ArticlemAbs
"Stapling" scFv for multispecific biotherapeutics of superior properties.
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Who cites it
16 citing papers in PubMed, 27 citations in OpenAlex.
- A p53Molecular therapy. Oncology · 2026Article
- Single-Chain Variable Fragment Fusion Proteins for Targeted Delivery and Therapy.Pharmaceuticals (Basel, Switzerland) · 2026Review
- Design optimization of antibody-ligand motifs to enhance CAR-T redirection activity against solid tumors.Cell reports. Medicine · 2026Article
- A side-by-side biophysical comparison of five single-domain antibody scaffolds used for synthetic display libraries.The Journal of biological chemistry · 2026Article
- TCR-mimic bispecific nanobody-based T cell engager targeting intracellular tumor antigens for cancer immunotherapy.Signal transduction and targeted therapy · 2026Article
- Two-in-one protein tag enables the soluble expression and calcium-dependent purification of single-chain antibody fragments.Communications biology · 2026Article
- A new era in CAR-NK cell therapy: from technological innovations to clinical applications.World journal of pediatrics : WJP · 2026Review
- scFv-based biologics in diabetes: from therapeutic potential to clinical prospects.Frontiers in immunology · 2026Review
- Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists.Antibodies (Basel, Switzerland) · 2025Article
- Genetically encoded intrabody probes for labeling and manipulating AMPA-type glutamate receptors.Nature communications · 2024Article
- Development of novel canine phage display-derived neutralizing monoclonal antibody fragments against rabies virus from immunized dogs.Scientific reports · 2024Article
- DARPin-fused T cell engager for adenovirus-mediated cancer therapy.Molecular therapy. Oncology · 2024Article
- Design and engineering of bispecific antibodies: insights and practical considerations.Frontiers in bioengineering and biotechnology · 2024Review
- Structural modeling of antibody variable regions using deep learning-progress and perspectives on drug discovery.Frontiers in molecular biosciences · 2023Review
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Corrections and comments
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Authors and funding
30 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.