Internalization via Antennapedia protein transduction domain of an scFv antibody toward c‐Myc protein

Abstract

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

“…Current approaches for intracellular delivery of antibodies, such as CPP-conjugated and anti-DNA scFvfused antibodies, are almost restricted to nuclear targets because of the intrinsic nuclear targeting property of the carrier. 1,2,8,10,14 On the other hand, cytotransmabs penetrated into the cytosol of living cells, enabling its direct targeting of cytosolic proteins.…”

“…1,2,7 Conjugation with protein transduction domains, which are represented by cell-penetrating peptides (CPPs) such as the HIV-1 TAT peptide, has been extensively attempted in order to facilitate the intracellular delivery of antibodies formatted as single chain variable fragments (scFvs), antigen-binding fragments (Fabs), and full-length IgGs. [8][9][10] However, most of the CPP-conjugated antibodies inherited the intrinsic intracellular trafficking of the parent CPPs, which were either entrapped inside endocytic vesicles, translocated into the nucleus, or eventually degraded in lysosomes without efficient endosomal release into the cytosol. 1,10,11 Some anti-DNA polyclonal antibodies or monoclonal antibodies (mAbs) predominantly found in humans and mice with autoimmune diseases have been shown to possess the ability to penetrate into living cells in their IgG format.…”

Single‐domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP‐ribosylating toxin of Salmonella typhimurium

“…Second, genetic fusion to translocation domains can serve as a mechanism to translocate proteins across the cell membrane into the cytosol (58,59). Such an approach has recently been used to introduce a singlechain variable fragment (scFv; i.e., the variable domains of the light and heavy chains of a conventional monoclonal antibody genetically fused via a linker peptide into a single polypeptide) directed against the myc oncogene into the cytosol of a human cell line (59). Similarly, fusion of a nanobody to a suitable protein or peptide-based translocation domain could provide a means of delivery for SpvB-neutralizing single-domain antibodies to the cytosol of epithelial cells.…”

A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

“…11 Over the years, the ETH2-GOLD library has proven to be a reliable source of good-quality human mAbs. 11,15,19,[25][26][27][28][29][30][31][32] However, as for other libraries, a degree of epitope bias may result from the library design. For example, we were so far unable to isolate further human mAbs from the ETH2-GOLD library that do not overlap with the clinical-stage F8 antibody.…”