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Published on: June 27, 2018
An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability
M Whitlow1, B A Bell, S L Feng
1Protein Engineering Department, Enzon, Incorporated, Piscataway, NJ 08854-3998.
Protein Engineering
|November 1, 1993
Summary
Optimizing linker length in single-chain variable fragments (sFvs) impacts binding affinity and aggregation. Longer linkers in antifluorescein and anticarcinoma sFvs improve stability and tumor targeting, while aggregation enhances avidity for repetitive antigens.
Area of Science:
- Biotechnology
- Immunology
- Protein Engineering
Background:
- Single-chain variable fragments (sFvs) are crucial in targeted therapies.
- Linker length significantly influences sFv stability, aggregation, and binding properties.
- Previous studies identified proteolytic susceptibility in certain sFv linker designs.
Purpose of the Study:
- To investigate the impact of linker length and modifications on antifluorescein and anticarcinoma sFv characteristics.
- To evaluate the influence of linker design on binding affinity, aggregation, and proteolytic stability.
- To assess the in vivo performance of modified sFvs in tumor models.
Main Methods:
- Design and synthesis of novel linker sequences, including the 218 linker with a proline modification.
- Characterization of sFv binding affinity, aggregation, and proteolytic stability in vitro.
- In vivo studies in mice to evaluate blood clearance, tumor uptake, and biodistribution of radiolabeled sFvs.
Main Results:
- Longer linkers in antifluorescein sFvs correlated with higher binding affinities and reduced aggregation.
- The CC49 sFv with the 218 linker exhibited reduced aggregation and enhanced in vitro proteolytic stability compared to the CC49/212 sFv.
- While CC49/218 sFv showed higher tumor uptake due to proteolysis resistance, aggregated CC49/212 sFv demonstrated higher affinity and tumor uptake attributed to multivalent avidity.
Conclusions:
- Linker engineering is a critical strategy for optimizing sFv performance in terms of stability and targeting.
- The 218 linker design offers improved stability and tumor targeting for the CC49 sFv.
- Aggregated sFvs can exhibit enhanced avidity, which may be beneficial for targeting repetitive antigens, despite potential stability trade-offs.
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