Development of a recombinant biosimilar single-chain variable fragment antibody targeting human estrogen receptor α36

The estrogen receptor alpha-36 (ER-α36) is an alternative splice variant of classical ER-α66 and is abundantly present in both ER-α66-positive and ER-α66-negative breast tumor cells. Given its clinical relevance, developing targeted strategies against this isoform is of particular significance to breast cancer research. This study aimed to develop an ER-α36-specific recombinant biosimilar single-chain variable fragment (scFv) antibody.

The primary amino acid sequence of the anti-ER-α36 scFv was retrieved from patent US20110311517A1. An expression cassette harboring the scFv coding sequence was designed and incorporated into the backbone of the pET-28a(+) expression vector for recombinant expression in Escherichia coli (E. coli) BL21(DE3) cells. Expression conditions were then optimized, and the protein was purified using immobilized metal affinity chromatography. The binding of the purified scFv to ER-α36-expressing breast cancer cells was assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry.

Characterization using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting experiments revealed a molecular weight of 29 kDa for the expressed scFv antibody. Relative quantification revealed the highest scFv protein expression level 16 h after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside at 25 °C. Flow cytometry and ELISA assays demonstrated specific binding of the scFv to ER-α36 protein on MDA-MB-231 breast cancer cells, while no interaction was detected with ER-α36-negative MCF-10A normal mammary epithelial cell line.

The anti-ER-α36 scFv antibody fragment was successfully expressed using the E. coli expression system, and the purified protein was able to specifically recognize and bind to ER-α36-expressing human breast cancer cells.