NANO-00875; No of Pages 11

Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxx – xxx nanomedjournal.com

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Nanobody–photosensitizer conjugates for targeted photodynamic therapy

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Raimond Heukers, MSc, Paul M.P. van Bergen en Henegouwen, PhD, Sabrina Oliveira, PhD⁎

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Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands Received 30 June 2013; accepted 23 December 2013

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Abstract

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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. © 2013 Published by Elsevier Inc.

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Key words: Photodynamic therapy; Targeted photosensitizer; Nanobody; VHH; Nanomedicine; Molecular imaging; EGFR

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Background

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Photodynamic therapy (PDT) makes use of three essential elements to induce cell death: a photosensitizer (PS), light of a particular wavelength, and oxygen. Since the first evidences of PDT-induced cell toxicity in the early 1900s, 1–3 many reports have been published on the usage of PDT to treat cancers of the bladder, skin, head and neck and of the ovaries, among others. 4–7 In general, the PS is administrated intravenously and, after a period of time, light of a particular wavelength is applied to the diseased area. The activated PS leads to type II photo-oxidative reactions, in which it reacts directly with oxygen to form the very toxic singlet oxygen ( 1O2) that damages lipids, proteins and/or nucleic acids 8. Type I reactions can also occur, in which reactive oxygen species are formed via intermediate reaction of PS with substrates other than oxygen. As these transient oxygen species

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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. ⁎Corresponding author at: Molecular Oncology, Division of Cell Biology Department of Biology, Faculty of Science, Utrecht University Padualaan 8, 3584 CH Utrecht, the Netherlands. E-mail address: [email protected] (S. Oliveira).

are short-lived molecules and have very short diffusion distances, 9 their toxicity is confined to the PS's localization upon light application. Subsequently, cells die through necrosis and/or apoptosis and tumor destruction occurs through microvasculature damage and involvement of both immune and inflammatory systems. 4 PSs clinically available are mainly derivatives of porphyrin (e.g. Photofrin®), chlorine (e.g. Foscan®), and phthalocyanine (e.g. Photosense®). 6 In general, the relatively high degree of hydrophobicity and lack of specificity of the PS result in illumination times 2 to 4 days after PS administration, in some off-target toxicity, and in a rather long period of patients' photosensitivity after PDT treatment. 6,7 Therefore, efforts have been made to render PS more hydrophilic and to target these molecules more selectively to tumors, through chemical modifications, delivery systems, and/or targeting molecules. 10–14 In particular, photoimmunotherapy (PIT) refers to the use of monoclonal antibodies (mAbs) for targeting of PS in PDT. 12 Although promising results have been reported with mAb–PS conjugates, 15–18 these conjugates have long halflives. Thus, further improvements would be valuable with respect of time needed for their tumor accumulation and the clearance of unbound conjugates. This has stimulated numerous studies on the usage of antibody fragments to target PS (e.g. Refs. 19–23). With the same aim, we have developed conjugates that combine for the first time nanobodies (NBs) with a PS.

1549-9634/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.nano.2013.12.007 Please cite this article as: Heukers R., et al., Nanobody–photosensitizer conjugates for targeted photodynamic therapy. Nanomedicine: NBM 2014;xx:1-11, http://dx.doi.org/10.1016/j.nano.2013.12.007

