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By-passing large screening experiments using sequencing as a tool to identify scFv fragments targeting
Kamel Deramchia1,2, Marie-Josee Jacobin-Valat1,2, Jeanny Laroche-Traineau1,2
1Magnetic Resonance Center of Biological Systems, UMR 5536, National Center for Scientific Research, Bordeaux Segalen University, 33076 Bordeaux Cedex, France.
Abstract:
Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. To interrogate the molecular components involved in this process, single-chain variable fragments (scFvs) from a semi-synthetic human antibody library were selected on the lesions induced in a rabbit model of atherosclerosis after two rounds of in vivo phage display. Homing Phage-scFvs were isolated from (1) the injured endothelium, (2) the underlying lesional tissue and (3) the cells within the intima. Clones selected on the basis of their redundancy or the presence of key amino acids, as determined by comparing the distribution between the native and the selected libraries, were produced in soluble form, and seven scFvs were shown to specifically target the endothelial cell surface and inflamed intima-related regions of rabbit tissue sections by immunohistology approaches. The staining patterns differed depending on the scFv compartment of origin. This study demonstrates that large-scale scFv binding assays can be replaced by a sequence-based selection of best clones, paving the way for easier use of antibody libraries in in vivo biopanning experiments. Future investigations will be aimed at characterizing the scFv/target couples by mass spectrometry to set the stage for more accurate diagnostic of atherosclerosis and development of therapeutic strategies.
Insights
Researchers developed a new method using antibody fragments (scFvs) to identify molecular targets in atherosclerosis. This approach simplifies in vivo biopanning for better diagnostics and therapies for this inflammatory disease.
Area of Science:
- Immunology
- Cardiovascular Research
- Molecular Biology
Background:
- Atherosclerosis is a chronic inflammatory disease characterized by vulnerable lesions and thrombosis.
- Identifying molecular components of atherosclerotic lesions is crucial for diagnosis and therapy.
Purpose of the Study:
- To develop a more efficient method for selecting antibody fragments targeting atherosclerotic lesions.
- To identify specific molecular targets on atherosclerotic lesions using in vivo phage display.
Main Methods:
- Selection of single-chain variable fragments (scFvs) from a human antibody library using phage display on induced atherosclerotic lesions in rabbits.
- Isolation of homing phage-scFvs from different lesion compartments: injured endothelium, underlying tissue, and intima.
- Sequence-based selection of promising clones and production of soluble scFvs.
- Immunohistological analysis to determine the specificity of selected scFvs against rabbit tissue sections.
Main Results:
- Seven scFvs were identified that specifically target the endothelial cell surface and inflamed intima.
- Distinct staining patterns were observed for scFvs based on their compartment of origin.
- Sequence-based selection proved effective, potentially replacing large-scale binding assays.
Conclusions:
- This study presents a streamlined approach for in vivo biopanning using antibody libraries.
- The identified scFvs show potential for diagnosing atherosclerosis and developing targeted therapies.
- Future work will focus on characterizing scFv/target interactions using mass spectrometry.
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