Abstract 6344: Recombinant Immunotoxin exhibited targeted killing of Trop2 overexpressing tumour cells lines

Abstract

Abstract Introduction: The high prevalence of cervical cancer remains a global challenge particularly in developing countries where its therapy is principally by chemoradiation marked by severe toxicities. Antibody-based therapy targeting specific overexpressed surface antigen has proven as a promising strategy. Tumor-associated calcium signal transducer 2 (Trop2), also known as epithelial glycoprotein-1 antigen, is a membrane protein found in epithelial tissues involved in cellular signalling, migration, proliferation, and differentiation in normal cells. Its overexpression is validated in several tumor types including basal, squamous and adenocarcinomas of the cervix, breast, colon, and gastrointestinal tracts. Due to its differential overexpression, Trop2 is a viable therapeutic marker whereby a recombinant immunotoxin composed of a single chain variable antibody fragment (scFv) armed with Pseudomonas exotoxin A (Anti-Trop2-ETA) may be delivered for targeted killing of such tumors. Methods: Plasmids encoding Anti-Trop2-ETA were designed in silico by fusion of anti-Trop2 single chain variable fragment (scFv) antibody to the N-terminus of Pseudomonas exotoxin A and then cloned into an pMT backbone. Expression was performed in BL21 E. Coli cells under osmotic stress and then Anti-Trop2-ETA was purified by IMAC via an N-terminus encoded polyhistidine tag. Purified fractions were structurally characterized by SDS PAGE gel and immunoblotting to confirm the presence of the Anti-Trop2-ETA protein to be used for treatment of tumor cell lines. Trop2 expression cervical cancer and breast tumor cell lines were confirmed, and cells were treated with Anti-Trop2-ETA. Results: Dose dependent killing by Anti-Trop2-ETA was observed in the Trop2 expressing tumor cell lines. IC50 values were significantly lower for Trop2 expressing cells as compared to control cells lacking Trop2 expression. Conclusion: Here, we demonstrated antigen specific killing of Trop2 overexpressing tumor cells in vitro. Targeted deployment of recombinant immunotoxin to specifically engage and kill Trop2 positive cells is a potential approach for cancer therapeutics. As such, the ability of the rIT to specifically target and kill Trop2 expressing tumors is an important initial finding towards the possibility of antigen-dependent elimination of Trop2 expressing cancer cells. Ongoing evaluations are aimed at antigen dependent biological activity as well as pharmacokinetics of the rIT in tumor bearing in vivo models. Citation Format: Dennis Makafui Dogbey, Sizalobuhle Masuku, Stefan Barth. Recombinant Immunotoxin exhibited targeted killing of Trop2 overexpressing tumour cells lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6344.