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NBs are the variable domain of a particular sort of antibodies, i.e. the heavy chain-only antibodies that were first discovered in dromedaries in 1993. 24 Nanobodies can be considered as the smallest naturally occurring binding domain, that is approximately 10 times smaller than conventional antibodies (NBs: 15 kDa, 2.5 nm × 4 nm 25; mAbs: 150 kDa, 14.2 nm × 8.5 nm × 3.8 nm 26 ). Despite their size, NBs can bind very specifically and tightly to their antigens (low nanomolar affinities), such as the epidermal growth factor receptor (EGFR), which is overexpressed in many types of human cancers. 27 Recently, we have demonstrated the advantages of NBs for optical molecular imaging of EGFRpositive tumors. 28 EGFR-targeted NBs showed a faster accumulation at the tumor, a more homogeneous distribution within the tumor, and a more rapid clearance of unbound molecules, compared to an anti-EGFR monoclonal antibody. In an attempt to translate these properties to the PDT context, we have conjugated the same NB targeting EGFR (7D12) to a PS. Furthermore, similarly to what was shown with internalizing mAbs, 29–31 we aimed to improve the potency of the PDT even further by stimulating intracellular delivery of the PS. For that, we used a biparatopic NB (7D12-9G8) that is known to be internalized via clustering-induced endocytosis of EGFR. 32 To further contribute to a more effective PDT, the PS used in this study is traceable through optical imaging, which enables light application at the most appropriate time and location. The idea of visualizing tumors through imaging of a PS dates back to the 1920s, 33 but the exploration of this feature is still in its infancy, 34 mainly due to the poor absorption of most PS in the near-infrared range, which is the most effective range of wavelength to penetrate through human tissues. The PS used in this study is the recently described, near-infrared fluorescent PS, IRDye700DX. 35 This silicon–phthalocyanine derivative is relatively hydrophilic, has the typical strong absorption band of phthalocyanines in the red region of the spectrum and the flexibility to be conjugated to proteins. 36 It has previously been conjugated to an EGFR-targeted mAb and was shown to be phototoxic when bound to the cell membrane or after internalization. Furthermore, tumor-specific PDT was shown, where shrinkage of tumors was only observed in those overexpressing EGFR. In this study, we have conjugated monovalent and biparatopic NBs targeting EGFR to the traceable PS IRDye700DX. These conjugates are characterized and their phototoxicity is evaluated in vitro. These NB–PS conjugates could have a significant impact on current PDT protocols, combining molecular imaging with therapy.

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Methods

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Nanobodies and PS conjugation

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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,

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purification and characterization of the NB–PS conjugates were performed as described in the Supplementary Materials.

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Cell lines and culture conditions

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The mouse fibroblast cell line NIH 3T3 2.2 (abbreviated 3T3 2.2) was described in Ref. 37; the human head and neck squamous cell carcinoma cell line UM-SCC-14C (abbreviated 14C) was kindly provided by Prof. Dr. G.A.M.S. van Dongen, (VUMC, Amsterdam, the Netherlands); the human epithelial carcinoma cell line A431 (CRL-1555) and the human cervical carcinoma cell line HeLa (CCL-2) were both obtained from ATCC (LGC Standards, Wesel, Germany). All cell lines were cultured as described in the Supplementary Materials.

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Cell binding assay

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Binding assays were performed on all cell lines, as described in detail in the Supplementary Materials. For evaluation of the association kinetics, 14C cells were incubated with 25 nm of NB–PS at 37 °C for up to 30 min. Thereafter, cells were washed twice and the fluorescence intensity (F.I.) of bound conjugates was detected with the Odyssey Infrared scanner, using the 700-nm channel.

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In vitro PDT

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One day after seeding 8000 cells per well of 96-wells plates (Greiner Bio-One, Alphen a/d Rijn, the Netherlands), cells are washed once with PDT medium (DMEM without phenol red supplemented with 8% FCS (vol/vol), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine). Then, a dilution range of NB–PS conjugates (or the 1:1 mixture of 7D12-9G8 with 7D12-9G8-PS) was added to the cells and incubated for 30 min at 37 °C. After the incubation (also referred to as pulse), cells were washed twice with PDT medium. Immediately after, the F.I. of the conjugates bound to and/or internalized by the cells was detected with the Odyssey scanner and the cells were illuminated immediately after, unless otherwise mentioned. Plates were illuminated with ~ 4-mW/ cm 2 fluence rate (measured with an Orion Laser power/energy monitor, Ophir Optronics LTD, Jerusalem, Israel), for a total light dose of 10 or 5 J/cm 2, using a device consisting of 96 LED lamps (670 ± 10 nm, 1 LED per well) described in Refs. 38,39 12After illumination, cells were placed back into the incubator, unless mentioned otherwise. In all experiments, a number of wells were covered during illumination as internal negative control. Experiments were repeated at least twice.

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Cell viability assays

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After overnight incubation of the cells treated as described above, cells were incubated with the Alamar Blue reagent, according to the manufacturer's protocol (AbD Serotec, Oxford, United Kingdom) and as described in the Supplementary Materials. Results are expressed as cell viability in percentage (%), thus relatively to the untreated cells, and the half maximal inhibitory concentration (IC50) are determined with using the GraphPad Prism 5.02 software.

