Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

Main entry route and mechanism of action of diphtheria toxin. 1. The toxin is secreted as one polypeptide which is composed of three functional domains: the N terminal catalytic domain ((C), also referred to as DTA/DT-A), the translocation domain (T) and the receptor binding domain (R) (see 3D structure (PDB Entry: 1f0l). In the left panel, the colors of the subunits correspond to those in the scheme). In addition, a disulfide bond bridges the C and T domains; 2. The toxin binds via its R domain to a cellular receptor (heparin binding epidermal growth factor precursor); 3. Cell-surface furin protease cleaves the polypeptide chain between the C and T domains that remain linked by a disulfide bond; 4. The toxin-receptor complex is internalized into clathrin coated pits; 5. In the lumen of the early endosome (EE), furin protease cleaves toxin molecules that escaped cell-surface cleavage. The T domain undergoes acidic-induced conformational change, inserted into the endosome membrane and forms a channel through which the catalytic domain can translocate into the cytoplasm where reduction of the interdomain bridging disulfide bond occurs; 6. In the cytoplasm, the catalytic domain inactivates eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation, which causes translation inhibition and consequently cell death. Denileukin Diftitox, which is also named "Ontak" or "DAB389 IL-2", is a fusion protein designed to direct a truncated form of diphtheria toxin to cells that express the high-affinity IL-2 receptor, (consisting of the following subunits: CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ)), which is present in many different hematologic malignancies like adult T cell leukemia (ATL), chronic lymphocytic leukemia, Hodgkin's and non-Hodgkin's lymphomas, cutaneous T cell lymphoma (CTCL) and other leukemias and lymphomas [10, [135] [136] [137] [138] [139] . The immunotoxin is comprised of a genetic fusion between a truncated form of DT (first 388 amino acids, "DAB 389 "), in which the natural receptor binding domain of the toxin was replaced by the cytokine interleukin-2 (IL-2) [140]. Phase I testing was conducted on patients with T-cell lymphoma (CTCL) (n = 35), other non-Hodgkin's lymphomas (NHL) (= 17), or Hodgkin's disease (HD) (n = 21). The drug, which was administrated by intravenous infusion, produced five complete (CR) and eight partial (PR) remissions in patients with CTCL with one CR and two PR occurring in NHL. No response was documented in patients with HD. The dose-limiting toxicity in these trials was asthenia [141] . In the pivotal phase III trial, 30% of 71 patients with CTCL treated with Denileukin Diftitox had an objective response (20% partial response; 10% complete response) [18] . since the FDA approval of ONTAK as the first immunotoxin for treatment of advanced CTCL in 1999, the drug was tested for treatment of other malignant and non-malignant diseases like B-cell NHL [23], B-cell chronic lymphocytic leukemia (CCL) [19] panniculitic lymphoma [142] , psoriasis [17, 143] and Graft-versus-host disease (GVHD) [21] . Responses were observed in all of these trials. The majority of acute myeloid leukemia (AML) blast cells express the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor [144] . In order to target these cancerous cells, the human GM-CSF was fused to DT388, a truncated DT toxin, replacing its natural receptor binding domain [145, 146] . The resulting molecule, DT388-GM-CSF (DTGM), was