Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

  • Christian Cremer
  • Grit Hehmann-Titt
  • Sonja Schiffer
  • Georg Melmer
  • Paolo Carloni
  • Stefan Barth
  • Thomas Nachreiner
Part of the Resistance to Targeted Anti-Cancer Therapeutics book series (RTACT, volume 6)


The use of therapies based on antibody fusion proteins for the selective elimination of tumor cells has increased markedly over the last two decades because the severe side effects associated with conventional chemotherapy and radiotherapy are reduced or even eliminated. However, the initial development of immunotoxins suffered from a number of drawbacks such as nonspecific cytotoxicity and the induction of immune responses because the components were non-human in origin. The most recent iteration of this approach is a new class of targeted human cytolytic fusion proteins (hCFPs) comprising a tumor-specific targeting component such as a human antibody fragment fused to a human effector domain with pro-apoptotic activity. Certain tumors resist the activity of hCFPs by upregulating the intracellular expression of native inhibitors, which rapidly bind and inactivate the human effector domains. Higher doses of the hCFPs are, therefore, required to improve therapeutic efficacy. To circumvent these inhibitory processes, novel isoforms of the enzymes granzyme B and angiogenin have been designed to increase their intrinsic activity and reduce their interactions with native inhibitors resulting in more potent hCFPs that can be applied at lower doses. This chapter summarizes the basic scientific knowledge that can facilitate the rational development of human enzymes with novel and beneficial characteristics, including the ability to avoid neutralization by native inhibitors.


Targeted therapy Human cytolytic fusion protein Apoptosis Effector domain Angiogenin Granzyme B Tumor-specific binding domain Natural inhibitor Serpin B9 PI-9 RNH1 





Antibody drug conjugate


Amyotrophic lateral sclerosis


Acute myeloid leukemia


Acute myelomonocytic leukemia


Apoptoticproteaseactivatingfactor 1


Annexin V


BH3 interacting domain death agonist


Computer-aided simulation modeling


Chronic myelomonocytic leukemia


Central nervous system


Cytotoxic T lymphocyte

Cyt c

Cytochrome C


Death-associated proteinkinase 2


Deoxyadenosine triphosphat


Dendritic cell


DNA fragmentation factor-45


Deutsche Forschungsgemeinschaft






DNA methyltransferase 2


Dipeptidyl peptidase 1


Epstein-Barr virus


Half maximal effective concentration


European Fund for Regional Development




Pseudomonas aeruginosaexotoxin A


Truncated version of the Pseudomonas aeruginosa exotoxin A


Food and Drug Administration


Granzyme B


Human anti-mouse antibody


Human cytolytic fusion protein


Human embryonic kidney


Human luteinizing hormone receptor


Inhibitor of caspase-activated DNase






Internal ribosome entrysite


Lewis Y antigen




Mitochondrial outer membrane permeabilization


Messenger RNA


mRNA-based ribonucleoproteins


Natural killer cells


Nuclear localization signal


North-Rhine Westphalia


Nuclear mitotic apparatus protein


Poly (ADP-ribose) polymerase


Polyethylene glycol


Propidium iodide

PI 9

Proteinase inhibitor-9


Rat pituitary gland


Reactive center loop


RNA-induced silencing complex


Ribonucleic acid


RNA interference


Ribonuclease/angiogen ininhibitor 1


Roswell Park Memorial Instiute


Ribosomal RNA


Single chain fragment variable


Sodium dodecyl sulfate


tRNA-derived stress-induced RNA


Tumor necrosis factor


Transfer RNA


X-linked inhibitor of apoptosis protein





This work was funded in part by a grant from the Germany province NRW from EFRE “European Fund for Regional Development” under the theme “Europe—Investment in our Future” and by grant BA 1772/18-1 from the Deutsche Forschungsgemeinschaft (DFG). The authors would like to thank Valeria Losasso and Xiaojing Cong for in silico simulations and Richard M. Twyman for critically reading the manuscript.


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Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Christian Cremer
    • 1
  • Grit Hehmann-Titt
    • 2
  • Sonja Schiffer
    • 1
  • Georg Melmer
    • 2
  • Paolo Carloni
    • 3
  • Stefan Barth
    • 1
    • 4
  • Thomas Nachreiner
    • 1
  1. 1.Department of Pharmaceutical Product DevelopmentFraunhofer-Institute for Molecular Biology and Applied EcologyAachenGermany
  2. 2.Pharmedartis GmbHAachenGermany
  3. 3.Computational BiophysicsGermany Research School for Simulation SciencesJülichGermany
  4. 4.Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical EngineeringUniversity Hospital RWTH AachenAachenGermany

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