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Alternatively, live and dead cells were distinguished with calcein AM (Invitrogen) and propidium iodide (Invitrogen) staining, according to the Supplementary Materials.

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Internalization assay

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Cells were pulsed with 25 nm of NB–PS conjugates for different time periods up to 30 min at 37 °C in PDT-medium, after which the cells were washed and fresh PDT-medium was added to the cells. Pulsed NB–PS was then chased for 210 min (time point 240 min) at 37 °C. At each time point, cells were washed twice and total F.I. was determined using the Odyssey Infrared scanner. In order to study the internalized fraction, surface bound NB–PS was removed by an acid wash of pH 2.5 for 2 × 10 min on ice, after which the residual (internalized) F.I. was measured.

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Co-culture assay

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A mixture consisting of 50% of HeLa and 50% of 14C cell lines was seeded in 96-wells plates (Greiner), pulsed with 25 nm of NB–PS and followed by 10 or 5 J/cm 2 of light dose. Either immediately after the light treatment or after overnight incubation (~ 16 h), dead cells were distinguished from living cells by propidium iodide and calcein AM staining (Supplementary Materials).

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Statistics

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Data were analyzed using the GraphPad Prism 5.02 software for Windows (GraphPad Software, San Diego, CA). To compare responses to treatments, analysis of significance was performed through unpaired t-tests and P b 0.05 was considered significant.

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Results

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Production and characterization of NB–PS conjugates

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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, conjugation of the NBs to the fluorescent PS was verified by SDS-PAGE and only traces of free PS were noticeable at the front of the gel (Figure 1, B). Determination of the degree of conjugation (D.O.C.) revealed that R2, 7D12 and 7D12-9G8 were on average conjugated to 1.0, 0.5 and 1.5 molecules of PS, respectively. The production of these NB– PS conjugates was reproducible.

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NB–PS conjugates bind specifically to EGFR

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In order to confirm that the conjugation of the PS to the NBs did not compromise their binding properties, binding assays were performed under non-internalizing conditions (4 °C) on cell lines expressing no and different levels of EGFR. Importantly, a clear correlation was observed between the fluorescence intensity (F.I.) of the conjugates detected and the cellular expression level of EGFR: A431 N 14C N HeLa N 3T3 2.2, where 3T3 2.2 is

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negative for EGFR expression (Figure 1, C). Thus, these fluorescent conjugates allow the distinction of cell lines with different expression levels of EGFR. Also, the differences in Bmax between the 7D12-PS and 7D12-9G8-PS correlated well with the difference in D.O.C. Binding of the NB–PS conjugates was also assessed by fluorescence microscopy, where a similar correlation between EGFR expression level and the F.I. at the cell membrane was noticeable (Figure 1, D). The apparent binding affinity of NBs was 1.4 ± 0.7 nm for 7D12-PS and 2.0 ± 0.2 nm for 7D12-9G8-PS, which is in the range of affinity values previously reported. 28,40

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NB–PS conjugates are potent PDT agents

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To explore the potential of these conjugates for PDT, we first investigated the association kinetics of the fluorescent EGFRtargeted NB–PS conjugates to EGFR overexpressing cells. Results showed that NB–PS conjugates bind very rapidly to EGFR receptors (Figure 2, A), early reaching approximate values of saturation, comparable to those determined by the binding assays at 4 °C (Figure 1, C, 14C cells). Subsequently, and in all the following experiments, cells were incubated for 30 min at 37 °C (indicated as pulse), with a concentration range of the three NB–PS conjugates or the PS alone. Also here, the detected maximum F.I. on cells for 7D12-PS and 7D12-9G8-PS correlated well with the D.O.C. (Figure 2, B). Importantly, no binding of free PS or R2-PS to 14C cells was detected, which indicates that there is no cell association of PS in the absence of EGFR-targeted NBs. After the pulse, cells were exposed to a light dose of 10 J/cm 2, to induce PDT and the cell viability was assessed the following day. Clearly, the EGFR-targeted NB–PS conjugates are very potent PDT agents, with IC50 values of 2.3 ± 0.7 nm for 7D12PS and of 0.6 ± 0.06 nm for 7D12-9G8-PS (Figure 2, C), where the latter is significantly more potent (P b 0.001). Importantly, free PS, R2-PS, and the non-illuminated EGFR-targeted NB–PS conjugates did not affect cell viability (IC50s could not be determined), which highlights the specificity of this PDT approach. PDT-induced toxicity was also observed by fluorescence microscopy, where dead cells, as indicated by propidium iodide staining of the nuclei, are distinguished from living cells, stained with calcein AM. Immediately after light application, all cells that were pulsed with 7D12-9G8-PS appeared dead, while in the case of 7D12-PS this was observed at a later time point. For both NB–PS conjugates, no cell death was observed when no light was applied after the pulse (Figure 2, D).

