Generation and in vitro functional characterization of anti-CD22 chimeric antigen receptor T cells derived from newly generated monoclonal antibody clones

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of B-cell malignancies, but relapse after CD19-directed therapy remains a major challenge, often because of loss or downregulation of CD19 expression. CD22 is an attractive alternative target because it is expressed on malignant B cells, including in many cases of CD19-negative disease. In this study, we generated and characterized novel anti-CD22 monoclonal antibody–derived CAR constructs for in vitro evaluation. Four anti-CD22 monoclonal antibody clones were initially screened, and two clones, 24A7G6 and 29H9C8, were selected for CAR development based on their favorable binding and CAR surface expression profiles. Second-generation CAR constructs containing either a 4−1BB or CD28 costimulatory domain were generated and introduced into primary human T cells. The resulting CAR T cells were evaluated for expansion, immunophenotype, activation and inhibitory marker expression, cytokine secretion, short-term cytotoxicity, long-term co-culture activity, and sequential tumor rechallenge against CD22-positive Raji and Nalm-6 cells and CD22-negative K562 cells. Both 24A7G6- and 29H9C8-derived CAR T cells expanded efficiently, showed predominant CD8 + populations with naïve and central memory phenotypes, and mediated potent antigen-specific cytotoxicity against CD22-positive target cells, while showing no activity against CD22-negative cells. Following antigen stimulation, CAR T cells upregulated activation markers and showed modest increases in inhibitory receptors. In long-term co-culture and sequential rechallenge assays, all constructs retained antitumor activity under prolonged antigen exposure. Notably, the 24A7G6-CD28 CAR demonstrated enhanced sustained tumor control compared with the corresponding 4−1BB construct in extended assays, whereas short-term cytotoxicity was broadly comparable across constructs. These findings show that the newly generated 24A7G6 and 29H9C8 scFvs can function as effective targeting domains for CD22-directed CAR T-cell development and provide an in vitro basis for prioritizing lead constructs for further preclinical evaluation.