Epidermal Growth Factor Receptor (EGFR)-targeted Photoimmunotherapy (PIT) for the Treatment of EGFR-expressing Bladder Cancer

Abstract

The use of light as a means of therapy for bladder cancer (BC) has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of photo-therapy called photoimmunotherapy (PIT) which targets EGFR-expressing BC. Anti-EGFR antibody panitumumab (pan) was labeled with the photo-absorber (PA), IRDye 700Dx (IR700), to create a pan IR700 antibody-PA conjugate which is activated by near-infrared radiation (NIR). BC tissue microarray (TMA) and BC cell lines were analyzed for expression of EGFR. Mechanism of PIT induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. EGFR staining of TMAs showed that while most BCs have expression of EGFR to a varying degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Pan IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Pan alone, pan IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed towards cell death. NIR-PIT with pan IR700 reduced growth compared to only pan IR700 treated UMUC-5 xenograft tumors. PIT is a new targeted treatment for bladder cancer. Pan IR700-induced PIT selectively kills EGFR-expressing BC cells in vitro and in vivo and therefore warrants further therapeutic studies in orthotopic xenografts of BC and ultimately in patients.

Changes in plasma membrane damage inducing cell death after treatment with near‐infrared photoimmunotherapy

“…Membrane lipid peroxidation is responsible for inducing membrane damage in PDT, 32,33 but any oxidative changes on major unsaturated lipid molecules after NIR-PIT have not been detected so far. 5,12 Unlike conventional plasma membrane-targeted photosensitizers, an mAb-IR700 is sufficiently far away from the plasma membrane and does not induce oxidative damage because of the very short half-life of singlet oxygen. 12,34 There is a report that free radical scavengers reduced the effect of NIR-PIT, 35 although this has not been generally accepted.…”

“…5,12 Unlike conventional plasma membrane-targeted photosensitizers, an mAb-IR700 is sufficiently far away from the plasma membrane and does not induce oxidative damage because of the very short half-life of singlet oxygen. 12,34 There is a report that free radical scavengers reduced the effect of NIR-PIT, 35 although this has not been generally accepted. 5,11 The molecular mechanism causing such minute plasma membrane damage in NIR-PIT remains unclear.…”

“…9,10 In contrast, several studies have shown that ROS is not the factor responsible for inducing membrane damage in NIR-PIT. 1,5,11,12 Thus, in order to understand the cytotoxic mechanism of NIR-PIT, it is important to elucidate how the damaged plasma membrane changes during the process of cell swelling and bleb formation. Therefore, in the present study, we aimed to investigate the changes in plasma membrane damage at early time points during NIR-PIT.…”

CD47-Targeted Near-Infrared Photoimmunotherapy for Human Bladder Cancer

“…The feasibility and safety of PDT using the photosensitizers HAL or Radiochlorine have been tested in small clinical trials (8,9). Because of lack of specificity of the photosensitizer, long irradiation times, and significant local side effects, PDT has not been widely adopted clinically for bladder cancer (8)(9)(10)(11)(12).…”

Site‐Specific Encoding of Photoactivity in Antibodies Enables Light‐Mediated Antibody–Antigen Binding on Live Cells

“…Unlike several other techniques used for measuring protein-protein interaction, this approach does not require sophisticated instrumentation or purified EGFR, and assesses the binding of antibody to EGFR on acell surface.T he technique is similar to whole cell enzyme-linked immunosorbent assay (ELISA), and on-cell western used for quantification of cell surface antigens. [25] As eries of control experiments were performed to demonstrate the viability of the on-cell assay used in this study.W hen7 D12 was incubated with MDA-MB-231, ac ell line with low levels of EGFR, often used as an egative control, [24] the chemiluminescence signal was significantly lower compared to the signal for A431 cancer cell line ( Figure S6). This supports our premise that the observed chemiluminescence is due to specific interaction between 7D12 and EGFR, and not due to non-specific binding of 7D12 to the cell surface.W ea lso measured the binding of an unrelated His6-tagged antibody fragment, RR6-VHH, [26] to A431 cells using this assay.N ear-background level of chemiluminescence was observed with RR6-VHH, demonstrating that the observed signal is not due to the non-specific interaction of antibody fragments with A431 cancer cells ( Figure S7).…”