IDENTIFICATION AND CHARACTERIZATION OF NOVEL HUMAN SINGLE CHAIN VARIABLE FRAGMENT AGAINST CD19 FOR ENGINEERED T CELL THERAPY
CD19 is the most promising target to generate chimeric antigen receptor (CAR) T cell therapy for B-cell malignancies. CAR is composed of antigen-binding domain usually derived from single chain variable fragment (scFv) and intracellular signaling domain of T cell receptor. Since the previous approved CD19 CAR T contains murine derived scFv, this might cause human anti-mouse antibody (HAMA) due to immunogenicity of murine part and reduce CAR T cells efficacy. In order to avoid this problem, we aimed to screen novel human scFv against CD19 and to characterize its function.
HeLa cells were engineered to overexpress CD19 or eYFP. HeLa-eYFP was incubated with human phage display library before incubating with HeLa-CD19. After subtraction and selection, expressed scFv proteins were examined for binding on native CD19 antigen expressed on Raji and/or HeLa-CD19 and K562 cells that did not express CD19 using flow cytometry.
Five scFv clones were found to express and secret proteins in bacterial culture. Only one clone of scFv, designated as 1E7, could bind to CD19 antigen expressed on Raji, K562-CD19, and HeLa-CD19, while it could not bind to K562 and HeLa cells that did not harbor CD19 antigen.
We could identify a novel human scFv against CD19 that exhibited specific binding to native CD19 antigen on cancer cells. Human CD19-specific CAR T cells will be generated and examined for their cytotoxicity, cytokine production, and immunophenotypes.