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Peer-reviewedCC BYFrontiers in immunology

Split intein-mediated reconstitution of regulatable CAR T-cells

  • Vladimir Mulens-Arias1
  • Marco Archinti2
  • Miriam Bastida2
  • Nuria Martínez2
  • Eleonora Pinto3
  • Víctor M. Díaz4
  • Pablo Menéndez1
4 affiliations
  1. 1Josep Carreras Leukemia Research Institute (IJC) , ,
  2. 2ZIP Solutions SL. , ,
  3. 3Department of Clinical Sciences and Translational Medicine, Universityof Rome “Tor Vergata” , ,
  4. 4OneChain Immunotherapeutics SL. , ,
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Abstract

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of B-cell malignancies. However, strategies that improve the modularity and controllability of CAR signaling could broaden its applicability and safety across additional clinical contexts. Here, we describe a regulable CAR T-cell platform based on split intein-mediated protein trans-splicing (PTS) to conditionally reconstitute functional receptors in primary T-cells.

Methods: As a proof of concept, we engineered a split CAR targeting CD1a (NCT05679895), a surface antigen expressed in T-cell acute lymphoblastic leukemia (T-ALL). The system consists of a membrane-anchored C-terminal scaffold containing the IMPDH.1 C split intein fused to 4-1BB and CD3ζ signaling domains, and a soluble N-terminal adaptor composed of an anti-CD1a single-chain variable fragment (scFv) fused to the complementary IMPDH.1 N split intein.

Results: Upon delivery of the adaptor module, the intein fragments undergo PTS, generating a functional membrane-bound CD1a CAR with splicing efficiencies of 85%. Reconstituted CAR T-cells exhibited antigen-specific cytotoxicity against CD1a ⁺ T-ALL cells coupled to high levels of interferon-γ secretion.

Discussion: These findings demonstrate that split intein-mediated CAR reconstitution enables a modular platform for conditional CAR reconstitution and transient control of receptor availability.

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Cite

BibTeXRIS
BibTeX
@article{mulensarias2026split,
  title = {{Split intein-mediated reconstitution of regulatable CAR T-cells}},
  author = {Vladimir Mulens-Arias and Marco Archinti and Miriam Bastida and Nuria Martínez and Eleonora Pinto and Víctor M. Díaz and Pablo Menéndez},
  journal = {Frontiers in immunology},
  year = {2026},
  month = oct,
  doi = {10.3389/fimmu.2026.1844972},
  url = {https://europepmc.org/article/PMC/PMC13672617},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Split intein-mediated reconstitution of regulatable CAR T-cells
AU  - Vladimir Mulens-Arias
AU  - Marco Archinti
AU  - Miriam Bastida
AU  - Nuria Martínez
AU  - Eleonora Pinto
AU  - Víctor M. Díaz
AU  - Pablo Menéndez
PY  - 2026
DA  - 2026/10/02
JO  - Frontiers in immunology
DO  - 10.3389/fimmu.2026.1844972
UR  - https://europepmc.org/article/PMC/PMC13672617
AB  - Introduction: Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of B-cell malignancies. However, strategies that improve the modularity and controllability of CAR signaling could broaden its applicability and safety across additional clinical contexts. Here, we describe a regulable CAR T-cell platform based on split intein-mediated protein trans-splicing (PTS) to conditionally reconstitute functional receptors in primary T-cells.  Methods: As a proof of concept, we engineered a split CAR targeting CD1a (NCT05679895), a surface antigen expressed in T-cell acute lymphoblastic leukemia (T-ALL). The system consists of a membrane-anchored C-terminal scaffold containing the IMPDH.1 C split intein fused to 4-1BB and CD3ζ signaling domains, and a soluble N-terminal adaptor composed of an anti-CD1a single-chain variable fragment (scFv) fused to the complementary IMPDH.1 N split intein.  Results: Upon delivery of the adaptor module, the intein fragments undergo PTS, generating a functional membrane-bound CD1a CAR with splicing efficiencies of 85%. Reconstituted CAR T-cells exhibited antigen-specific cytotoxicity against CD1a ⁺ T-ALL cells coupled to high levels of interferon-γ secretion.  Discussion: These findings demonstrate that split intein-mediated CAR reconstitution enables a modular platform for conditional CAR reconstitution and transient control of receptor availability.
SN  - 1664-3224
ER  -