Related Experiment Video
Updated: Aug 6, 2026
09:56
A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy
Published on: February 21, 2025
Developability engineering of scFvs enables robust CAR function under transient mRNA expression
Maurizio Mangolini1, Aubin Ramon2,3, Bingqian Li2
1Chimeris UK, The Works, Unity Campus, London Road, CB22 3EE, Cambridge, UK.
Scientific Reports
|July 18, 2026
Summary
Engineering the quality of single-chain variable fragments (scFvs) is crucial for mRNA-delivered chimeric antigen receptor (CAR) T-cell therapies. Optimizing scFv biophysical properties enhances CAR expression and T-cell function, improving therapeutic potential.
Area of Science:
- Biotechnology
- Immunology
- Molecular Engineering
Background:
- Lipid nanoparticle (LNP) delivery of messenger RNA (mRNA) allows in vivo generation of chimeric antigen receptor (CAR)-T cells, bypassing viral vector limitations.
- Transient expression from mRNA necessitates high developability of CAR components, especially single-chain variable fragments (scFvs).
Purpose of the Study:
- To investigate how intrinsic biophysical quality of scFvs impacts CAR expression and function when delivered via mRNA.
- To engineer and assess scFv variants for improved developability in the context of mRNA-based CAR-T cell therapy.
Main Methods:
- Employed a humanization and optimization workflow using computational developability metrics.
- Engineered scFv variants from three antibody families (FMC63, 14G2a, MGA271) with varying stability and aggregation properties.
- Conducted functional testing in primary human T cells after mRNA-LNP delivery.
Main Results:
- Optimized scFvs demonstrated increased humanness, improved thermal stability, and reduced non-specific interactions, while maintaining antigen recognition.
- Functional outcomes strongly depended on the starting scFv quality; stable scFvs showed minimal change, while stabilizing aggregation-prone scFvs significantly boosted CAR expression and cytotoxic activity.
- Optimization of MGA271-derived binders reduced non-specific interactions and off-target killing, even with decreased monovalent affinity.
Conclusions:
- Developability engineering of scFvs is a critical factor for successful transient mRNA-encoded CAR therapies.
- Tailoring scFv biophysical properties can enhance CAR-T cell efficacy and safety for mRNA-based therapeutic approaches.
Related Concept Videos
02:28Leaky Scanning
During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA. Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
01:57Protein Complexes with Interchangeable Parts
Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
02:22Regulated mRNA Transport
In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing specific...
02:22Regulated mRNA Transport
In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing specific...
02:15Experimental RNAi
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...