A Model for Targeting Colon Carcinoma Cells Using Single-Chain Variable Fragments Anchored on Virus-Like Particles via Glycosyl Phosphatidylinositol Anchor

Abstract

The current study demonstrated the display, expression and purification of VLP-rscFvs efficiently. As a test model VLP-rscFv-SRB were prepared which can be used for immunotherapy. rscFvs provide the specificity needed to target tumors and VLPs serve as carrier transporting the dye to target.

Virus-Like Particles Displaying Recombinant Short-Chain Fragment Region and Interleukin 2 for Targeting Colon Cancer Tumors and Attracting Macrophages

“…The preparation of recombinant bacmids carrying gag-and GPI-anchored rscFvs was previously described. 17,19 89…”

“…Silkworm larvae were reared and fed as previously reported 17 and injected with 40 μl of 91 recombinant bacmid DNA solutions containing 5 μg of BmNPV-gag577 bacmid, 19 5 μg of 92 BmNPV-rscFv bacmid, 17 and 5 μg of BmNPV-rhIL2 bacmid in 10% (v/v) DMREI-C reagent 93 (Invitrogen, Carlsbad, CA, USA) in PBS using a 1 ml syringe with a 26G×1/2 needle (Terumo, 94 Somerset, New Jersey, USA). 95…”

“…17 The presence of the GPI anchor was confirmed by digestion with 0.1 U of phosphatidyl-inositol specific Phospholipase C (PI-PLC) (Sigma-Aldrich) in 200 μl/well of PBS (pH 7.5) for 2 h at 27°C as previously reported. 17 The function of rhIL2s was confirmed using 100 nanograms per well of soluble-IL-2Rα (Wako) in 100 μl of HEPES buffer pH 7.5 in triplicate incubated overnight at 4°C on an immunoplate. The plate was washed and blocked as previously reported 17 , and 5 μg or 2.5 μg of VLP-rscFv-rhIL2 was added.…”

“…17 The function of rhIL2s was confirmed using 100 nanograms per well of soluble-IL-2Rα (Wako) in 100 μl of HEPES buffer pH 7.5 in triplicate incubated overnight at 4°C on an immunoplate. The plate was washed and blocked as previously reported 17 , and 5 μg or 2.5 μg of VLP-rscFv-rhIL2 was added. As a negative control, 5 μg of VLP was added per well and incubated for 1 h at room temperature.…”

“…VLP-rscFv-rhIL2s were produced in silkworm by the co-expression of the gag, rscFv and rhIL2 proteins using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid. To display on the surface of RSV VLPs, rscFv and rhIL2 were anchored using glycophosphoinositol (GPI) 17 and hemagglutinin transmembrane region (HA-TM) 18 , respectively. Each of these anchors can be easily embedded in a lipid bilayer to display the proteins, but here we focus on simultaneously displaying two anchors.…”