Construction And Identification Of Anti-EGFR Single Chain Fragment Antibody(scFv)and Inhibits The Proliferation Of NSCLC Cells And Tumor Growth By ScFv-mediate SiRNA

Objective:EGFR-TKIs resistance is the reason of tumor progression and disease relapse in EGFR-mutated NSCLC patients. siRNA can silence the expression of resistance-related genes. In order to targetedly deliver siRNA into tumor cells, we designed and constructed anti-EGFR scFv/s-9R fusion proteins, expressed them in E.coli, and explored their binding, internalization and anti-tumor abilities both in vitro and in vivo. Methods:1. The plasmids contained with scFv and s-9R sequences were inserted into the expression vector pGEX-4T-1. After being induced in E.coli BL21(DE3) by IPTG and purified by GST affinity column, the two fusion proteins were detected by SDS-PAGE and Western blot. The antigen-binding activity of the fusion proteins were analyzed by ELISA assay, and the binding of s-9R with nucleotide fragments were verified by gel shift assay.2. The cellular internalization of the fusion proteins or s-9R/siRNA complexes was analyzed by LSCM and flow cytometry; the expression of target genes were detected either by RT-qPCR or Western blot; cell proliferation was analyzed by MTT and Clonogenic assay; cell apoptosis was analyzed by FCM.3. In vivo flurescent living images of xenografted tumors and FAM-siRNA-targeted delivery were obtained and analyzed after injection of fluorescein-labeled fusion proteins or s-9R/Cy5-siRNA/FAM-siRNA. To evaluate the in vivo anti-tumor activity, s-9R/EGFR-siRNA complexes were injected via mouse tail, and gefitinib was administered by oral gavage; the s-9R/HER2-siRNA complexes were injected via mouse tail as well. Tumor volumes were calculated regularly. At last, tumor xenografts were harvested, weighed and snap-frozen for cryosectioning or formalin fixed for paraffin sectioning. Formalin-fixed, paraffin-embedded tumor tissue sections were stained by immunohistochemistry for EGFR, HER2 and Ki-67; for in situ detection of apoptosis, paraffin-embedded sections of tumor tissues were labeled using a fluorescein-TUNEL kit. Results:1. After induced by IPTG and purified by affinity chromatography, the expression of the fusion proteins was confirmed by SDS-PAGE and Western blot; ELISA assay showed that the fusion proteins maintained antigen binding activitiy; gel shift assay verified that s-9R has nucleic acid fragment binding activity; indirect immunofluorescence staining revealed that they were able to internalize into EGFR-positive cells but not into EGFR-negative cells. Moreover, s-9R can deliver siRNA into EGFR-positive NSCLC cells and silence target genes expression.2. In vitro, the sensitivity of NSCLC cells to gefitinib was restored after being treated with s-9R/siRNA complexes, and the apoptosis rates significantly increased. As showed by RT-qPCR and Western blot assay, the expression of HER2 in SPC-A1 cells was efficiently downregulated by s-9R/HER2-siRNA, consequently cell proliferation significantly decreased and cell cycle arrested at G1 phase.3. In vivo, whole-body near-infrared fluorescence living imaging revealed that scFv and s-9R were enriched in the tumor tissues after being injected via the mice tail vein; and s-9R can deliver Cy5/FAM-labeled siRNA to tumor sites. Co-treatment of s-9R/siRNA with Gefitinib successfully suppressed the H1975 xenograft tumor progression and prolonged the life span of nude mice; protein-delivered HER2-siRNA could efficiently suppress tumor growth of nude mice; the expression of target proteins and Ki67 were decreased, while the TUNEL positive cells were increased. Conclusion:We developed two EGFR-specific scFvs and confirmed their binding and internalization capacity. Moreover, we showed that s-9R, as a siRNA carrier, can effectively deliver siRNA into NSCLC cells and silence the expression of target genes. With the co-treatment of s-9R/siRNAs with gefitinib, we successfully induced cancer cells apoptosis in vitro and suppressed tumor progression in vivo. This scFv-mediated siRNA delivery system and the co-treatment strategy can increase the feasibility of using siRNA therapeutically to overcome EGFR-TKIs resistance in clinic. Our study also provides preclinical support for the therapeutic potential of scFv-based RNAi therapy targeting HER2 in NSCLC management.