Abstract
The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VHH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 108/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.
Highlights
Engineered antibodies represent a new generation of biological drugs that have been increasingly used in clinics
To produce single-domain antibodies specific for endothelin receptor type B (EDNRB), alpacas were immunized with cultured human melanoma cells used as antigens
Using the serial dilution method, we estimated the size of the anti-human melanoma nanobodies library as 1.2 × 108/ml
Summary
Engineered antibodies represent a new generation of biological drugs that have been increasingly used in clinics. The variable domain of a heavy-chain (VHH) antibody has a simple structure with only approximately 400 bp gene fragments. It can be obtained in vitro in both prokaryotic and eukaryotic expression systems, which is the major technical advantage of nanobodies over conventional genetically engineered antibodies [2, 3]. Endothelin (EDN), a major paracrine factor in melanocyte biology, plays a role in proliferation and differentiation of melanocytes in response to ultraviolet radiation and is involved in the pathogenesis of melanoma [8]. EDN and its receptor (EDNRB) on the melanocyte surface form the EDN signaling system [9] implicated in survival, proliferation, differentiation, and migration of melanocytes [10].
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