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

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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.

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Abbreviations

AML:

Acute myeloid leukemia

Ang:

Angiogenin

CMML:

Chronic myelomonocytic leukemia

DNA:

Deoxyribonucleic acid

EC50 :

Half maximal effective concentration

Gb:

Granzyme B

GFP:

Green fluorescent protein

hCFP:

Human cytolytic fusion protein

hIFNγ:

Human interferon gamma

hM1Φ:

Human pro-inflammatory macrophages

HEK293T:

Human embryonic kidney cells

IMAC:

Immobilized metal ion affinity chromatography

K i :

Inhibitory constant

MOG:

Myelin oligodendrocyte glycoprotein

PBMC:

Peripheral blood mononuclear cell

PCR:

Polymerase chain reaction

PI:

Propidium iodide

RNA:

Ribonucleic acid

RNH1:

Ribonuclease/angiogenin inhibitor 1

RPMI:

Roswell Park Memorial Institute

SEM:

Standard error of the mean

SOE:

Splicing by overlap extension

tRNA:

Transfer RNA

tiRNA:

tRNA-derived stress-induced RNA

XTT:

2,3-bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

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Acknowledgments

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.

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    Authors and Affiliations

    1. Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074, Aachen, Germany

      Christian Cremer, Hanna Braun, Lea Schenke, Stefan Barth & Thomas Nachreiner

    2. Department of Computational Biophysics, German Research School for Simulation Sciences (Joint Venture of RWTH Aachen University and Forschungszentrum Jülich), 52428, Jülich, Germany

      Xiaojing Cong & Paolo Carloni

    3. Institute for Advanced Simulations IAS-5, Computational Biomedicine, Forschungszentrum, Jülich, Germany

      Xiaojing Cong & Paolo Carloni

    4. Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074, Aachen, Germany

      Radoslav Mladenov & Rainer Fischer

    5. Department of Hematology and Oncology (Internal Medicine IV), University Hospital RWTH Aachen, Pauwelsstr. 30, 52074, Aachen, Germany

      Edgar Jost & Tim H. Brümmendorf

    6. Institute for Molecular Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany

      Rainer Fischer

    7. South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine (IDM), Anzio Road, Observatory, Cape Town, 7925, South Africa

      Stefan Barth

    8. Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town, 7925, South Africa

      Stefan Barth

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    1. Christian Cremer
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    Correspondence to Thomas Nachreiner.

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    Stefan Barth and Thomas Nachreiner contributed equally to this manuscript.

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    Cremer, C., Braun, H., Mladenov, R. et al. Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo. Cancer Immunol Immunother 64, 1575–1586 (2015). https://doi.org/10.1007/s00262-015-1763-8

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