Inhibition of human lung cancer cells by anti-p21Ras scFv mediated by the activatable cell-penetrating peptide

Abstract

Activatable cell-penetrating peptide (ACPP) is a tumour-targeting cell-penetrating peptide. Here, we used ACPP to carry anti-p21Ras scFv for Ras-driven cancer therapy. The ACPP-p21Ras scFv fusion protein was prepared by a prokaryotic expression system and Ni-NTA column purification. The human tumour cell lines A549, SW480, U251 and Huh7 and the normal cell line BEAS 2B were used to study the tumor-targeting and membrane-penetrating ability of ACPP-p21Ras scFv. The antitumour activity of ACPP-p21Ras scFv on A549 cells and H1299 cells in vitro was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, scratch wound healing, plate cloning and apoptosis assays. The penetration pathway of ACPP was determined by enhanced green fluorescent protein. The ACPP-p21Ras scFv fusion protein was successfully obtained at a concentration of 1.8 mg/ml. We found that ACPP-p21Ras scFv could penetrate tumour cell membranes with high expression of matrix metalloproteinase-2 (MMP-2), effectively inhibit the migration and proliferation of A549 cells and H1299 cells, and promote the apoptosis of A549 cells and H1299 cells. The membrane penetration experiment demonstrated that ACPP could enter A549 cells by direct penetration. The ability of ACPP to penetrate the membrane was affected by the addition of a membrane affinity inhibitor and a change in the potential difference across the cell membrane but not by the addition of endocytosis inhibitors and a change in temperature. The ACPP-p21Ras scFv fusion protein can penetrate tumour cells with MMP-2 expression and has antitumour activity against A549 cells and H1299 cells in vitro . This molecule is expected to become a potential antitumour drug for Ras gene-driven lung cancer.

Toxicity Studies of Cardiac-Targeting Peptide Reveal a Robust Safety Profile

“…These hurdles were initially ameliorated by topical use of Tat conjugated to a therapeutic [ 25 , 26 , 27 ], or in a compartment-restricted manner such as intra-articular [ 28 , 29 , 30 ] or intra-ocular administration [ 31 , 32 , 33 ]. Another use of non-specific CPP was the use of activatable peptides given a certain tissue milieu in oncological applications [ 34 , 35 , 36 , 37 , 38 ] or enzyme-activatable cell penetrating peptides [ 39 , 40 , 41 ]. Phage display to identify specific tumor- or tissue-targeting CPPs was yet another strategy in attempts to harness CPP’s transduction abilities for clinical application [ 9 , 42 , 43 , 44 ].…”

Mechanisms of Action and Limitations of Monoclonal Antibodies and Single Chain Fragment Variable (scFv) in the Treatment of Cancer

“…Other alternative vector strategies for delivering scFvs to the tumor cells included “cell-penetrating peptides” (CPPs), which are natural or synthetic peptides with the ability to interact with cellular membranes for the internalization of cells and effective intracellular delivery. CPPs had low cytotoxicity and immunogenicity [ 160 ] and have been widely used as vehicles for the administration of scFv with a Ras-blocking effect in colon cancer [ 161 ] and lung cancer [ 162 ]; these peptides improved the intracellular administration and orientation, thus favoring the antitumoral activity of the scFv.…”

Antibody variable region engineering for improving cancer immunotherapy

“…The solid tumor is relatively inaccessible to whole antibodies because of several biological barriers [ 21 ]. In contrast, scFv and nanobodies have superior tissue penetration properties compared with mAbs: they demonstrate a higher perfusion rate across endothelial and epithelial barriers and diffuse faster through the tumor stroma because of their smaller size [ 22 , 23 , 24 , 25 ]. Nevertheless, the small size of scFv and nanobodies also has its drawbacks.…”