Isolation and characterization of a novel scFv antibody fragments specific for Hsp70 as a tumor biomarker

• Published in: Journal of cellular biochemistry 2019-09, Vol.120 (9), p.14711-14724
• Main Authors: Vostakolaei, Mehdi Asghari, Molavi, Ommoleila, Hejazi, Mohammad Saeid, Kordi, Shirafkan, Rahmati, Saman, Barzegari, Abolfazl, Abdolalizadeh, Jalal
• Format: Article
• Language: English
• Subjects: Antibodies; Bioinformatics; Biomarkers; Display devices; DNA sequencing; E coli; Electrophoresis; Gel electrophoresis; Heat shock proteins; Hsp70 protein; human Hsp70; Immunofluorescence; Immunotherapy; in silico CDR interaction analysis; Lung carcinoma; Monoclonal antibodies; monoclonal antibody; Organic chemistry; Peptides; Phage display; Phages; Polymerase chain reaction; single‐chain fragment variable; Sodium; Sodium dodecyl sulfate; Sodium lauryl sulfate; Surface plasmon resonance; Therapeutic applications; Tumors
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.28732

Many studies have shown that more than 50% of tumors express heat shock protein 70 kDa (Hsp70) at the plasma membrane surface while not seen in normal cells, therefore it is a promising therapeutic target in human cancers. Hence, we used phage display technology to produce a single‐chain fragment variable (scFv) antibody against human Hsp70. For this, a target peptide from human Hsp70 was designed using bioinformatics studies and was chemically synthesized. Then, the selection was performed using four rounds of biopanning with a stepwise decreased amount of the target peptide. Fourteen positive scFv clones were selected using monoclonal phage enzyme‐linked immunosorbent assay screening, which was further characterized by means of the polymerase chain reaction and DNA sequencing. Among them, the G6 clone was selected to express scFv into the Escherichia coli. Expression and purification of the scFv shown by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and confirmed by Western blot analysis. In silico analysis confirmed specific binding of the scFv to Hsp70 in CDR regions. The specificity of the scFv measured by surface plasmon resonance and immunofluorescence of the A549 human lung carcinoma cell line confirmed the in vitro function of the scFv. Based upon these findings, we propose a novel anti‐human Hsp70 scFv as potential immunotherapy agents that may be translated into preclinical/clinical applications. In our bench‐scale approach using phage display technology (PDT), we were able to isolate and purify an anti‐human Hsp70 single‐chain fragment variable (scFv) fragment. Since the functionality of the novel present scFv was confirmed using three criteria including in silico docking, surface plasmon resonance (SPR) and IF, it could be used as tools to detect Hsp70 as a new effective therapeutic target in the human cancers. However, to use this scFv in cancer immunotherapy, we shall focus on affinity maturation and in vivo models using computational design/protein engineering and animal models, respectively.