TY - JOUR
T1 - Generation and characterization of avian single chain variable fragment against human Alpha-Enolase
AU - Lee, Chi Hsin
AU - Tsai, Chu Hsuan
AU - Leu, Sy Jye
AU - Liu, Ko Jiunn
AU - Wang, Wei Chu
AU - Tsai, Bor Yu
AU - Chiang, Liao Chun
AU - Mao, Yan Chiao
AU - Benedict Dlamini, Nhlanhla
AU - Tsai, Chen Hsin
AU - Yang, Yi Yuan
N1 - Publisher Copyright:
© 2023 Elsevier B.V.
PY - 2023/7
Y1 - 2023/7
N2 - Overexpression of human alpha-enolase (hEno1) has been reported in a wide range of cancers and is tightly associated with poor prognosis, making it a remarkable biomarker and therapeutic target. In this study, polyclonal yolk-immunoglobulin (IgY) antibodies purified from hEno1-immunized chickens showed a noticeable specific humoral response. Phage display technology was used to construct two antibody libraries of IgY gene-derived single-chain variable fragments (scFvs) containing 7.8 × 107 and 5.4 × 107 transformants, respectively. Phage-based ELISA indicated that specific anti-hEno1 clones were significantly enriched. The nucleotide sequences of scFv-expressing clones were determined and classified into seven groups either in the short linker or the long linker. Moreover, higher mutation rates were revealed in the CDR regions, especially in the CDR3. Three distinguish antigenic epitopes were identified on the hEno1 protein. The binding activities of selected anti-hEno1 scFv on hEno1-positive PE089 lung cancer cells were confirmed using Western blot, flow cytometry, and immunofluorescence assay. In particular, hEnS7 and hEnS8 scFv antibodies significantly suppressed the growth and migration of PE089 cells. Taken together, these chicken-derived anti-hEno1 IgY and scFv antibodies have great potential to develop diagnostic and therapeutic agents for the treatment of lung cancer patients with high expression levels of hEno1 protein.
AB - Overexpression of human alpha-enolase (hEno1) has been reported in a wide range of cancers and is tightly associated with poor prognosis, making it a remarkable biomarker and therapeutic target. In this study, polyclonal yolk-immunoglobulin (IgY) antibodies purified from hEno1-immunized chickens showed a noticeable specific humoral response. Phage display technology was used to construct two antibody libraries of IgY gene-derived single-chain variable fragments (scFvs) containing 7.8 × 107 and 5.4 × 107 transformants, respectively. Phage-based ELISA indicated that specific anti-hEno1 clones were significantly enriched. The nucleotide sequences of scFv-expressing clones were determined and classified into seven groups either in the short linker or the long linker. Moreover, higher mutation rates were revealed in the CDR regions, especially in the CDR3. Three distinguish antigenic epitopes were identified on the hEno1 protein. The binding activities of selected anti-hEno1 scFv on hEno1-positive PE089 lung cancer cells were confirmed using Western blot, flow cytometry, and immunofluorescence assay. In particular, hEnS7 and hEnS8 scFv antibodies significantly suppressed the growth and migration of PE089 cells. Taken together, these chicken-derived anti-hEno1 IgY and scFv antibodies have great potential to develop diagnostic and therapeutic agents for the treatment of lung cancer patients with high expression levels of hEno1 protein.
KW - alpha–enolase (Eno1)
KW - Phage display technology
KW - single-chain variable fragment (scFv)
KW - yolk-immunoglobulin (IgY)
UR - http://www.scopus.com/inward/record.url?scp=85159455502&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85159455502&partnerID=8YFLogxK
U2 - 10.1016/j.intimp.2023.110277
DO - 10.1016/j.intimp.2023.110277
M3 - Article
C2 - 37196558
AN - SCOPUS:85159455502
SN - 1567-5769
VL - 120
JO - International Immunopharmacology
JF - International Immunopharmacology
M1 - 110277
ER -
