HER2-siRNA delivered by EGFR-specific single chain antibody inhibits NSCLC cell proliferation and tumor growth

Overexpression of human epidermal growth factor receptor type2 (HER2) is closely associated with aggressive progression and poor prognosis in non-small cell lung cancer (NSCLC). Here, we generated an EGFR-scFv-arginine nonamer peptide fusion protein (scFv-9R) as a cargo to deliver HER2 specific siRNA into HER2-positive NSCLC cells both in vitro and in vivo. HER2-siRNAs delivered by scFv-9R effeciently silenced HER2 expression in EGFR-positive NSCLC cells, and consequently resulted in G1 arrest and cell growth inhibition. Importantly, intravenous injection of scFv-9R/HER2-siRNA complex markedly suppressed growth of EGFR-positive NSCLC xenograft in nude mice, resulting from downregulated HER2 expression, reduced cell proliferation and enhanced cell apoptosis. Collectively, our study provides a novel therapeutic strategy for the treatment of EGFR-positive, HER2-overexpressed NSCLC.


Plasmid construction and protein purification
The genes encoding scFv and scFv-9R were designed according to the amino acid sequences of Nimotuzumab with a linker (Gly4Ser3) connected its VH and VL chains, and the nine-mer arginine encoding sequence designed in the 3′ scFv sequence to obtain the scFv-9R fusion gene. In order to label the scFvs, we also designed an 6×His.tag-encoding sequence at the 3′ scFv sequences, which was synthesized by Augct Co. (Beijing, China). For bacterial expression, the scFv and scFv-9R sequence were isolated as a BamHI/XhoI fragment from T vector and inserted the sequence into a BamHI/ XhoI-digested plasmid, pGEX-4T-1 (Novagen, Uppsala, Sweden). Single colonies of E. coli BL21 (DE3) carrying the manufactured plasmid pGEX-4T-1-scFv for the expression of EGF receptor-specific scFv protein, were grown overnight at 37°C in 2×YT medium supplemented with 100 μg/ml ampicillin. We diluted the cultures 100fold in the same medium, and incubated them at 37°C to an OD600 of 0.4-0.6, followed by inducing with 0.1 mM isopropyl-1-thio-β-galactopyranoside (IPTG) at 30°C for 5 h. Via centrifugation (5,000 g, 4°C, 15 min), cells were harvested, washed in ice-cold phosphate buffer saline (PBS), then re-suspended them in binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Soluble cell lysates was centrifuged at 12,000 g for 20 min after 30 min of sonication. Following the manufacturerʼs instructions, we then applied a glutathionesepharose affinity column (GE Healthcare, Uppsala, Sweden) to the supernatant. The fusion proteins was eluted from the GST-affinity column using 10 mM reduced glutathione dissolved in 50 mM Tris-HCl (pH 8.0) at room temperature. We performed dialysis on the purified sample against PBS (PH 8.0) for 24 h at 4°C, then incubated the sample with 25-fold diluted thrombin (Novagen, Darmstadt, Germany) at room temperature for 8 h. Finally, the solutions were filtered in a glutathione-sepharose affinity column to remove the GST.tag. The resultant fusion proteins were quantified by a bicinchoninic acid assay (Pierce, Rockford, IL USA) and immuno-blotting with an anti-6×His.tag mouse monoclonal antibody (1:1000, #34660, Qiagen, CA, USA).
The actual sequence of the scFv peptide is as follows: LQQSGAEVKKPGSSVKVSCKASGYTFTNYYI YWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVT ITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDS DGRGFDFWGQGTTVTVSGGGGSGGGGSGGGGSIQ MTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLD WYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGT DFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQI

Affinity of the fusion proteins to EGFR measured by ELISA
The affinity of the scFv and scFv-9R fusion protein for EGFR was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, recombinant EGFR (Proteintech, Wuhan, China; 1 μg per well) was immobilized on 96-well microplates and incubated with 100 nM of each the fusion proteins or BSA at 37°C for 1 h. Successively immunobloted with anti-6×His.tag antibody for 2 h and HRP-conjugated Goat Anti-Rabbit IgG (Boster, Wuhan, China) for 1 h. After extensive washing, a 3, 3′, 5, 5′-Tetramethylbenzidine (TMD) substrate was added and reaction was terminated after 30 min by adding 2 M sulfuric acid. The plates were read in the microplate reader at the absorbance of 450 nm. (Bio-Rad iMark680, California, America).

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR)
The qRT-PCR analysis was performed in a total volume of 20 μL with the following amplification steps: an initial denaturation step at 95°C for 3 min, followed by 54 cycles of denaturation at 95°C for 10 s, anneal at 56°C for 15 s and extention at 72°C for 15 s. Data were analyzed according to the comparative Ct method and were normalized by β-actin expression in each sample. Data shown are representative of the mean values and standard deviations from three independent experiments performed in triplicate.

Expression and clinico-pathological correlation of HER2 in human NSCLC specimens
A total of 75 human paraffin-embedded NSCLC tissue sections storage at the Department of Pathology in Xijing Hospital, were investigated for HER2 expression by immunohistochemistry. The deparaffinized specimens were incubated with rabbit anti-human HER2 monoclonal antibody (1:100, #BA0015-1, Boster, Wuhan, China) overnight at 4°C and detected by the IHC rabbit DAB kit (NeoBioscience, Beijing, China).
HER2-positive NSCLC specimens were further selected for clinico-pathological correlation analysis. Clinical parameters such as gender, age, lymph node metastasis and TNM stage were collected. The association between HER2 expression and clinico-pathological parameters was examined by χ 2 test. A p value of <0.05 was considered significant. Patients offering samples for the study signed informed consent forms. This study was