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The biparatopic NB–PS leads to more effective PDT

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7D12-9G8 induces EGFR internalization, via receptor mediated endocytosis, and is taken up by cells more efficiently than 7D12 (Figure S1). 32 This suggests that 7D12-9G8 can lead to a more effective intracellular delivery of PS, resulting in a more potent PDT (in line with studies of internalizing mAbs 29–31). The efficacy of the PDT correlated well with the amount of PS that was present on the cells at the time of illumination (Figure 2, B and C), resulting in the lowest IC50 (i.e. highest toxicity) for the biparatopic NB–PS conjugate. However, the D.O.C. (i.e. average PS conjugated) of 7D12-9G8-PS is higher than that of 7D12-PS. So, to study the contribution of PS

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Figure 1. (A) Schematic representation of the NBs targeting EGFR and of the negative control. (B) Fluorescent NB–PS conjugates separated by SDS-PAGE. Conjugates are identified as R2-PS, 7D12-PS and 7D12-9G8-PS. Free PS is observed at the gel front (arrow). (C) NB–PS conjugates bind to three cell lines according to their EGFR expression level: A431 N 14C N HeLa and not to 3T3 2.2 cells (no EGFR). Mean F.I. ± SEM. (D) Images of fluorescence microscopy obtained with an EVOS microscope. 268 269 270 271 272 273 274 275 276 277 278

internalization via the biparatopic NB more carefully, we have made a mixture of 7D12-9G8 with 7D12-9G8-PS (7D12-9G8Mix). This approach resulted in a similar amount of fluorescent PS associated with the cells for both NB formats (Figure 3, A, open squares of 7D12-PS and filled diamonds of 7D12-9G8Mix). Subsequently, acid washes were conducted to determine the F.I. of the PS located intracellularly, which showed that the 7D12-9G8-Mix accumulated in cells to a greater extent than 7D12-PS (Figure 3, B). These data confirm that 7D12-9G8-PS is internalized more efficiently than 7D12-PS. More importantly, even though the total PS associated with cells is comparable, the

mixture was significantly more toxic than 7D12-PS (IC50 1.2 ± 0.3 nm and IC50 2.3 ± 0.7 nm, respectively, P b 0.05). As for the 7D12-9G8-PS, which resulted in more PS associated with cells than 7D12-9G8-Mix, the toxicity was evidently higher (IC50 0.6 ± 0.06 nm, P b 0.01) (Figure 3, C).

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Illumination right after incubation leads to stronger phototoxicity

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The fact that NB–PS conjugates are internalized by cells could result in residualization of the conjugates. As this would be relevant for the subsequent illumination time point for PDT, we

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Figure 2. (A) Interaction of EGFR-targeted NB–PS conjugates with cells over time. (B) Total F.I. associated with 14C cells after 30-min pulse with a concentration range of the three NB–PS conjugates and PS alone. (C) Percentage (%) of cell viability after a 10-J/cm 2 light dose relative to untreated cells. Data are means ± SEM. (D) Fluorescence microscopy for the detection of NB–PS (in red), of dead cells stained with propidium iodide (in blue) and of intact cells stained with calcein AM (in green).

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have investigated this aspect in more detail employing a “pulsechase” experimental set-up. During the chase, the amount of PS bound to the tumor cells decreased over time for both formats (41% ± 6% for 7D12-PS and 43% ± 2% for 7D12-9G8-PS),

while the fraction of internalized NB–PS remained constant (Figure 4, A). These data suggest that no recycling of the NBs occurs and that both formats of NB–PS conjugates residualize within the cells.

