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

Abstract 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.