Nanobody-targeted photodynamic therapy for the treatment of feline oral carcinoma: a step towards translation to the veterinary clinic

Abstract Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and tumor-selective treatment, using nanobodies (NBs) to deliver a photosensitizer (PS) specifically to cancer cells. Upon local light application, reactive oxygen species are formed and consequent cell death occurs. NB-PDT has preclinically shown evident success and we next aim to treat cats with oral squamous cell carcinoma (OSCC), which has very limited therapeutic options and is regarded as a natural model of human head and neck SCC. Immunohistochemistry of feline OSCC tissue confirmed that the epidermal growth factor receptor (EGFR) is a relevant target with expression in cancer cells and not in the surrounding stroma. Three feline OSCC cell lines were employed together with a well-characterized human cancer cell line (HeLa), all with similar EGFR expression, and a low EGFR-expressing human cell line (MCF7), mirroring the EGFR expression level in the surrounding mucosal stroma. NBA was identified as a NB binding human and feline EGFR with comparable high affinity. This NB was developed into NiBh, a NB-PS conjugate with high PS payload able to effectively kill feline OSCC and HeLa cell lines, after illumination. Importantly, the specificity of NB-PDT was confirmed in co-cultures where only the feline OSCC cells were killed while surrounding MCF7 cells were unaffected. Altogether, NiBh can be used for NB-PDT to treat feline OSCC and further advance NB-PDT towards the human clinic.

days later, cells were incubated for 2 hours at 4 °C with a concentration range of NBA-IRDye800CW (conjugated as in [1]) in binding medium (DMEM without phenol red, supplemented with 25 mM HEPES and 1 % BSA, pH 7.2). Unbound NB was washed off and the bound fraction was brought in suspension by incubating the cells with acid wash buffer (0.2 M glycine-HCl and 150 mM NaCl, pH 2.3) twice, for 6 and 3 minutes. Both fractions were transferred to a new plate, neutralized (1 M Tris-HCl, pH 9), and scanned with an Odyssey infrared scanner (LI-COR). Data was analyzed with GraphPad Prism software to determine the Bmax, i.e. total receptor density. This value was interpolated in a titration curve made with NBA-IRDye800CW, to obtain molecules of nanobody per well using the Avogadro constant. Assuming binding of one nanobody per EGFR molecule and counting the cells per well, the number of EGFR molecules per cell was calculated. In this assay, A431 cells were included as a known EGFR-overexpressing cell line (1-2 million receptors per cell [2]) to confirm the setup of the assay.

Characterization of NB-PS conjugates
The purity and integrity of the different NB-PS conjugates and unconjugated NB were assessed by size separation via 15 % SDS-PAGE. The gel was scanned at 700 nm with an Odyssey scanner (LI-COR) to detect the PS fluorescence. Afterwards, total protein was stained with Page Blue (Thermo Fisher Scientific) and detected again with the infrared scanner. In addition, the absorption spectra (230 -750 nm) of each NB and conjugate was measured with a NanoDrop spectrophotometer (NanoDrop Technologies).
EGFR knockdown and binding assay SCCF1 cells were seeded in a 96-well plate (10,000 cells/well) and incubated at 37 °C. The next day, cells were transfected with EGFR Stealth siRNA (Thermo Fisher, EGFR-3236) following the standard protocol for Lipofectamine 2000 (Invitrogen, 11668-027). Non-transfected cells were taken along, and non-targeting siRNA (Qiagen, SI03650318) was employed as negative control. After 48 hours, cells were collected and membrane EGFR stained to verify the knockdown. Staining was performed by incubation for 45 minutes at 4 °C with mouse anti-EGFR antibody (ThermoFisher, MA5-13269) diluted 1:75, followed by goat anti-mouse Alexa 488 (Invitrogen, A11029) for 30 minutes at 4 °C (diluted 1:200). HeLa cells as reference cell line and unstained controls were taken along. Measurements were performed with a FACS Canto II (BD) and further analyzed with FlowLogic software (Inivai Technologies).
The binding of NBA-PS(1) on EGFR knockdown SCCF1 cells was assessed. Two days before the assay, SCCF1 cells were left untreated or transfected with EGFR gene siRNA as explained above. After 48 hours, cells were incubated for 2 hours at 4 °C with a concentration range of NBA-PS(1) in binding medium (DMEM without phenol red, supplemented with 25 mM HEPES and 1 % BSA, pH 7.2). Unbound conjugate was washed off and PS fluorescence detected on cells with an Odyssey scanner (LI-COR) at 700 nm.

Antioxidant capacity
Cells were collected (200,000 cells per cell line), pelleted and resuspended in MQ water with 0.5 % Triton X-100 (Sigma-Aldrich). After 10 minutes on ice, cell debris was removed by centrifugation and supernatant used for the assay. Samples were processed as indicated by the manufacturer with a Total Antioxidant Capacity Assay Kit (Sigma-Aldrich, #MAK187), without using the optional Protein Mask reagent. Absorbance at 550 nm was measured with a FLUOstar Optima microplate reader (BMG Labtech) and data analyzed with GraphPad Prism software to calculate concentration of antioxidant in each sample. Figure S1. Detection of membrane EGFR on several human and feline cell lines. (a) EGFR was detected by flow cytometry on three feline OSCC cell lines (SCCF1, SCCF2 and SCCF3) and two human cancer cell lines (HeLa and MCF7) using a commercial anti-EGFR, species cross-reactive antibody. Histogram shows the fluorescence intensity corresponding to EGFR for each cell line. (b) The number of membrane EGFR molecules per cell was quantified based on the saturation binding of NBA-IRDye800CW. Average results are shown, calculated from at least three independent experiments. A431 cells were included as a known EGFR-overexpressing cell line. MCF7 cells are reported to have 10,000 EGF receptors per cell [3], but such low numbers are below the detection limit of the assay.