Abstract
Glioblastomas (GBMs) are high‐grade brain tumors, differentially driven by alterations (amplification, deletion or missense mutations) in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12–15 months following standard therapy. A combination of interventions targeting tumor‐specific cell surface regulators along with convergent downstream signaling pathways may enhance treatment efficacy. Against this background, we investigated a novel photoimmunotherapy approach combining the cytotoxicity of photodynamic therapy with the specificity of immunotherapy. An EGFR‐specific affibody (ZEGFR:03115) was conjugated to the phthalocyanine dye, IR700DX, which when excited with near‐infrared light produces a cytotoxic response. ZEGFR:03115–IR700DX EGFR‐specific binding was confirmed by flow cytometry and confocal microscopy. The conjugate showed effective targeting of EGFR positive GBM cells in the brain. The therapeutic potential of the conjugate was assessed both in vitro, in GBM cell lines and spheroids by the CellTiter‐Glo® assay, and in vivo using subcutaneous U87‐MGvIII xenografts. In addition, mice were imaged pre‐ and post‐PIT using the IVIS/Spectrum/CT to monitor treatment response. Binding of the conjugate correlated to the level of EGFR expression in GBM cell lines. The cell proliferation assay revealed a receptor‐dependent response between the tested cell lines. Inhibition of EGFRvIII+ve tumor growth was observed following administration of the immunoconjugate and irradiation. Importantly, this response was not seen in control tumors. In conclusion, the ZEGFR:03115–IR700DX showed specific uptake in vitro and enabled imaging of EGFR expression in the orthotopic brain tumor model. Moreover, the proof‐of‐concept in vivo PIT study demonstrated therapeutic efficacy of the conjugate in subcutaneous glioma xenografts.
Highlights
Glioblastomas (GBMs) are high-grade brain tumors, differentially driven by alterations in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12–15 months following standard therapy
The specificity of ZEGFR:03115–IR700DX binding in vitro correlated with the expression level of EGFR as seen by flow cytometry and, importantly, pre-blocking the cells with 100fold excess of unlabeled affibody molecules effectively reduced the median fluorescence in all tested cell lines (Fig. 1b)
Afterwards, in order to assess the penetration depth of the ZEGFR:03115–IR700DX, which is critical for successful PIT, 3D U87-MGvIII or WSz4 spheroids that more closely resemble in vivo tissue in terms of cellular communication, were cultured with media containing either the conjugate, IR700DX or mAbEGFR-FITC (Fig. 1f, Supporting Information Fig. 2b, Supporting Information Movie 2–4)
Summary
Glioblastomas (GBMs) are high-grade brain tumors, differentially driven by alterations (amplification, deletion or missense mutations) in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12–15 months following standard therapy. A combination of interventions targeting tumor-specific cell surface regulators along with convergent downstream signaling pathways may enhance treatment efficacy. Against this background, we investigated a novel photoimmunotherapy approach combining the cytotoxicity of photodynamic therapy with the specificity of immunotherapy. An EGFR-specific affibody (ZEGFR:03115) was conjugated to the phthalocyanine dye, IR700DX, which when excited with near-infrared light produces a cytotoxic response. Developing novel, nearinfrared (NIR) molecularly targeted probes for intraoperative treatment of high-grade gliomas is an attractive prospect.[13,14]
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