A strategy for searching tumor-specific VHH antibodies derived through phage display library combined with high throughput sequencing

To isolate precise tumor cell-specific VHH antibodies, we implemented a method using a tumor-specific VHH phage library where the phage library was enriched against 4 different cancer cell lines through a cell flow cytometer. Repeating the operations several times against each tumor-specific cell to confirm phage library enrichment, pooled phage libraries were applied to the next-generation sequencer. The top 50 sequences according to amplification factors from each library were aligned and constructed phylogenetic tree but unfortunately, identical sequences were observed from different libraries with high amplification factors. To solve this issue, we combinedly aligned all 200 (50 seq. from each library) and constructed a molecular phylogenetic tree with 10 clusters. We regenerated one clone from each group using the PCR technique and determined the binding specificity against tumor cells, among them highly enriched clones from each library showed unique binding, and moderately enriched clones were multiple cross-reactive binders.