Small Cleavable Adapters Enhance the Specific Cytotoxicity of a Humanized Immunotoxin Directed Against CD64-positive Cells

Abstract

The most potent immunotoxins (ITs) developed to date contain bacterial or plant cytotoxic components. As these are potentially immunogenic in man, human proteins are preferred for the long-term treatment of cancer. We have developed the first humanized IT for the treatment of CD64 malignancies such as acute myeloid leukemia. The bacterially expressed IT is composed of a humanized anti-CD64 single chain fragment [h22(scFv)] genetically fused to the human RNase angiogenin. As angiogenin lacks a dedicated translocation domain responsible for the higher potency of bacterial and plant-derived toxins, we have incorporated a recombinant adapter that contains a synthetic translocation domain flanked by proteolytically cleavable endosomal and cytosolic consensus sites. Although insertion of the adapter increased the cytotoxicity by up to 20-fold, serum stability was markedly reduced. Therefore, we designed a modified adapter variant with the endosomal-cleavable peptide deleted. The IT containing the truncated adapter showed significantly higher cytotoxicity than the adapter-free IT and superior serum stability to facilitate the potential applications in patients.

Targeting antibodies to the cytoplasm

“…The NEMO-binding peptide was successfully delivered into activated endothelial cells and interfered with the nuclear factor kappaB pathway. 206 Translocation adapter sequences, which are composed of cytosolic and endosomal cleavage sites flanking a viral membrane-penetrating peptide, improved the activity of immunotoxins and targeted RNases more than 100-fold 207,208 and may also be suitable to deliver larger non-toxin protein moieties, including antibody fragments, into the cytosol of cells. Another new approach, receptor-mediated delivery (RMD), utilizes a variant of substance P, a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R), and was able to transfer a chemical conjugated cargo protein into the cytosol.…”

EpCAM-Selective Elimination of Carcinoma Cells by a Novel MAP-Based Cytolytic Fusion Protein

“…One tentative hypothesis discussed in the literature is that upon the pH shift in the endosome, the conformation of the protein is changed, leading to the exposure of potential processing and/or signal sequences (45). However, the cytotoxic activity of human CFPs can be improved further by the insertion of adapter sequences that facilitate the translocation of the effector molecule from the endosome to the cytosol (29). Therefore, our future research will focus on improving the translocation efficiency of anti-EpCAM (scFv)-MAP, to further enhance its therapeutic potential.…”

“…Several human enzymes, including the protease granzyme B, RNase angiogenin, or death-associated protein kinase 2, have been demonstrated to induce apoptosis in target cells, when delivered by antigen-specific ligands or antibody fragments (26)(27)(28)(29). Similarly, death ligands of the tumor necrosis factor (TNF) family have been genetically fused to anticancer scFvs to selectively induce apoptosis in various forms of cancer (30,31).…”

Human microtubule‐associated protein tau mediates targeted killing of CD30+ lymphoma cells in vitro and inhibits tumour growth in vivo

“…In addition to human granzyme B, human RNases, such as angiogenin, have been used to replace nonhuman toxins (Hetzel et al, 2008;Stahnke et al, 2008;Mathew & Verma, 2009;Schiffer et al, 2013). The specific cytotoxicity of human angiogenin towards CD30-overexpressing HL-derived cell lines has been demonstrated by fusion with the CD30 ligand (Huhn et al, 2001).…”

“…In addition, the cytotoxic activity of human CFPs could be enhanced by improving the translocation efficiency from the endosome to the cytosol through engineering a translocation-promoting adapter peptide between the binding and the cytotoxic domain (Hetzel et al, 2008).…”