A general chemical synthesis platform for crosslinking multivalent single chain variable fragments.
Joan G Schellinger1, Avinash Kudupudi, Arutselvan Natarajan
1Chemistry Department, University of California Davis, One Shields Avenue, Davis, CA 95616, USA.
Organic & Biomolecular Chemistry
|December 2, 2011
Summary
Multivalent single-chain variable fragments (scFv) offer enhanced tumor binding compared to traditional antibodies. This study presents a versatile chemical crosslinking strategy for creating stable, multivalent immunoconjugates for cancer therapy and imaging.
Area of Science:
- Biotechnology
- Immunology
- Chemical Biology
Background:
- Multivalent single-chain variable fragments (scFv) exhibit superior affinity for tumor-associated antigens over monovalent scFv and intact monoclonal antibodies (mAb).
- Covalent multivalent constructs offer enhanced in vivo and in vitro stability compared to self-associating variants.
- Genetically engineered expression vectors enable site-specific cysteine functionalization of scFv for covalent attachment.
Purpose of the Study:
- To develop and validate a versatile chemical crosslinking strategy for constructing stable, multivalent immunoconjugates.
- To utilize di-scFv-C constructs with site-specific cysteine for conjugation with polyethylene glycol (PEG) linkers.
- To evaluate the efficacy of copper(I) catalyzed azide alkyne 1,3-dipolar cycloaddition (CuAAC) chemistry for creating these conjugates.
Main Methods:
- Genetically engineered expression vectors to produce di-scFv-C with central cysteine functionality.
- Chemical crosslinking of scFv thiol groups using azide and multi-alkyne functionalized PEG linkers.
- Copper(I) catalyzed azide alkyne 1,3-dipolar cycloaddition (CuAAC) for covalent ligation of protein conjugates.
- SDS-PAGE and densitometry to determine ligation yields; ELISA to assess tumor binding.
Main Results:
- Chemoselective ligation via CuAAC achieved >70% yield with optimized PEG linkers.
- Developed di-scFv-C constructs derived from anti-MUC1 mAb for targeting cancer cells.
- ELISA demonstrated significantly increased tumor binding of the resulting tetravalent scFv construct.
Conclusions:
- A versatile chemical crosslinking strategy enables the construction of biologically active multivalent and bi-specific immunoconjugates.
- The developed method provides stable, high-affinity constructs with potential applications in pre-targeted radioimmunotherapy and imaging.
- This approach offers a robust platform for advancing targeted cancer therapies and diagnostics.
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