Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59

Abstract

BackgroundTherapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC).MethodsWe isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay.ResultsThe scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells.ConclusionA 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

The Complement System: A Potential Therapeutic Target in Liver Cancer

“…Currently, anti-C5aR1 in combination with anti-PD-1/PD-L1 is being studied in clinical trials for the treatment of patients with advanced solid tumors (NCT03665129). Adjuvant cancer therapy is utilizing monoclonal antibodies (mAbs) directed against complement regulatory proteins (CRPs), including CD46, CD55, and CD59 [ 115 ]. Sherbenou and colleagues reported that CD46 antibodies kill tumor cells by apoptosis while protecting benign cells [ 116 ].…”