Nanobody–photosensitizer conjugates for targeted photodynamic therapy

doi:10.1016/j.nano.2013.12.007

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 10, Issue 7, October 2014, Pages 1441-1451

https://doi.org/10.1016/j.nano.2013.12.007 Get rights and content

Abstract

Photodynamic therapy (PDT) induces cell death through light activation of a photosensitizer (PS). Targeted delivery of PS via monoclonal antibodies has improved tumor selectivity. However, these conjugates have long half-lives, leading to relatively long photosensitivity in patients. In an attempt to target PS specifically to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies (NB) targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). These fluorescent conjugates allow the distinction of cell lines with different expression levels of EGFR. Results show that these conjugates specifically induce cell death of EGFR overexpressing cells in low nanomolar concentrations, while PS alone or the NB–PS conjugates in the absence of light induce no toxicity. Delivery of PS using internalizing biparatopic NB–PS conjugates results in even more pronounced phototoxicities. Altogether, EGFR-targeted NB–PS conjugates are specific and potent, enabling the combination of molecular imaging with cancer therapy.

From the Clinical Editor

This study investigates the role of EGFR targeting nanobodies to deliver traceable photosensitizers to cancer molecules for therapeutic exploitation and concomitant imaging. Altogether, EGFR-targeted NB–PS conjugates combine molecular imaging with cancer therapy, the method is specific and potent, paving the way to clinical application of this technology.

Graphical Abstract

Conjugates consisting of nanobodies (NB) targeting the epidermal growth factor receptor (EGFR) and a traceable photosensitizer (PS) are here described and evaluated in vitro. EGFR-targeted NB–PS conjugates retain their binding specificity after PS conjugation and the biparatopic NB leads to a more effective intracellular delivery of PS. These NB–PS conjugates specifically induce cell death of EGFR overexpressing cells, in the low nanomolar concentration, while PS alone or the NB–PS conjugates in the absence of light induce no toxicity. These new NB–PS conjugates are promising in the field of targeted photodynamic therapy (PDT).

Section snippets

Nanobodies and PS conjugation

Nanobodies (NBs) 7D12, R2, and 7D12-9G8 were produced as described in the Supplementary Materials. The photosensitizer IRDye700DX (here named PS) was purchased from LI-COR (LI-COR Biosciences, Lincoln, Nebraska) as an N-hydroxysuccinimidine (NHS) ester. Conjugation of the PS to the NBs, purification and characterization of the NB–PS conjugates were performed as described in the Supplementary Materials.

Cell lines and culture conditions

The mouse fibroblast cell line NIH 3T3 2.2 (abbreviated 3T3 2.2) was described in Ref. ³⁷; the

Production and characterization of NB–PS conjugates

The EGFR-specific NB 7D12 is employed for targeting the PS to EGFR expressing cells for PDT, and the non-relevant NB R2 is used as a negative control (Figure 1, A). In addition, the internalizing biparatopic NB 7D12-9G8 is used to investigate whether more effective internalization of PS would further increase the toxicity of these conjugates. Similarly to the previous study,²⁸ NBs were conjugated to the PS (IRDye700DX) via random NHS-mediated coupling to lysine amino acids. After purification,

Discussion

Over the years, many efforts have been made in the field of photodynamic therapy (PDT) to target photosensitizers (PS) specifically to the site of interest (i.e. tumor).11, 12, 13, 14 Particularly in the context of photoimmunotherapy (PIT), monoclonal antibodies (mAbs) have been employed for this purpose. However, these conjugates have long half-lives and, consequently, antibody fragments have been used for more rapid and efficient clearance of PS from blood and normal tissues. In this study,

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Citation Excerpt :
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: Note: S. Oliveira is supported by the STW-NWO Veni grant number (11878). R. Heukers is supported by the Focus & Massa project of the Utrecht University and by a grant from arGEN-X, Gent, Belgium. The authors declare no conflict of interest.

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Original ArticleNanobody–photosensitizer conjugates for targeted photodynamic therapy

Abstract

From the Clinical Editor

Graphical Abstract

Section snippets

Nanobodies and PS conjugation

Cell lines and culture conditions

Production and characterization of NB–PS conjugates

Discussion

Photodiagnosis Photodyn Ther

Adv Drug Deliv Rev

Adv Drug Deliv Rev

Adv Drug Deliv Rev

Cancer Lett

Structure

J Biol Chem

Exp Cell Res

J Control Release

Toxicol Appl Pharmacol

J Biol Chem

Ueber die Wirkung fluorescirender Stoffe auf Infusorien

Z Biol

Therapeutische versuche mit floureszierenden stoffen

Muench Med Wschrf

Die Sensibilisierende Wirkung Fluorescierender Substanzer. Untersuchungen Uber die Photodynamische Erscheinung

FCW Vogel Leipzig

Photodynamic therapy

J Natl Cancer Inst

The history of photodetection and photodynamic therapy

Photochem Photobiol

Photodynamic therapy of cancer: an update

CA Cancer J Clin

The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen

Photochem Photobiol

Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy

Photochem Photobiol

Preclinical examination of first and second generation photosensitizers used in photodynamic therapy

Photochem Photobiol

Photosensitiser delivery for photodynamic therapy. Part 2: systemic carrier platforms

Expert Opin Drug Deliv

Development of meta-tetrahydroxyphenylchlorin-monoclonal antibody conjugates for photoimmunotherapy

Cancer Res

Preparation, phototoxicity and biodistribution studies of anti-carcinoembryonic antigen monoclonal antibody–phthalocyanine conjugates

Photochem Photobiol

Epidermal growth factor receptor-targeted immunophotodiagnosis and photoimmunotherapy of oral precancer in vivo

Cancer Res

Biodistribution of charged F(ab′)2 photoimmunoconjugates in a xenograft model of ovarian cancer

Br J Cancer

Original Article
Nanobody–photosensitizer conjugates for targeted photodynamic therapy