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Nagaya et al. Published OnlineFirst June 6, 2017; DOI: 10.1158/1541-7786.MCR-17-0164 patients with inoperable head and neck cancer targeting EGFR was approved by the FDA, and started in June 2015 (https://clinical trials.gov/ct2/show/NCT02422979) and finished in August 2016. In this trial, patients were injected with cextuximab–IR700 con- jugate, (referred to as RM1929 in the study), that binds to target EGFR molecules on the cell membrane of head and neck cancers. About 24 hours later, the tumor is exposed to NIR light by means of a laser at a wavelength of 690 nm that is absorbed by IR700. NIR-PIT induces nearly immediate necrotic cell death rather than apoptotic cell death that is most commonly induced by other cancer therapies (20). NIR-PIT has been shown to be effective with a variety of different antibodies but has not been previously tested with fully human anti-PSMA antibody (21–26). In this study, we investi- gated anti-PSMA antibody-IR700 as a candidate APC for NIR-PIT. Using a PSMA-expressing PC3 cell line in vitro binding, in vivo tumor accumulation and intratumoral distribution were evalu- ated. NIR-PIT was then performed with anti-PSMA-IR700 in vitro and repeated NIR-PIT was performed in a tumor-bearing mouse model in vivo. Materials and Methods Reagents Water soluble, silica-phthalocyanine derivative, IRDye 700DX NHS ester was obtained from LI-COR Biosciences. A fully human IgG1 anti-human prostate-specific membrane antigen (PSMA) was kindly provided by Progenics Pharmaceuticals, Inc. patent; US 8114965 B2; ref. 27). All other chemicals were of reagent grade. Synthesis of IR700-conjugated anti-PSMA Conjugation of dyes with mAb was performed according to a previous report (19). In brief, anti-PSMA mAb (1.0 mg, 6.7 nmol) was incubated with IR700 NHS ester (65.1 mg, 33.3 nmol) in 0.1 mol/L Na2HPO4 (pH 8.6) at room temperature for 1 hour. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare). The protein concentration was determined with Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific Inc) by measuring the absorption at 595 nm with UV-Vis (8453 Value System; Agilent Technologies). The concentration of IR700 was measured by absorption at 689 nm to confirm the number of fluorophore molecules per mAb. The synthesis was controlled so that an average of two IR700 molecules was bound to a single antibody. We abbreviate IR700 conjugated to anti-PSMA mAb as anti-PSMA-IR700. As a quality control for the conjugate, we performed SDS-PAGE. Conjugate was separated by SDS-PAGE with a 4%–20% gradient polyacrylamide gel (Life Technologies). A standard marker (Crystalgen Inc.) was used as a protein molec- ular weight marker. After electrophoresis at 80 V for 2.5 hours, the gel was imaged with a Pearl Imager (LI-COR Biosciences) using a 700-nm fluorescence channel. We used diluted anti-PSMA anti- body as a control. The gel was stained with Colloidal blue staining to determine the molecular weight of conjugate. Cell culture PC3-PSMAþ (PC3pip) cell line generated by transduction of PC3 cells using VSV-G pseudotyped lentiviral vector expressing human PSMA and a control blank vector–transfected PC3 cell line, PC3-PSMA- (PC3flu; refs. 28, 29) were used for PSMA- targeting studies with anti-PSMA-IR700. Both cells were estab- 1154 Mol Cancer Res; 15(9) September 2017 lished at the Cleveland Clinic Foundation. A luciferase stably expressed PC3pip cell line was also established with the trans- fection of pGL4.51 luc2/CMV/Neo Vector (Promega). High lucif- erase expression was confirmed with 10 passages. We abbreviate this cell line as PC3pip-luc. Cells were grown in RPMI1640 (Life Technologies) supplemented with 10% FBS and 1% penicillin/ streptomycin (Life Technologies) in tissue culture flasks in a humidified incubator at 37C in an atmosphere of 95% air and 5% carbon dioxide. Flow cytometry To verify in vitro anti-PSMA-IR700 binding, fluorescence from cells after incubation with anti-PSMA-IR700 was measured using a flow cytometer (FACSCalibur, BD Biosciences) and CellQuest software (BD Biosciences). PC3flu and PC3pip-luc cells (2 105) were seeded into 12-well plates and incubated for 24 hours. Medium was replaced with fresh culture medium containing 3 mg/mL of anti-PSMA-IR700 and incubated for 6 hours at 37C. To validate the specific binding of the conjugated antibody, excess antibody (30 mg) was used to block 3 mg of anti- PSMA-IR700. Fluorescence microscopy To detect the antigen-specific localization and effect of NIR-PIT, fluorescence microscopy was performed (BX61; Olympus Amer- ica, Inc.). Ten-thousand PC3flu and PC3pip-luc cells were seeded on cover-glass–bottomed dishes and incubated for 24 hours. Anti- PSMA-IR700 was then added to the culture medium at 3 mg/mL and incubated for 6 hours at 37 C. After incubation, the cells were washed with PBS. The filter set to detect IR700 consisted of a 590– 650 nm excitation filter, a 665–740 nm band-pass emission filter. Transmitted light DIC images were also acquired. In vitro NIR-PIT The cytotoxic effects of NIR-PIT with anti-PSMA-IR700 were determined by flow cytometric propidium iodide (PI; Life Technologies) staining, which can detect compromised cell membranes. A total of 2 105 PC3flu and PC3pip-luc cells were seeded into 12-well plates and incubated for 24 hours. Medium was replaced with fresh culture medium containing 3 mg/mL of anti-PSMA-IR700 and incubated for 6 hours at 37C. After washing with PBS, PBS was added, and cells were irradi- ated with a red light-emitting diode (LED), which emits light at 690 20 nm wavelength (L690-66-60; Marubeni America Co.) at a power density of 50 mW/cm2 as measured with an optical power meter (PM 100, Thorlabs). Cells were scratched 1 hour after treatment. PI was then added in the cell suspension (final 2 mg/mL) and incubated at room temperature for 30 minutes, followed by flow cytometry. Each value represents mean SEM of five experiments. For bioluminescence imaging (BLI), either 2 105 PC3pip-luc cells were seeded into 12-well plates or 2 107 PC3pip-luc cells were seeded onto a 10-cm dish; both were preincubated for 24 hours. After replacing the medium with fresh culture medium containing 3 mg/mL of anti-PSMA-IR700, the cells were incubated for 6 hours at 37C in a humidified incubator. After washing with PBS, phenol-red–free culture medium was added. Then, cells were exposed with a LED or a NIR laser which emits light at 690 5 nm wavelength (BWF5-690-8-600-0.37; B&W TEK INC.). The output power density in mW/cm2 was measured with an optical power meter (PM 100, Thorlabs). For luciferase activity, 150 mg/ mL D-luciferin–containing media (Gold Biotechnology) was Molecular Cancer Research Downloaded from mcr.aacrjournals.org on December 1, 2020. © 2017 American Association for Cancer Research.PDF Image | Near-Infrared Photoimmunotherapy Targeting Prostate Cancer
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