A novel multimeric sCD19 ‐streptavidin fusion protein for functional detection and selective expansion of CD19 ‐targeted CAR‐T cells

Background CARs are engineered receptors comprising an immunoglobulin single-chain variable fragment (scFv) that identifies and binds to the target antigen, a transmembrane domain, and an intracellular T-cell signaling domain. CD19 is a B lineage-specific transmembrane glycoprotein and is expressed in more than 95% of B-cell malignancies. Streptavidin (SA) is a homo-tetrameric protein derived from Streptomyces avidinii, which can bind four biotin molecules with an extremely high affinity at a Kd value of 10(-15) M. Aims The aim of the study is to generate a novel soluble multimeric fusion protein, sCD19-streptavidin (sCD19-SA) for functional detection and selective expansion of CD19-targeted CAR-T cells. Methods The fusion proteins CD19-SA was expressed in CHO cells and purified by use of Ni-nitrilotriacetic acid agarose beads. Results A novel fusion protein (sCD19-SA) was generated, consisting of the extracellular domain of human CD19 and the core region of SA, and could be used to functionally detect CD19-targeted CAR-T cells. Furthermore, this protein was demonstrated to form multimers to activate CAR-T cells to induce their selective expansion. Importantly, sCD19-SA-stimulated CD19-targeted CAR-T cells could improve antitumor effects in vivo. Conclusions Our study has highlighted the potential of utilizing antigen-SA fusion proteins such as sCD19-SA for CAR-T therapy for the functional detection of CAR expression and selective expansion of CAR-T cells.