Notch signaling pathway comprises of four different Notch receptors (Notch-1-4) and five ligands (Delta-1, -3, -4 and Jagged1, 2). Receptor-ligand interaction on the cell surface results in a conformational change in the negative regulatory region (NRR) domain, which exposes a proteolytic cleavage site on the Notch receptor to ᵧ -secretase. Therefore, NRR is a target for antibody blocking that confer stability of NRR and block the cleavage of Notch receptor. Notch signaling regulates proliferation, differentiation, and apoptosis of various cell types. Aberrant increases or deficiencies in Notch signaling lead to cancer development of various origins. Small molecule ᵧ -secretase inhibitor (GSI) is generally used for inhibiting Notch signaling but it has undesirable side effects because ᵧ -secretase has multiple substrates. scFv-Fc is a fusion protein of the single-chain variable fragments (scFv) and human immunoglobulin G (IgG) Fc domain. scFv-Fc has potential in clinical application over the whole monoclonal antibody for target therapy. The purpose of this research is to produce the recombinant antibody scFv-Fc against NRR of Notch1 and 2 in HEK-293T cell line and characterize the recombinant antibody in human leukemia cell line Jurkat. The results showed that recombinant anti-Notch1 and 2 scFv-Fc were successfully expressed in HEK-293T cell line when scFv-Fc gene harboring plasmid was transfected into the cells. Proteins from culture supernatant was purified by affinity chromatography column. The purified protein showed a single band protein, on SDS-PAGE after purification by two-step affinity chromatography method of His-tag and protein A affinity column. To test whether the recombinant antibody scFv-Fc recognizes the NRR, anti-Notch1 scFv-Fc and anti-Notch2 scFv-Fc were incubate to Jurkat cell line at 1, 10 µg, respectively. The results by flow cytometry showed specific binding of antibody. The effect on cleaved Notch and experession of the Notch target gene, HES1, HEY1 were examined. Anti-Notch1 scFv-Fc and anti-Notch2 scFv-Fc were used at 1, 10 µg, respectively. The results showed that the recombinant antibody decreased the level of cleaved Notch1 and reduced the expression of HES1 and HEY1. Therefore, the recombinant antibody anti-Notch1 scFv-Fc and anti-Notch2 scFv-Fc specifically inhibited Notch signaling at equivalent level as to that of GSI. Specific inhibition of Notch signaling by the recombinant antibodies may present a novel therapeutic approach in treatment of cancer in the future
