A roadmap for systematic humanization of a chimeric antigen receptor: preclinical validation of a humanized CD22 scFv as a model.

The humanization of chimeric antigen receptor (CAR) T-cell constructs is essential for reducing immunogenicity while preserving optimal antigen-binding affinity. However, this process poses significant challenges, as alterations within the single-chain variable fragments (scFvs) can adversely affect specificity, stability, and functional efficacy. In this study, we presented a structure-guided humanization strategy for the development of CD22-targeted CAR T cells to treat relapsed or refractory B-cell malignancies. Our approach involved rational grafting of murine complementarity-determining regions (CDRs) onto human antibody frameworks, followed by comprehensive in silico and biophysical evaluations to identify and mitigate potential liabilities, including aberrant glycosylation sites and structural instability. The resulting humanized CD22-CAR constructs retained antigen recognition, specificity, and cytotoxic potency comparable with their murine counterparts, as demonstrated in extensive in vitro and in vivo validation studies. Collectively, these findings established a robust, structure-informed humanization pipeline that supports the translation of next-generation CAR-T therapies with reduced immunogenicity and sustained therapeutic efficacy.