors, trophoblast cell surface antigen 2 (Trop2) is regarded as a potential therapeutic target. Trop2-directed antibody-drug conjugates have been developed for treatment of solid tumors; however, their clinical benefits can be limited by the associated undesirable effects which is likely attributed to the on-target off-tumor effect arising from the low basal Trop2 expression on the surface of multiple normal epithelial tissues. The present study aims to develop a humanized Trop2/CD3 bispecific T-cell engager antibody that can promote immunological synapse formation with tumor cells overexpressing Trop2, redirecting T cell-mediated killing specifically to cancers with reduced risk of on-target off-tumor effects. Methods: A Trop2/CD3 bispecific antibody was generated by linking a humanized anti-Trop2 monoclonal antibody with a CD3-binding scFv. To tune the affinity of the bispecific antibody for tumor target, in silico paratope prediction was firstly done using ProABC-2 (Ambrosetti F et al., 2020), following by alanine substitution of the identified paratope residues individually. A total of 6 CDR variants were generated with their binding to Trop2 examined using biolayer interferometry. Selected candidates were further characterized for their binding to Trop2-positive cancer cells and activation of T cells upon target engagement. Redirection of T cell-mediated killing on cancer cells expressing high Trop2 level was also studied in vitro. Results: Substitution of a complementarity-determining residue (CDR) on the heavy chain of anti-Trop2 antibody with an alanine significantly reduced binding affinity to Trop2. This CDR variant was selected for further functional characterization. Apart from weaker binding to Trop2-positive tumor cells, the variant also demonstrated a slower cytokine induction, a less acute T-cell activation, and a cytotoxic effect which was more restricted to Trop2-high cancer cells. Conclusions: Our new Trop2/CD3 bispecific antibody demonstrates potent anti-tumor activity, while at the same time shows a reduced risk of on-target off-tumor toxicity. The drug candidate warrants further investigations including toxicity study in monkeys. [Table: see text]