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Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

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Abstract

Trastuzumab (Herceptin®), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.

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Abbreviations

HER2/neu :

Human epidermal growth factor receptor 2

ADCC:

Antibody-dependent cell-mediated cytotoxicity

ADCP:

Antibody-dependent cell-mediated phagocytosis

FBP:

Folate binding protein

sFcεRIα:

Soluble FcεRI α-chain

ECDHER2 :

HER2 protein extracellular domain

CM:

Complete medium

PI:

Propidium iodide

CFSE:

Carboxy-fluorescein diacetate, succinimidyl ester

NIP:

4-Hydroxy-3-nitro-phenacetyl

PI3K:

Phosphoinositide 3-kinase

TGF-a:

Tumour growth factor α

VEGF:

Vascular endothelial growth factor

TNF-α:

Tumour necrosis factor-α

MTS:

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt)

PMS:

Phenazine methosulfate

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Acknowledgments

This work was generously supported by the the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) at Guy’s and St. Thomas’ NHS Foundation Trust/King’s College London, United Kingdom; the Austrian Science Fund (FWF) (P-18238-B13); the European Molecular Biology Organisation (EMBO) (fellowship ASTF258.00-2008); Hans und Blanca Moser Stiftung (AP00326OFF), Austria; NIH/NCI R01 supplement CA107023-02S1, Susan G. Komen Breast Cancer Foundation grant (BCTR0706771) and the 2007–2008 University of California Cancer Research Coordinating Committee seed grant, USA. We thank Dr. Rebecca Beavil, Dr. Pooja Takhar and Mr. Richard Brunner for their helpful comments and Ms. Kate Kirwan for expert assistance with the figures. We are grateful to Dr. Jean-Pierre Kinet and to Dr. Silvana Canevari for the generous provision of advice and materials.

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Authors and Affiliations

  1. Cutaneous Medicine and Immunotherapy Unit, St. John’s Institute of Dermatology, Division of Genetics and Molecular Medicine, King’s College London School of Medicine, 9th Floor, Guy’s Tower, Guy’s Hospital, London, SE1 9RT, UK

    Panagiotis Karagiannis, Frank O. Nestle & Sophia N. Karagiannis

  2. Randall Division of Cell and Molecular Biophysics, King’s College London, 3rd Floor, New Hunt’s House, Guy’s Campus, London, SE1 1UL, UK

    James Hunt, Samuel K. E. Gan, Philip S. Hobson, Andrew J. Beavil & Hannah J. Gould

  3. Department of Academic Oncology, King’s College London, 3rd Floor, Bermondsey Wing, Guy’s Hospital, London, SE1 9RT, UK

    Sarah M. Rudman & James Spicer

  4. Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, USA

    Tracy R. Daniels & Manuel L. Penichet

  5. Departments of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, USA

    Manuel L. Penichet

  6. The Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, USA

    Manuel L. Penichet

  7. IPP, Department of Pathophysiology, Center of Physiology, Pathophysiology and Immunology, Medical University Vienna, Waehringer G. 18, 1090, Vienna, Austria

    Panagiotis Karagiannis, Josef Singer, Diana Mechtcheriakova, Regina Knittelfelder & Erika Jensen-Jarolim

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  1. Panagiotis Karagiannis
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  2. Josef Singer
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  3. James Hunt
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  4. Samuel K. E. Gan
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  7. Regina Knittelfelder
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  8. Tracy R. Daniels
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  9. Philip S. Hobson
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  13. Manuel L. Penichet
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  14. Hannah J. Gould
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  15. Erika Jensen-Jarolim
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  16. Sophia N. Karagiannis
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Correspondence to Sophia N. Karagiannis.

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Panagiotis Karagiannis and Josef Singer contributed equally to this work.

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Amino Acid Sequence of Trastuzumab IgE (PDF 67 kb)

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Karagiannis, P., Singer, J., Hunt, J. et al. Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells. Cancer Immunol Immunother 58, 915–930 (2009). https://doi.org/10.1007/s00262-008-0607-1

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