Site-Specific Dual-Labeling of a VHH with a Chelator and a Photosensitizer for Nuclear Imaging and Targeted Photodynamic Therapy of EGFR-Positive Tumors.

Emma Renard,Sanne A M Van Lith,Martin Gotthardt,Estel Collado Camps,Franck Denat,Victor Goncalves,Mark Rijpkema,Coline Canovas,Annemarie Kip

doi:10.3390/cancers13030428

Abstract

Simple SummaryVariable domains of heavy chain only antibodies are small proteins that can be used for tumor imaging and therapy upon conjugation of functional groups. As frequently used random conjugation techniques can decrease binding to the target of interest, site-specific conjugation of these functional groups is preferred. Here, we optimized site-specific conjugation of both a chelator for binding of a radiometal and a photosensitizer to epidermal growth factor receptor (EGFR) binding VHH 7D12. We characterized this dual-labeled VHH for nuclear imaging and targeted photodynamic therapy of EGFR-expressing tumors.Variable domains of heavy chain only antibodies (VHHs) are valuable agents for application in tumor theranostics upon conjugation to both a diagnostic probe and a therapeutic compound. Here, we optimized site-specific conjugation of the chelator DTPA and the photosensitizer IRDye700DX to anti-epidermal growth factor receptor (EGFR) VHH 7D12, for applications in nuclear imaging and photodynamic therapy. 7D12 was site-specifically equipped with bimodal probe DTPA-tetrazine-IRDye700DX using the dichlorotetrazine conjugation platform. Binding, internalization and light-induced toxicity of DTPA-IRDye700DX-7D12 were determined using EGFR-overexpressing A431 cells. Finally, ex vivo biodistribution of DTPA-IRDye700DX-7D12 in A431 tumor-bearing mice was performed, and tumor homing was visualized with SPECT and fluorescence imaging. DTPA-IRDye700DX-7D12 was retrieved with a protein recovery of 43%, and a degree of labeling of 0.56. Spectral properties of the IRDye700DX were retained upon conjugation. 111In-labeled DTPA-IRDye700DX-7D12 bound specifically to A431 cells, and they were effectively killed upon illumination. DTPA-IRDye700DX-7D12 homed to A431 xenografts in vivo, and this could be visualized with both SPECT and fluorescence imaging. In conclusion, the dichlorotetrazine platform offers a feasible method for site-specific dual-labeling of VHH 7D12, retaining binding affinity and therapeutic efficacy. The flexibility of the described approach makes it easy to vary the nature of the probes for other combinations of diagnostic and therapeutic compounds.

Highlights

The variable domains of heavy chain only antibodies (VHHs or nanobodies®) were discovered in the early 1990s
We evaluated the function of the Variable domains of heavy chain only antibodies (VHHs) for single photon emission computed tomography (SPECT) imaging and targeted photodynamic therapy (tPDT) of epidermal growth factor receptor (EGFR)-expressing tumors
The cysteine residue introduced at the C-terminus of anti-EGFR VHH 7D12 was equipped with BCN-PEG2-maleimide to ensure selective labeling with the bimodal tetrazine probe

Summary

Introduction

The variable domains of heavy chain only antibodies (VHHs or nanobodies®) were discovered in the early 1990s They are an interesting class of molecules for tumor imaging and therapy, mainly because of their small size (15 kDa versus 150 kDa for monoclonal antibodies) and fast clearance, leading to efficient tissue penetration [1] and good tumorto-background ratios at early time points post injection [2,3,4,5,6,7,8,9,10]. Sitespecific labeling was shown to lead to preferable pharmacokinetics and tumor targeting potential [12]

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Discussion

Conclusion