title: Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo.
abstract: Immunotoxins are fusion proteins that combine a targeting component such as an antibody fragment or ligand with a cytotoxic effector component that induces apoptosis in specific cell populations displaying the corresponding antigen or receptor. Human cytolytic fusion proteins (hCFPs) are less immunogenic than conventional immunotoxins because they contain human pro-apoptotic enzymes as effectors. However, one drawback of hCFPs is that target cells can protect themselves by expressing endogenous inhibitor proteins. Inhibitor-resistant enzyme mutants that maintain their cytotoxic activity are therefore promising effector domain candidates. We recently developed potent variants of the human ribonuclease angiogenin (Ang) that were either more active than the wild-type enzyme or less susceptible to inhibition because of their lower affinity for the ribonuclease inhibitor RNH1. However, combining the mutations was unsuccessful because although the enzyme retained its higher activity, its susceptibility to RNH1 reverted to wild-type levels. We therefore used molecular dynamic simulations to determine, at the atomic level, why the affinity for RNH1 reverted, and we developed strategies based on the introduction of further mutations to once again reduce the affinity of Ang for RNH1 while retaining its enhanced activity. We were able to generate a novel Ang variant with remarkable in vitro cytotoxicity against HL-60 cells and pro-inflammatory macrophages. We also demonstrated the pro-apoptotic potential of Ang-based hCFPs on cells freshly isolated from leukemia patients.
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074, Aachen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Cremer
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074, Aachen, Germany.
authors.forename: Hanna
authors.initials: H
authors.lastname: Braun
authors.affiliation: Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074, Aachen, Germany.
authors.forename: Radoslav
authors.initials: R
authors.lastname: Mladenov
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074, Aachen, Germany.
authors.forename: Lea
authors.initials: L
authors.lastname: Schenke
authors.affiliation: Department of Computational Biophysics, German Research School for Simulation Sciences (Joint Venture of RWTH Aachen University and Forschungszentrum Jülich), 52428, Jülich, Germany.|Institute for Advanced Simulations IAS-5, Computational Biomedicine, Forschungszentrum, Jülich, Germany.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Cong
authors.affiliation: Department of Hematology and Oncology (Internal Medicine IV), University Hospital RWTH Aachen, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Edgar
authors.initials: E
authors.lastname: Jost
authors.affiliation: Department of Hematology and Oncology (Internal Medicine IV), University Hospital RWTH Aachen, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Tim H
authors.initials: TH
authors.lastname: Brümmendorf
authors.affiliation: Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074, Aachen, Germany.|Institute for Molecular Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Department of Computational Biophysics, German Research School for Simulation Sciences (Joint Venture of RWTH Aachen University and Forschungszentrum Jülich), 52428, Jülich, Germany.|Institute for Advanced Simulations IAS-5, Computational Biomedicine, Forschungszentrum, Jülich, Germany.
authors.forename: Paolo
authors.initials: P
authors.lastname: Carloni
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074, Aachen, Germany.|South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine (IDM), Anzio Road, Observatory, Cape Town, 7925, South Africa.|Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town, 7925, South Africa.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
authors.affiliation: Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074, Aachen, Germany. nachreiner@hia.rwth-aachen.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Nachreiner
mesh_terms: D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003603:Cytotoxins / Q000235:genetics / Q000633:toxicity; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000473:pathology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000235:genetics* / Q000633:toxicity
chemical_list: D002352:Carrier Proteins; D003603:Cytotoxins; C501467:RNH1 protein, human; C046366:angiogenin; D012259:Ribonuclease, Pancreatic
keywords: Angiogenin; Human cytolytic fusion protein; Leukemia; RNH1; Site-directed mutagenesis; Targeted therapy