Cancer Immunology, Immunotherapy

, Volume 64, Issue 12, pp 1575–1586 | Cite as

Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo

  • Christian Cremer
  • Hanna Braun
  • Radoslav Mladenov
  • Lea Schenke
  • Xiaojing Cong
  • Edgar Jost
  • Tim H. Brümmendorf
  • Rainer Fischer
  • Paolo Carloni
  • Stefan Barth
  • Thomas Nachreiner
Original Article


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.


Angiogenin RNH1 Human cytolytic fusion protein Site-directed mutagenesis Targeted therapy Leukemia 



Acute myeloid leukemia




Chronic myelomonocytic leukemia


Deoxyribonucleic acid


Half maximal effective concentration


Granzyme B


Green fluorescent protein


Human cytolytic fusion protein


Human interferon gamma


Human pro-inflammatory macrophages


Human embryonic kidney cells


Immobilized metal ion affinity chromatography


Inhibitory constant


Myelin oligodendrocyte glycoprotein


Peripheral blood mononuclear cell


Polymerase chain reaction


Propidium iodide


Ribonucleic acid


Ribonuclease/angiogenin inhibitor 1


Roswell Park Memorial Institute


Standard error of the mean


Splicing by overlap extension


Transfer RNA


tRNA-derived stress-induced RNA





This project was funded by the Deutsche Forschungsgemeinschaft (DFG). The authors would like to thank Dr. Christoph Stein (Institute for Applied Medical Engineering, University Hospital RWTH Aachen/Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of Pharmaceutical Product Development, Aachen) for helpful discussions on leukemia specimen handling, Judith Niesen (Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of Pharmaceutical Product Development, Aachen) for providing the fusion protein 2112(scFv)-Ang GGRRmut, Anh-Tuah Pham (Institute for Applied Medical Engineering, University Hospital RWTH Aachen) for providing her2(scFv)-Ang GGRRmut and Fanny Frenzel (University Hospital RWTH Aachen, Department of Hematology and Oncology, Internal Medicine IV, Aachen, Germany) for providing patient data and specimens. Finally, we are very grateful to Dr. Richard M Twyman for critically reading this manuscript. Radoslav Mladenov was supported by a scholarship from the Jürgen Manchot Foundation.

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to disclose.

Supplementary material

262_2015_1763_MOESM1_ESM.pdf (240 kb)
Supplementary material 1 (PDF 239 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Christian Cremer
    • 1
  • Hanna Braun
    • 1
  • Radoslav Mladenov
    • 4
  • Lea Schenke
    • 1
  • Xiaojing Cong
    • 2
    • 3
  • Edgar Jost
    • 5
  • Tim H. Brümmendorf
    • 5
  • Rainer Fischer
    • 4
    • 6
  • Paolo Carloni
    • 2
    • 3
  • Stefan Barth
    • 1
    • 7
    • 8
  • Thomas Nachreiner
    • 1
  1. 1.Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical EngineeringUniversity Hospital RWTH AachenAachenGermany
  2. 2.Department of Computational BiophysicsGerman Research School for Simulation Sciences (Joint Venture of RWTH Aachen University and Forschungszentrum Jülich)JülichGermany
  3. 3.Institute for Advanced Simulations IAS-5Computational BiomedicineJülichGermany
  4. 4.Department of Pharmaceutical Product DevelopmentFraunhofer Institute for Molecular Biology and Applied EcologyAachenGermany
  5. 5.Department of Hematology and Oncology (Internal Medicine IV)University Hospital RWTH AachenAachenGermany
  6. 6.Institute for Molecular BiotechnologyRWTH Aachen UniversityAachenGermany
  7. 7.South African Research Chair in Cancer BiotechnologyInstitute of Infectious Disease and Molecular Medicine (IDM)Cape TownSouth Africa
  8. 8.Department of Integrative Biomedical Sciences, Faculty of Health SciencesUniversity of Cape TownCape TownSouth Africa

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