Development and in vivo evaluation of novel humanized CD19 CAR-T cells for advanced B cell malignancies

  • Front Immunol. 2026 May 26:17:1798748. doi: 10.3389/fimmu.2026.1798748.
Clara de Oliveira Andrade  1 ,  Marcus Rafael Lobo Bezerra  2 ,  Isabel Garcia Sousa  3 ,  Eduardo Mannarino Correia  1 ,  Ana Julia Ferreira Lima  2  4 ,  Luiza Abdo  1 ,  Emmanuel Arthur Albuquerque Aragão  1 ,  Ronny Petterson Dos Santos Araujo  2 ,  Daniel Pasqualino Pinheiro  2 ,  Igor Cabral Studart  2  3  4 ,  João Vitor Steimbach  1 ,  Marco Antônio Pretti  1 ,  Gilvan Pessoa Furtado  2  4 ,  Marcos Roberto Lourenzoni  2  4 ,  Marcelo Macedo Brigido  3 ,  Andréa Queiroz Maranhão  #  3  5 ,  Martín Hernán Bonamino  #  1  5  6
Affiliations
  • 1. Cell and Gene Therapy Program, Research Coordination, National Cancer Institute (INCA), Rio de Janeiro, Brazil.
  • 2. Protein Engineering and Health Solutions Group (GEPeSS) - Oswaldo Cruz Foundation (Fiocruz) - Eusébio, Ceará, Brazil.
  • 3. Molecular Immunology Group, Biology Institute, University of Brasilia, Brasília, Brazil.
  • 4. Graduate Program in Biotechnology of Natural Resources (PPGBiotec) - Department of Fisheries Engineering - Federal University of Ceará - Fortaleza, Ceará, Brazil.
  • 5. Pasteur Fiocruz Center on Immunology and Immunotherapy, Eusébio, Ceará, Brazil.
  • 6. Vice-Presidency of Research and Biological Collections (VPPCB), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil.
  • # Contributed equally.
PMID: 42273674 DOI: 10.3389/fimmu.2026.1798748
Abstract

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. In vitro cytotoxicity assays demonstrated comparable tumor cell killing and cytokine secretion across all constructs, including against CD19low leukemia targets. In vivo xenograft models of standard and advanced B-cell Acute Lymphoblastic Leukemia revealed that the H1 CAR-T cells achieved durable tumor control and overall survival comparable to FMC63, whereas the lower-affinity H2 construct showed reduced persistence and increased exhaustion marker expression.

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.

Keywords
CAR-T cell therapy; CD19; antibody humanization; exhaustion markers; non-viral gene delivery; sleeping beauty.
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