Developability engineering of scFvs enables robust CAR function under transient mRNA expression
- 1Chimeris UK
- 2Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge
Abstract
Lipid nanoparticle delivery of mRNA enables in vivo generation of CAR-T cells while avoiding viral vector manufacturing constraints. However, transient expression places stringent requirements on the developability of CAR components, particularly single-chain variable fragments (scFvs). Here we examine how intrinsic scFv biophysical quality governs CAR expression and function under mRNA delivery. Using a humanisation and optimisation workflow guided by computational developability metrics, we engineered scFv variants from three clinically relevant antibody families (FMC63, 14G2a and MGA271/chBRCA84D) spanning a range of baseline stability and aggregation propensity. Optimised variants showed increased humanness and, in most cases, improved thermal stability and reduced non-specific interactions while broadly preserving antigen recognition. Functional testing in primary human T cells following mRNA–LNP delivery revealed strong dependence on starting binder quality: optimisation produced minimal functional change for the stable, predominantly monomeric FMC63 scFv, whereas stabilisation of the aggregation-prone 14G2a scFv markedly increased CAR surface expression and cytotoxic activity. For MGA271-derived binders, optimisation reduced non-specific interactions and off-target killing despite reduced monovalent affinity. Together, these data establish developability engineering of scFvs as a critical enabling step for transient mRNA-encoded CAR therapies.
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BibTeX
@article{mangolini2026developability,
title = {{Developability engineering of scFvs enables robust CAR function under transient mRNA expression}},
author = {Maurizio Mangolini and Aubin Ramon and Bingqian Li and Emily Souster and Jasvinder Hayre and Rajesh Karattil and Pietro Sormanni and Shimobi Onuoha},
journal = {Scientific Reports},
year = {2026},
month = jul,
doi = {10.1038/s41598-026-60295-2},
url = {https://doi.org/10.1038/s41598-026-60295-2},
note = {CC BY},
}
RIS
TY - JOUR
TI - Developability engineering of scFvs enables robust CAR function under transient mRNA expression
AU - Maurizio Mangolini
AU - Aubin Ramon
AU - Bingqian Li
AU - Emily Souster
AU - Jasvinder Hayre
AU - Rajesh Karattil
AU - Pietro Sormanni
AU - Shimobi Onuoha
PY - 2026
DA - 2026/07/01
JO - Scientific Reports
DO - 10.1038/s41598-026-60295-2
UR - https://doi.org/10.1038/s41598-026-60295-2
AB - Lipid nanoparticle delivery of mRNA enables in vivo generation of CAR-T cells while avoiding viral vector manufacturing constraints. However, transient expression places stringent requirements on the developability of CAR components, particularly single-chain variable fragments (scFvs). Here we examine how intrinsic scFv biophysical quality governs CAR expression and function under mRNA delivery. Using a humanisation and optimisation workflow guided by computational developability metrics, we engineered scFv variants from three clinically relevant antibody families (FMC63, 14G2a and MGA271/chBRCA84D) spanning a range of baseline stability and aggregation propensity. Optimised variants showed increased humanness and, in most cases, improved thermal stability and reduced non-specific interactions while broadly preserving antigen recognition. Functional testing in primary human T cells following mRNA–LNP delivery revealed strong dependence on starting binder quality: optimisation produced minimal functional change for the stable, predominantly monomeric FMC63 scFv, whereas stabilisation of the aggregation-prone 14G2a scFv markedly increased CAR surface expression and cytotoxic activity. For MGA271-derived binders, optimisation reduced non-specific interactions and off-target killing despite reduced monovalent affinity. Together, these data establish developability engineering of scFvs as a critical enabling step for transient mRNA-encoded CAR therapies.
SN - 2045-2322
ER -