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Figure 4. (A) Total (light gray) and internal (dark gray) fluorescence of NB–PS conjugates, during 30-min pulse and the following 210-min chase. PS that is released from the cell surface (1) and residualizing PS (2) are indicated with arrows. (B) Left: Total F.I. after 14C cells were pulsed with NB–PS conjugates or after a 210-min chase (240 min) Mean F.I. ± SEM. Right: Cell viability after illumination with 10 J/cm 2, right after pulse or after the chase period. Mean % ± SEM.

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To gain more insight in the fate of the NB–PS conjugates after internalization, we studied the possibility of lysosomal degradation. After 30-min pulse and 210 min of chase, free PS

was only observed in the cellular fraction and this was significantly more for the 7D12-9G8-PS (20% ± 3% of the internalized fraction), as compared to 7D12-PS (6% ± 2%)

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(Figure S2A, P b 0.05). The lack of free PS observed in the medium demonstrates the stability of the conjugates in culture medium. Further stability studies have shown that NB–PS is stable for at least 4 h in full human serum (Supplementary Materials). Chloroquin (CQ) prevented the degradation of 7D129G8-PS, as well as of the control conjugate EGF-IRDye800CW (P b 0.01 and P b 0.001, respectively), indicating that the conjugate is degraded in the lysosomes. Moreover, intracellular trafficking toward the lysosomes was confirmed by fluorescence microscopy (Figure S2B). Besides lysosomal degradation of the NB–PS conjugates, the biparatopic NB also induced degradation of the EGFR (Figure S2C). To investigate how a delay in light application would affect PDT potency, 14C cells were treated as described earlier for PDT (Figure 4, B, left). Cells were illuminated either right after the 30-min pulse or after the chase period of 210 min (i.e. 240-min time point). After the chase, there was for both NB–PS conjugates a significant decrease in potency (7D12-PS IC50 10.1 ± 4.4 nm, P b 0.001; 7D12-9G8-PS IC50 1.2 ± 0.07 nm, P b 0.01), though 7D12-9G8-PS remained toxic in the very low nanomolar concentration range (Figure 4, B, right). NB–PS-mediated PDT is tuneable for EGFR overexpressing tumor cells The selectivity of the NB–PS induced PDT was assessed by performing the PDT on different cell lines with varying expression levels of EGFR. Similarly to the correlation observed between the level of binding of the fluorescent conjugates and the expression level of these cells (Figure 1, C), the toxicity of the conjugates was higher (lower IC50) for the cell line with higher EGFR expression level: on A431 cells, 7D12-PS led to IC50 1.3 ± 0.06 nm and 7D12-9G8-PS to 0.52 ± 0.10 nm, compared to the IC50s determined on 14C cells of 2.3 ± 0.7 nm with 7D12-PS and 0.6 ± 0.06 nm with 7D12-9G8-PS. Importantly, the viability of the low EGFR expressing HeLa cells was only affected by 7D12-9G8-PS (IC50 2.29 ± 0.68 nm) (Figure 5, A). In order to reduce the toxicity on low EGFR expressing cells, similar PDT assays were performed but with half the light dose (5 J/cm 2). For the three cell lines this resulted in lower toxicity, especially for HeLa cells in which only a moderate effect was observed with 7D12-9G8-PS (IC50 22.8 ± 7.5 nm) (Figure 5, B). For the other two cell lines, the reduced light dose resulted in the following IC50s: on 14C cells 39 ± 14.5 nm with 7D12-PS and 1.2 ± 0.26 nm with 7D12-9G8-PS; and on A431 cells 2.3 ± 0.34 nm with 7D12-PS and 1.1 ± 0.47 nm with 7D129G8-PS. Selectivity of the NB–PS conjugates was also demonstrated using co-culture experiments performed with the 14C cell line overexpressing EGFR and the HeLa cell line expressing low levels of EGFR. These two cell lines can be distinguished in cocultures by their different morphology and by the different EGFR expression levels (Figure 5, C, top), as validated by the fluorescence intensities of cell bound EGF-Alexa-555 and NB– PS conjugates (Figure S3). With 7D12-PS, immediately after the light treatment with 10 J/cm 2, no cell damage was yet observed through fluorescence microscopy. But, the next day, a clear

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propidium iodide staining was visible, specifically in 14C cells, whereas HeLa cells remained intact (Figure 5, C, middle). Importantly, the PS (red color) overlapped very well with propidium iodide-stained cells, which confirms the selective cell death of 14C cells. These observations suggest that PDT mediated by 7D12-PS requires more time before the effect is detectable through fluorescence microscopy (also suggested earlier, Figure 2, D). Interestingly, for 7D12-9G8-PS, immediately after the light treatment with 5 J/cm 2, cell damage was observed by the appearance of propidium iodide staining, specifically in 14C cells, while HeLa cells were stained with calcein AM (Figure 5, C, right).

