Selection of a CD۱۹ specific scfv by phage display with future prospect in CAR-T cell therapy of leukemia cancer

Backgrounds: Targeting tumor cells through artificially modified T-cells showed promisingresults in preclinical and clinical studies during last decades. CD۱۹ is the most used target forchimeric antigen receptor T-cell against B cell leukemia. Chimeric antigen receptor throughwitch targeting occurs, is composed of a targeting moiety often a single chain antibody, acostimulatory domain and a CD۳ζ domain of a T-cell receptor. scfv antibodies have been usedextensively for targeting surface markers of cancer cells. Their small size, easier production andtheir high affinity make them an ideal substitute for monoclonal antibodies.Materials and Methods: In this study, we selected a CD۱۹ scFv from a human scFv libraryusing phage display. Briefly, we amplified the library culture, and then we added the commercialhelper phage to the culture then incubated the culture three days for bacteriophage production.We then purified phages using PEG ۸۰۰۰ by ultracentrifugation and their titer was calculatedbefore selection. In each round, titrated phage was subjected to CD۱۹ coated beads in solution.After washing of unbound phages, bound ones were recovered and their affinities were analyzedusing ELISA against CD۱۹ and then transformed for another round of phage production andselection. After three rounds of selections, clones with the highest affinity were selected,sequenced and then cloned into pET۲۸a.Results: Three scFvs were selected in this step, expressed in BL۲۱ and analyzed in SDS-PAGE.They are then extracted using IMAC and affinity of them was measured by ELISA. A thinprotein band of ~۲۷ kDa was observed in SDS-PAGE and visualized in western blot using HRPconjugatedanti-his tag antibody.Conclusion: Our results showed that clone ۲۶ is able to recognize CD۱۹ better than two otherclones.

کلیدواژه ها:

CD۱۹ ، scFv ، CART cell ، Phage display