Binding specificity of HuScFv to cholangiocarcinoma cells; New avenues for diagnosis and treatment

Nathawadee Sawatpiboon,¹ Monrat Chulanetra,² Wanpen Chaicumpa,² Sunisa Duang sa-ard,¹ Kanda Kasetsinsombat,¹ Adisak Wongkajornsilp¹

Affiliations:
¹ Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
² Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

Abstract

Background: Cholangiocarcinoma (CCA) is a very aggressive cancer of the bile ducts. Recent advances in
immunotherapy, particularly with human single-chain variable fragments (HuScFv), have shown promise in the treatment of solid tumors by targeting cancer cells or improving the immune response.
Objective: This study aimed to select and produce human single-chain antibody fragments (HuScFv) specific to CCA cells (HubCCA1, RMCCA) from phage display HuScFv libraries with minimum or no binding to cholangiocytes (MMNK1).
Methods: Phages that displayed HuScFv to CCA cells were selected by bio-panning and each phagemid was transduced to HB2151 E. coli. The presence of huscfv was determined by direct colony PCR. HuScFv was produced in the E. coli and detected by Western blot analysis and confirmed their specificity and binding capacity to CCA by flow cytometry.
Results: From biopanning, 196 of 350 colonies (56%) of HB2151 E. coli harboring the huscfv gene, and 106 of these (30%) produced the protein. Flow cytometry testing with 14 clones confirmed the presence of the HuScFv protein. The result showed that five HuScFv clones (25, 33, 61, 68, and 80) exhibited stronger binding to CCA cell lines (HubCCA1, RMCCA) compared to cholangiocytes. Furthermore, each clone possessed a distinct amino acid sequence, suggesting unique binding specificities.
Conclusion: HuScFv specific to CCA cells were successfully selected from phage display HuScFv libraries which offer new revenues to develop a pan-CCA immunotherapy and diagnosis of CCA
Key words: Single-chain variable fragment, flow cytometry, CCA, immunotherapy, targeted cancer therapy