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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). 11–14 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, we have developed an alternative that combines the small size and high binding affinity of nanobodies (NBs) with a relatively hydrophilic and traceable PS. The NBs here employed are robust and their binding properties are not affected by the random conjugation of the PS. Even though the conjugation reactions were performed similarly for each NB, different D.O.C. were obtained, as a result of differences in amino acid sequences of the NBs. This could possibly be avoided in the future through site-directed conjugation, when this PS is available with a maleimide reactive group, as recently shown with anti-HER2 nanobodies for optical imaging. 41 The PS conjugated to these NBs is the silicon–phthalocyanine derivative IRDye700DX, which is relatively more hydrophilic compared to the most common PS employed and is traceable by near-infrared fluorescence imaging. 35 The first property is reflected in easiness of conjugation and purification of the NB–PS conjugates, while the second property facilitates in vitro testing by allowing its detection and quantification, bound to or inside cells. In addition, it allows the distinction of cell lines that differ in EGFR expression level. This is of particular interest in vivo, since EGFR is expressed in normal tissues, albeit at a much lower level compared to cancer cells. Importantly, the association of the EGFR-targeted NB–PS conjugates and the subsequent toxicity induced on the different cell lines correlated well with their EGFR expression level. For the EGFR-targeted NB–PS conjugates to be cytotoxic to tumor cells in vivo, they have to bind specifically to tumor cells and be retained at the tumor, before they are cleared from the bloodstream through the kidneys. From previous studies, 28 it is known that the highest concentration of NB in the blood stream is found up to 15–30 min post-injection. Therefore, it was important to observe the rapid association kinetics of these conjugates (Figure 2, A). This time period differs from what is

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generally used with mAb–PS conjugates (e.g. 6 h, 12 h, or longer 29–31), as in that case longer serum half-lives are expected. Importantly, the amount of NB–PS associated with the cells during the 30-min pulse was sufficient to result in strong toxicity after light application, emphasizing the selectivity and potency of these conjugates for PDT (Figure 2, C and D). Besides different time intervals for exposure of cells to conjugates, PDT protocols may also vary in time interval between exposure and the application of light. We therefore investigated the retention of the conjugates in cells in an in vitro pulse-chase assay, which showed that dissociation of membranebound conjugates occurs over time. Consequently, less potent PDT was observed, therefore encouraging illumination as early as possible. Nevertheless, the internalization and subsequently the retention of the conjugates within cells were clearly improved by employing 7D12-9G8-PS. Although the D.O.C was different for the 7D12-PS and 7D12-9G8-PS, our experiment employing the mixture of 7D12-9G8 with 7D12-9G8-PS (i.e 7D12-9G8Mix) enabled a comparison between the two NB formats. We realize that this comparison is not absolutely fair, as the PS molecules will certainly be differently distributed (intra-, intermolecularly/cellularly) when conjugated to 7D12 or 7D12-9G8. Nevertheless, while delivering a similar amount of fluorescent PS to the cells, the internalizing biparatopic NB induced a significantly more potent PDT, which is in agreement with previous studies employing internalizing mAbs for PDT. 29–31 Notably, our results are in contrast with those of Mitsunaga et al 35 which reported no additional effect upon internalization of a mAb-IRDye700DX conjugate. However, in that study no details on dose–response curves were presented, which could be the reason for missing the additional effect of internalization. In any case, one can also expect a different mechanism of internalization between biparatopic NBs and a mAb. Unlike a single mAb, biparatopic NBs 32 or a combination of different mAbs 42 induce receptor clustering and consequently faster endocytosis. This clustering might also affect the consequent downstream trafficking. The improved efficacy and faster cell death induced by the internalizing NB might result from the destruction of more vital structures or organelles in the cell. Whether the mechanism through which NB–PS conjugates induce cell death is involving singlet oxygen only or its combination with photothermal activity, as proposed by Mitsunaga et al, is currently unclear, though preliminary data have confirmed the production of singlet oxygen upon PS illumination. The propidium iodide staining and the absence of annexin-V staining upon NB–PS illumination, classically indicate cell death through necrosis (Figure S4). Though necrosis has not been indicated as the main mechanism of action of other silicon–phthalocyanines, 43–45 we believe that the mechanism is determined by the quantity and location where the singlet oxygen is formed (i.e. location of the PS). In the case of our NB–PS, due to the relative hydrophilicity of IRDye700DX, the location of this PS is determined by the NB.

