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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

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.

Keywords

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

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

Ki

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

Notes

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.

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)

References

  1. 1.
    Weidle UH, Georges G, Brinkmann U (2012) Fully human targeted cytotoxic fusion proteins: new anticancer agents on the horizon. Cancer Genomics Proteomics 9:119–133PubMedGoogle Scholar
  2. 2.
    Monti DM, D’Alessio G (2004) Cytosolic RNase inhibitor only affects RNases with intrinsic cytotoxicity. J Biol Chem 279:39195–39198. doi: 10.1074/jbc.C400311200 CrossRefPubMedGoogle Scholar
  3. 3.
    Leland PA, Raines RT (2001) Cancer chemotherapy–ribonucleases to the rescue. Chem Biol 8:405–413PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Rutkoski TJ, Raines RT (2008) Evasion of ribonuclease inhibitor as a determinant of ribonuclease cytotoxicity. Curr Pharm Biotechnol 9:185–189PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Cremer C, Vierbuchen T, Hein L et al (2015) Angiogenin mutants as novel effector molecules for the generation of fusion proteins with increased cytotoxic potential. J Immunother 38:85–95. doi: 10.1097/CJI.0000000000000053 CrossRefPubMedGoogle Scholar
  6. 6.
    Czech A, Wende S, Morl M et al (2013) Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress. PLoS Genet 9:e1003767. doi: 10.1371/journal.pgen.1003767 PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Pizzo E, Sarcinelli C, Sheng J et al (2013) Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival. J Cell Sci 126:4308–4319. doi: 10.1242/jcs.134551 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Russo N, Shapiro R, Acharya KR et al (1994) Role of glutamine-117 in the ribonucleolytic activity of human angiogenin. Proc Natl Acad Sci USA 91:2920–2924PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Chen CZ, Shapiro R (1999) Superadditive and subadditive effects of “hot spot” mutations within the interfaces of placental ribonuclease inhibitor with angiogenin and ribonuclease A. Biochemistry 38:9273–9285. doi: 10.1021/bi990762a CrossRefPubMedGoogle Scholar
  10. 10.
    Chen CZ, Shapiro R (1997) Site-specific mutagenesis reveals differences in the structural bases for tight binding of RNase inhibitor to angiogenin and RNase A. Proc Natl Acad Sci USA 94:1761–1766PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Acharya KR, Shapiro R, Allen SC et al (1994) Crystal structure of human angiogenin reveals the structural basis for its functional divergence from ribonuclease. Proc Natl Acad Sci USA 91:2915–2919PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Leonidas DD, Shapiro R, Subbarao GV et al (2002) Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency. Biochemistry 41:2552–2562CrossRefPubMedGoogle Scholar
  13. 13.
    de Kruif J, Tijmensen M, Goldsein J et al (2000) Recombinant lipid-tagged antibody fragments as functional cell-surface receptors. Nat Med 6:223–227. doi: 10.1038/72339 CrossRefPubMedGoogle Scholar
  14. 14.
    Graziano RF, Tempest PR, White P et al (1995) Construction and characterization of a humanized anti-gamma-Ig receptor type I (Fc gamma RI) monoclonal antibody. J Immunol 155:4996–5002PubMedGoogle Scholar
  15. 15.
    Ho SN, Hunt HD, Horton RM et al (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59CrossRefPubMedGoogle Scholar
  16. 16.
    Stocker M, Tur MK, Sasse S et al (2003) Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. Protein Expr Purif 28:211–219CrossRefPubMedGoogle Scholar
  17. 17.
    Gallagher R, Collins S, Trujillo J et al (1979) Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54:713–733PubMedGoogle Scholar
  18. 18.
    Diehl V, Kirchner HH, Schaadt M et al (1981) Hodgkin’s disease: establishment and characterization of four in vitro cell lies. J Cancer Res Clin Oncol 101:111–124CrossRefPubMedGoogle Scholar
  19. 19.
    Schiffer S, Hristodorov D, Mladenov R et al (2013) Species-dependent functionality of the human cytolytic fusion proteins granzyme B-H22(scFv) and H22(scFv)-angiogenin in macrophages. Antibodies 2:9–18CrossRefGoogle Scholar
  20. 20.
    Kampmeier F, Ribbert M, Nachreiner T et al (2009) Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase. Bioconjug Chem 20:1010–1015. doi: 10.1021/bc9000257 CrossRefPubMedGoogle Scholar
  21. 21.
    Niesen J, Stein C, Brehm H et al (2015) Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities. J Cancer Res Clin Oncol. doi: 10.1007/s00432-015-1975-5 Google Scholar
  22. 22.
    Nachreiner T, Kampmeier F, Thepen T et al (2008) Depletion of autoreactive B-lymphocytes by a recombinant myelin oligodendrocyte glycoprotein-based immunotoxin. J Neuroimmunol 195:28–35. doi: 10.1016/j.jneuroim.2008.01.001 CrossRefPubMedGoogle Scholar
  23. 23.
    Ribbert T, Thepen T, Tur MK et al (2010) Recombinant, ETA′-based CD64 immunotoxins: improved efficacy by increased valency, both in vitro and in vivo in a chronic cutaneous inflammation model in human CD64 transgenic mice. Br J Dermatol 163:279–286. doi: 10.1111/j.1365-2133.2010.09824.x CrossRefPubMedGoogle Scholar
