An Anti‐Mesothelin Recombinant Immunotoxin Based on Deoxyribonuclease‐I

Abstract

Recombinant immunotoxins (RITs) are fusion proteins consisting of a toxin attached to an antibody fragment. Unlike traditional chemotherapy, RITs used in cancer therapy can be specifically targeted to tumor cells while sparing healthy cells. One shortcoming of RITs is their immunogenicity, which is the immune response of patients to treatment with RITs. Patients with intact immune systems often produce antibodies to the toxin that neutralize its function. For this experiment, we attempted to prepare a RIT using deoxyribonuclease‐ I (DNase‐I) instead of a typical bacterial or plant‐derived toxin. DNase‐I is an endogenous human protein that catalyzes the degradation of DNA into its component nucleotides. Previous research shows that DNase‐I can be toxic to cells if delivered through targeted endocytosis. Furthermore, we predict that DNase‐I will be less immunogenic than other toxins, because it is a human protein. We successfully cloned an expression vector joining DNase‐I to an anti‐mesothelin antibody fragment and expressed it in E. coli. The resulting protein, however, was unstable and we were unable to purify it after refolding from inclusion bodies. We concluded that the design of the fusion protein needs improvement to enhance its stability. It is also possible that the refolding conditions need further optimization. Future directions include exploring both of these possibilities.Support or Funding InformationI would like to thank the Towson Bridges Program and CCBC for the opportunity. Thank you to Dr. Laing, Dr. Snyder, Dr. Berlyn and Trudymae for guidance throughout the program. Thank you to the NIH Bridges Grant 5 R25 GM058264 16 for funding. Special thanks to Dr. Weldon, Jillian Baker and all students that have helped throughout the project.