NBs differ from mAbs, namely in the absence of the Fc tail and inability to trigger antibody-dependent cell cytotoxicity. Nevertheless, 7D12-9G8-PS induces the lysosomal trafficking and degradation of both NB and EGFR. This correlates well with the natural negative feedback mechanism employed by EGFRs natural ligand EGF (reviewed in Ref. 46), and is also in agreement with previous studies suggesting inhibition of endosomal recycling by antibody-induced EGFR internalization. 42,47 Importantly, despite the lysosomal degradation, PS remains inside the cells, which in case of a necessary delay in light application, would not drastically compromise the efficacy, particularly in case of 7D12-9G8-PS. These NB–PS conjugates need to be evaluated in vivo. Nevertheless, based on previous molecular imaging studies, 28,41,48 we can expect that these NB–PS conjugates will rapidly and homogenously distribute through tissues and bind tightly to the EGFR on tumor cells, while unbound conjugates will be rapidly cleared through the kidneys. If this is indeed the case for the NB– PS conjugates, these NB–PS conjugates could be an important contribution to the current field of targeted PDT. Yet, for this to happen, not only the specific delivery of PS is important, but also the success of the light application. The important advantage of PDT is that light can be applied restrictedly to the sites of interest, thus, tumor cells will receive the highest light dose, compared to the surrounding tissues. In order to gain more insights on a situation where cancer cells overexpressing EGFR are closely located to (normal) cells with low EGFR expression, co-culture experiments were performed with the 14C cell line and the HeLa cell line. These co-culture studies showed that by adjusting the light dose, the treatment could be 100% specific to the EGFR overexpressing 14C cells and safe to the low EGFR expressing HeLa cells. These data are in agreement with the concept of the threshold dose, which implies that a minimum concentration of phototoxic product (e.g. 1O2) is needed to result in cellular or tissue destruction. 49,50 The challenge in vivo will be to determine the appropriate settings to surpass the threshold dose, solely in targeted tissues, such as ovarian or head and neck tumors. Penetration of light through tissues is also known to be limited due to the optical properties of tissues, which in this case also renders PDT safer for (deeper) normal tissues. In case of surgery, these traceable NB–PS conjugates could be employed at first for imaging and to guide the tumor resection (image-guided surgery). Thereafter, a certain light dose could be applied in the resected area for PDT of the remaining tumor cells. This approach would likely contribute to more radical tumor resections. In conclusion, we have demonstrated that NBs conjugated to a PS are suitable for specific and targeted PDT in vitro. Secondly, it was shown that the potency of PDT is enhanced by intracellular delivery of PS through the biparatopic NB. The next step is to evaluate these new conjugates in vivo, in order to determine the impact and contribution of these NB–PS conjugates in the field of targeted PDT, which combines tumor molecular imaging with therapy.

Figure 5. (A) Comparison of PDT on different cell lines pulsed with 7D12-PS (open squares) or 7D12-9G8-PS (closed squares) and illuminated with 10-J/cm 2 or (B) 5-J/cm 2 light dose. Mean % cell viability ± SEM. (C) Fluorescence microscopy of co-cultures of HeLa and 14C cells, for the detection of NB–PS (in red), of dead cells stained with propidium iodide (in blue) and of intact cells stained with calcein AM (in green).

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Appendix A. Supplementary Data

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References

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Nanobody-photosensitizer conjugates for targeted photodynamic therapy.

Photodynamic therapy (PDT) induces cell death through light activation of a photosensitizer (PS). Targeted delivery of PS via monoclonal antibodies ha...
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