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. 2019 Jul 1;16(7):3145-3156.
doi: 10.1021/acs.molpharmaceut.9b00360. Epub 2019 Jun 19.

Nanobody-Targeted Photodynamic Therapy Selectively Kills Viral GPCR-Expressing Glioblastoma Cells

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Free PMC article

Nanobody-Targeted Photodynamic Therapy Selectively Kills Viral GPCR-Expressing Glioblastoma Cells

Timo W M De Groof et al. Mol Pharm. .
Free PMC article

Abstract

Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with near-infrared light. One of the aspects hampering the clinical use of PDT is the poor selectivity of the photosensitizer. To improve this, we have recently introduced a new approach for targeted PDT by conjugating photosensitizers to nanobodies. Diverse G protein-coupled receptors (GPCRs) show aberrant overexpression in tumors and are therefore interesting targets in cancer therapy. Here we show that GPCR-targeting nanobodies can be used in targeted PDT. We have developed a nanobody binding the extracellular side of the viral GPCR US28, which is detected in tumors like glioblastoma. The nanobody was site-directionally conjugated to the water-soluble photosensitizer IRDye700DX. This nanobody-photosensitizer conjugate selectively killed US28-expressing glioblastoma cells both in 2D and 3D cultures upon illumination with near-infrared light. This is the first example employing a GPCR as target for nanobody-directed PDT. With the emerging role of GPCRs in cancer, this data provides a new angle for exploiting this large family of receptors for targeted therapies.

Keywords: G protein-coupled receptors; US28; cancer; glioblastoma; nanobody; photodynamic therapy; targeted photosensitizer.

Conflict of interest statement

The authors declare the following competing financial interest(s): R.H. is affiliated with QVQ Holding BV, a company offering VHH services and VHH-based imaging molecules. All other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
VUN100 binds the HCMV-encoded US28 with high affinity. (A) Binding of the nanobodies VUN100 and US28 Nb to US28-expressing membranes, as determined by ELISA. (B,C) Displacement of 125I-CCL5 (B) and 125I-CX3CL1 (C) from US28-expressing membranes by unlabeled ligand or the nanobodies VUN100 and US28 Nb. (D) Effect of nanobodies on the US28-mediated phospholipase C activation. No Nb, No nanobody; Irr. Nb, Irrelevant nanobody; biv. US28 Nb, bivalent US28 nanobody.
Figure 2
Figure 2
VUN100 binds to the N-terminus and ECL3 loop of US28. (A) Immunofluorescence microscopy of the binding of VUN100 to Mock, US28 wild-type (US28 WT), N-terminus truncated US28 (US28-Δ2-22), and US28 with mutation of the tyrosine at position 16 to a phenylalanine (US28 Y16F). US28 was detected using an anti-US28 antibody (US28). VUN100 binding was detected using the Myc-tag and an anti-Myc antibody (VUN100). (B) Detection of surface and total expression of HA-tagged US28 wildtype (US28 WT) and HA-tagged US28 chimeras with the CL1–3 loop being substituted by the corresponding loops of CCR5. Receptor expression was detected by the N-terminal HA-tag. (C) Immunofluorescence microscopy of the binding of VUN100 to Mock transfected or US28 chimeras with the ECL1–3 loop being substituted by the corresponding loops of CCR5. US28 was detected using an anti-US28 antibody (US28). VUN100 binding was detected using the Myc-tag and an anti-Myc antibody (VUN100). (D) Binding ELISA of different concentrations of VUN100 to membranes of HEK293T cells transfected with wild-type US28 (WT), US28 Y16F (Y16F), US28 ECL1-CCR5 chimera (ECL1), US28 ECL3-CCR5 chimera (ECL3), and US28-Δ2-22 (Δ2-22).
Figure 3
Figure 3
VUN100 binds US28 in glioblastoma cells and glioblastoma patient material. (A) Immunofluorescence microscopy of the binding of VUN100 to glioblastoma cells (US28 negative) and glioblastoma cells expressing US28 (US28 positive). US28 was detected using an anti-US28 antibody (US28). VUN100 binding was detected using the Myc-tag and an anti-Myc antibody (VUN100). (B) Detection of US28 in parallel sections of glioblastoma patient material. Nuclei were stained using Hoechst staining (blue). US28 was detected using an anti-US28 antibody (US28). Nanobodies were detected via their Myc-tag (brown). An IgG isotype control and irrelevant nanobody (Irr. Nb) were used as controls.
Figure 4
Figure 4
Binding of VUN100-PS conjugates to US28. (A) SDS-PAGE of the VUN100-IRDye 700DX conjugate (VUN100-PS). A small quantity of free photosensitizer is observed at the gel front (arrow). (B) Binding of VUN100-PS to US28 negative (US28 negative) and US28 positive U251 glioblastoma cells (US28 positive). U251-iUS28 were induced for 48 h with doxycycline resulting in US28 positive glioblastoma cells US28 negative glioblastoma cells (if not induced). VUN100-PS was visualized with a widefield fluorescent microscope. (C) Binding of different concentrations of VUN100-PS to US28 negative and positive cells on ice. Fluorescence of VUN100-PS bound to cells was detected using an Odyssey infrared scanner at 700 nm.
Figure 5
Figure 5
VUN100-PS selectively kills US28-expressing cells upon illumination. (A) Staining of US28 after 48 h of doxycycline-induction of US28-expressing glioblastoma cells. US28 was visualized using anti-US28 antibody and the percentage of US28-positive cells was determined using ImageJ. (B) Detection of different concentrations of bound and internalized VUN100 to US28 positive and negative cells. Binding was determined using Odyssey infrared scanner at 700 nm. (C) Determination of cell viability after incubation with different concentrations of VUN100-PS and illumination 10 J/cm2 light dose. Cell viability was determined using Alamar blue reagent (*, p < 0.05; **, p < 0.01, t test). (D) Staining of dead cells with propidium iodide (PI) and living cells (calcein) 24 h after nanobody-targeted PDT using 50 nM VUN100-PS, performed as described above.
Figure 6
Figure 6
VUN100-PS selectively binds to US28-expressing spheroids and induces cell toxicity upon illumination. (A) Staining of dead cells with propidium iodide (PI) and living cells (calcein) of US28 negative spheroids (US28 negative) and US28 positive spheroids (US28 positive). (B) Incubation of VUN100-PS with US28 negative and positive spheroids. Spheroids and VUN100-PS were visualized with an EVOS microscope. (C) Determination of cell viability after incubation with different concentrations of VUN100-PS and illumination with a 10 J/cm2 light dose. Cell viability was determined using CellTiter-Glo 3D reagent (*, p < 0.05; **, p < 0.01, t test).

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