Computer-aided design of novel immunotoxins for the treatment of malignant diseases

• Applicants: Professor Dr. Stefan Barth; Professor Dr. Paolo Carloni
• Subject Area: Hematology, Oncology
• Term: from 2014 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 243918739

Conventional treatments for cancer including chemotherapy, radiation and surgery have been limited by several factors such as drug resistance, non-specificity deleterious to normal cells, relapse due to the often-remaining metastatic cancer cells and failure in non-resectable tumors. Immunotoxins (ITs) as a target therapy can combat these problems by specifically killing cancer cells. The current third-generation ITs are recombinant proteins composed of a cell-selective recombinant antibody fragment genetically fused to a mutated bacterial or plant toxin. The antibody directs the IT to bind its target antigen, which is selectively expressed on cancer cells. The IT is subsequently internalized in the cells, which enables the toxin to kill the target cells. Several such ITs are in clinical trials and denileukin difitox (trade name Ontak) has been approved by FDA for treatment of cutaneous T-cell lymphoma. However, third-generation ITs suffer from major drawbacks due to the plant or bacteria toxins used: (I) their potential immunogenicity: upon repeated application, the immune system of the patients can react with the generation of neutralizing antibodies leading to reduced therapeutic efficiency. (II) Their potential in inducing vascular leak syndrome, due to non-specific binding to endothelial cells. (III) Their off-target activities towards healthy cells. To circumvent these problems, a promising IT strategy is to develop a new generation of ITs containing human enzymes instead of plant or bacteria toxins as the cytotoxic moiety. Previously, we have developed the first human cytolytic fusion proteins (hCFP) with promising specific cytotoxicity for CD30-positive and CD64-positive malignancies in vitro. These hCFP contain the human pancreatic ribonuclease (RNase) angiogenin (HuAng), which eliminates the target cells by degrading their RNA. These HuAng-containing hCFP may lead to novel immunotherapeutic agents with an extensive therapeutic index, provided that two major issues are resolved: (I) The hCFPs must be able to evade the inhibition of intracellular human placental RNase inhibitor RNH1 which binds with high affinity to human RNases. (II) The low catalytic activity of HuAng must be improved to enable high efficiency of the hCFPs. In this project, we will address these two issues by modifying the molecular structure of HuAng to obtain variants with both high catalytic activity and weak binding to RNH1. Based on known experimental data, computational approaches will enable us to investigate the molecular mechanism of such catalysis and binding. This will lead to rational design of the desired HuAng variants. Novel hCFP will be generated using these variants. Their specific cytotoxicity towards target cancer cells will be examined in vitro using our well-established experimental procedure. By combining the advanced computational and experimental techniques, this project will lead to successful development of HuAng-derived anti-cancer agents.

• DFG Programme: Research Grants
• Participating Person: Dr. Thomas Nachreiner