Quantitation of the Tumor-Targeting Properties of Antibody Fragments Conjugated to Cell-Permeating HIV-1 TAT Peptides

Abstract

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.

Cell-penetrating and cell-targeting peptides in drug delivery

“…Much effort has been put into improving the cell uptake of antibodies coupled to various CPPs since the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass [68]. As already discussed in a previous review [69], the coupling of cell specific antibodies to CPPs did increase their cellular internalization in vitro as expected [70].…”

“…In this study, the cell-penetrating "driving force" predominated over the specific antigen binding of the Mab fragments. Similarly, Niesner et al observed a complete loss of in vivo cellular specificity when a tumor-targeting scFv fragment was coupled to the Tat peptide [68].…”

Sequence-specific Peptide Aptamers, Interacting with the Intracellular Domain of the Epidermal Growth Factor Receptor, Interfere with Stat3 Activation and Inhibit the Growth of Tumor Cells

“…Protein transduction is therefore a suitable method to introduce inhibitory molecules into target cells. Recent studies showed that a PTD consisting of nine D-arginine residues shows even better transduction efficiencies than the L-amino acids and that the degradation of the recombinant protein is decreased (41). Although such a PTD could be linked chemically to the peptide aptamer protein, the genetic fusion and the ease of recombinant protein production are practical advantages.…”

Site of Docking and Fusion of Insulin Secretory Granules in Live MIN6 β Cells Analyzed by TAT-conjugated Anti-syntaxin 1 Antibody and Total Internal Reflection Fluorescence Microscopy

“…The powerful strategy of a protein transduction system has been reported previously (11,12) in studies of the protein transduction domain of HIV-1 TAT protein, which has been shown to cross biological membranes efficiently and to promote delivery of peptides and proteins into cells. Moreover, intracellular delivery of antibodies by chemically conjugating with TAT has been reported (13)(14)(15). We have succeeded previously in delivering TAT-fused peptide into live MIN6 cells (16).…”