Development and

Background: Chimeric antigen receptor T-cell therapy targeting CD19 has revolutionized the treatment of B-cell malignancies; however, limitations related to immunogenicity, persistence, and manufacturing costs remain significant barriers to broader clinical application. Most approved CD19-directed CAR-T products rely on murine-derived single-chain variable fragments, which may elicit anti-mouse immune responses and compromise long-term efficacy. Methods: Here, we report the design, characterization, and preclinical validation of two novel humanized anti-CD19 CAR constructs derived from the FMC63 antibody and generated using a non-viral Sleeping Beauty transposon system. Two humanized scFv variants, H1 and H2, sharing the same humanized light chain but distinct heavy chain frameworks, were evaluated for binding affinity, structural stability, and functional performance. Results: Although both humanized variants displayed reduced affinity relative to FMC63, they retained specific CD19 binding and supported robust CAR expression, activation, and memory differentiation in primary human T cells. Conclusions: Collectively, these results demonstrate that rational humanization and harmonization of anti-CD19 scFvs can preserve antitumor efficacy, while mitigating functional exhaustion. This work supports the H1 construct as a promising candidate for further clinical development and highlights a scalable, cost-effective strategy for advancing locally manufactured CAR-T therapies in resource-limited settings.