| Summary: | Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.
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