Developability engineering of scFvs enables robust CAR function under transient mRNA expression

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.