  24. 24.
    Stöcker M, Pardo A, Hetzel C et al (2008) Eukaryotic expression and secretion of EGFP-labeled annexin A5. Protein Expr Purif 58:325–331. doi: 10.1016/j.pep.2007.12.009 CrossRefPubMedGoogle Scholar
  25. 25.
    Verhoven B, Schlegel RA, Williamson P (1995) Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182:1597–1601CrossRefPubMedGoogle Scholar
  26. 26.
    Krauss J, Arndt MA, Vu BK et al (2005) Targeting malignant B-cell lymphoma with a humanized anti-CD22 scFv-angiogenin immunoenzyme. Br J Haematol 128:602–609. doi: 10.1111/j.1365-2141.2005.05356.x CrossRefPubMedGoogle Scholar
  27. 27.
    Arndt MA, Krauss J, Vu BK et al (2005) A dimeric angiogenin immunofusion protein mediates selective toxicity toward CD22+ tumor cells. J Immunother 28:245–251CrossRefPubMedGoogle Scholar
  28. 28.
    Hendrzak JA, Wallace PK, Morahan PS (1994) Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages. Cytometry 17:349–356. doi: 10.1002/cyto.990170412 CrossRefPubMedGoogle Scholar
  29. 29.
    Leonidas DD, Shapiro R, Allen SC et al (1999) Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth. J Mol Biol 285:1209–1233. doi: 10.1006/jmbi.1998.2378 CrossRefPubMedGoogle Scholar
  30. 30.
    Dickson KA, Kang DK, Kwon YS et al (2009) Ribonuclease inhibitor regulates neovascularization by human angiogenin. Biochemistry 48:3804–3806. doi: 10.1021/bi9005094 PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Stahnke B, Thepen T, Stocker M et al (2008) Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes. Mol Cancer Ther 7:2924–2932. doi: 10.1158/1535-7163.MCT-08-0554 CrossRefPubMedGoogle Scholar
  32. 32.
    Schiffer S, Letzian S, Jost E et al (2013) Granzyme M as a novel effector molecule for human cytolytic fusion proteins: CD64-specific cytotoxicity of Gm-H22(scFv) against leukemic cells. Cancer Lett 341:178–185. doi: 10.1016/j.canlet.2013.08.005 CrossRefPubMedGoogle Scholar
  33. 33.
    Dunphy CH, Tang W (2007) The value of CD64 expression in distinguishing acute myeloid leukemia with monocytic differentiation from other subtypes of acute myeloid leukemia: a flow cytometric analysis of 64 cases. Arch Pathol Lab Med 131:748–754. doi:10.1043/1543-2165(2007)131[748:TVOCEI]2.0.CO;2PubMedGoogle Scholar
  34. 34.
    Santos IM, Franzon CM, Koga AH (2012) Laboratory diagnosis of chronic myelomonocytic leukemia and progression to acute leukemia in association with chronic lymphocytic leukemia: morphological features and immunophenotypic profile. Rev Bras Hematol Hemoter 34:242–244. doi: 10.5581/1516-8484.20120058 PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Schiffer S, Rosinke R, Jost E et al (2014) Targeted ex vivo reduction of CD64-positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B-based cytolytic fusion proteins. Int J Cancer 135:1497–1508. doi: 10.1002/ijc.28786 CrossRefPubMedGoogle Scholar
  36. 36.
    Karehed K, Dimberg A, Dahl S et al (2007) IFN-gamma-induced upregulation of Fcgamma-receptor-I during activation of monocytic cells requires the PKR and NFkappaB pathways. Mol Immunol 44:615–624. doi: 10.1016/j.molimm.2006.01.013 CrossRefPubMedGoogle Scholar
  37. 37.
    Wei H, Bera TK, Wayne AS et al (2013) A modified form of diphthamide causes immunotoxin resistance in a lymphoma cell line with a deletion of the WDR85 gene. J Biol Chem 288:12305–12312. doi: 10.1074/jbc.M113.461343 PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Conway O’Brien E, Prideaux S, Chevassut T (2014) The epigenetic landscape of acute myeloid leukemia. Adv Hematol. 2014:103175. doi: 10.1155/2014/103175 PubMedCentralPubMedGoogle Scholar
  39. 39.
    Jankowska AM, Makishima H, Tiu RV et al (2011) Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood 118:3932–3941. doi: 10.1182/blood-2010-10-311019 PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Schnerch D, Yalcintepe J, Schmidts A et al (2012) Cell cycle control in acute myeloid leukemia. Am J Cancer Res 2:508–528PubMedCentralPubMedGoogle Scholar
  41. 41.
    Scholl C, Gilliland DG, Frohling S (2008) Deregulation of signaling pathways in acute myeloid leukemia. Semin Oncol 35:336–345. doi: 10.1053/j.seminoncol.2008.04.004 CrossRefPubMedGoogle Scholar
  42. 42.
    Gao X, Xu Z (2008) Mechanisms of action of angiogenin. Acta Biochim Biophys Sin (Shanghai) 40:619–624CrossRefGoogle Scholar
  43. 43.
    Lu Z, Xu S (2006) ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life 58:621–631. doi: 10.1080/15216540600957438 CrossRefPubMedGoogle Scholar
  44. 44.
    Peng Y, Li L, Huang M et al (2014) Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells. Cell Signal 26:2782–2792. doi: 10.1016/j.cellsig.2014.08.021 CrossRefPubMedGoogle Scholar
  45. 45.
    Sadagopan S, Veettil MV, Chakraborty S et al (2012) Angiogenin functionally interacts with p53 and regulates p53-mediated apoptosis and cell survival. Oncogene 31:4835–4847. doi: 10.1038/onc.2011.648 PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Ivanov P, Emara MM, Villen J et al (2011) Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 43:613–623. doi: 10.1016/j.molcel.2011.06.022 PubMedCentralCrossRefPubMedGoogle Scholar

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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