VHH-Photosensitizer Conjugates for Targeted Photodynamic Therapy of Met-Overexpressing Tumor Cells.

Raimond Heukers,Paul Van Bergen En Henegouwen,Vida Mashayekhi,Mercedes Ramirez-Escudero,Hans De Haard,Theo Verrips,Sabrina Oliveira

doi:10.3390/antib8020026

Abstract

Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.

Highlights

Photodynamic therapy (PDT) is a type of cancer treatment in which tumor cells are killed by reactive oxygen species, such as singlet oxygen, formed by the local and light-induced activation of a photosensitizer (PS) [1]
We have described the nanobody-PS or VHH-PS conjugates targeting the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) that is found overexpressed on a large variety of cancers, such as head and neck, lung, or colon cancer [17,18]
Three previously selected VHHs recognizing the Met ectodomain were taken for further characterization, i.e., E9, G2 and F5 [25]

Summary

Introduction

Photodynamic therapy (PDT) is a type of cancer treatment in which tumor cells are killed by reactive oxygen species, such as singlet oxygen, formed by the local and light-induced activation of a photosensitizer (PS) [1]. By locally reacting with proteins, lipids, and nucleic acids, the reactive oxygen species generated can induce cell death, vascular damage, and an inflammatory response [2]. It is for this mode of action that PDT is used in clinic to treat malignancies. The PSs generally used in the clinic are relatively hydrophobic, are systemically applied, and are non-targeted. These factors combined can result in off-target toxicity and long lasting photosensitivity [2,3].

Methods

Results

Conclusion