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

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Resistance to Immunotoxins in Cancer Therapy

Part of the book series: Resistance to Targeted Anti-Cancer Therapeutics ((RTACT,volume 6))

Abstract

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.

These authors contributed equally to this work

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Abbreviations

Å:

Angström

ADC:

Antibody drug conjugate

ALS:

Amyotrophic lateral sclerosis

AML:

Acute myeloid leukemia

AMML:

Acute myelomonocytic leukemia

APAF 1:

Apoptoticproteaseactivatingfactor 1

AV:

Annexin V

BID:

BH3 interacting domain death agonist

CASM:

Computer-aided simulation modeling

CMML:

Chronic myelomonocytic leukemia

CNS:

Central nervous system

CTL:

Cytotoxic T lymphocyte

Cyt c:

Cytochrome C

DAPK2:

Death-associated proteinkinase 2

dATP:

Deoxyadenosine triphosphat

DC:

Dendritic cell

DFF45:

DNA fragmentation factor-45

DFG:

Deutsche Forschungsgemeinschaft

DNA:

Deoxyribonucleicacid

DNA PK:

DNA-dependentproteinkinase

DNMT2:

DNA methyltransferase 2

DPPI:

Dipeptidyl peptidase 1

EBV:

Epstein-Barr virus

EC50:

Half maximal effective concentration

EFRE:

European Fund for Regional Development

ER:

Endoplasmicreticulum

ETA:

Pseudomonas aeruginosaexotoxin A

ETA’:

Truncated version of the Pseudomonas aeruginosa exotoxin A

FDA:

Food and Drug Administration

GrB:

Granzyme B

HAMA:

Human anti-mouse antibody

hCFP:

Human cytolytic fusion protein

HEK:

Human embryonic kidney

hLHR:

Human luteinizing hormone receptor

ICAD:

Inhibitor of caspase-activated DNase

IFN:

Interferon

IL:

Interleukin

IRES:

Internal ribosome entrysite

LeY:

Lewis Y antigen

LPS:

Lipopolysaccharide

MOMP:

Mitochondrial outer membrane permeabilization

mRNA:

Messenger RNA

mRNP:

mRNA-based ribonucleoproteins

NKcells:

Natural killer cells

NLS:

Nuclear localization signal

NRW:

North-Rhine Westphalia

NuMA:

Nuclear mitotic apparatus protein

PARP:

Poly (ADP-ribose) polymerase

PEG:

Polyethylene glycol

PI:

Propidium iodide

PI 9:

Proteinase inhibitor-9

raPIT5a:

Rat pituitary gland

RCL:

Reactive center loop

RISC:

RNA-induced silencing complex

RNA:

Ribonucleic acid

RNAi:

RNA interference

RNH1:

Ribonuclease/angiogen ininhibitor 1

RPMI:

Roswell Park Memorial Instiute

rRNA:

Ribosomal RNA

scFv:

Single chain fragment variable

SDS:

Sodium dodecyl sulfate

tiRNA:

tRNA-derived stress-induced RNA

TNF:

Tumor necrosis factor

tRNA:

Transfer RNA

XIAP:

X-linked inhibitor of apoptosis protein

XTT:

2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide

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Acknowledgements

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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Authors and Affiliations

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

    Christian Cremer, Sonja Schiffer, Stefan Barth & Thomas Nachreiner

  2. Pharmedartis GmbH, Forckenbeckstr. 6, 52074, Aachen, Germany

    Grit Hehmann-Titt & Georg Melmer

  3. Computational Biophysics, Germany Research School for Simulation Sciences, Wilhelm-Johnen-Straße, 52428, Jülich, Germany

    Paolo Carloni

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

    Stefan Barth

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  1. Christian Cremer
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Correspondence to Stefan Barth .

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  1. Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India

    Rama Shanker Verma

  2. Department of Microbiology, Immunology,, University of California of Los Angeles, Los Angeles, California, USA

    Benjamin Bonavida

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Cremer, C. et al. (2015). Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins. In: Verma, R., Bonavida, B. (eds) Resistance to Immunotoxins in Cancer Therapy. Resistance to Targeted Anti-Cancer Therapeutics, vol 6. Springer, Cham. https://doi.org/10.1007/978-3-319-17275-0_8

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