Near-Infrared Photoimmunotherapy Targeting Prostate Cancer with Prostate-Specific Membrane Antigen (PSMA) Antibody

Abstract

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for molecular therapy. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photoabsorber conjugate (APC). Here, we describe the efficacy of NIR-PIT, using a fully human IgG₁ anti-PSMA monoclonal antibody (mAb), conjugated to the photoabsorber, IR700DX, in a PSMA-expressing PC3 prostate cancer cell line. Anti-PSMA-IR700 showed specific binding and cell-specific killing was observed after exposure of the cells to NIR light in vitro In the in vivo study, anti-PSMA-IR700 showed high tumor accumulation and high tumor-background ratio. Tumor-bearing mice were separated into 4 groups: (i) no treatment; (ii) 100 μg of anti-PSMA-IR700 i.v.; (iii) NIR light exposure; (iv) 100 μg of anti-PSMA-IR700 i.v., NIR light exposure was administered. These were performed every week for up to 3 weeks. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other control groups (P < 0.001), and significantly prolonged survival was achieved (P < 0.0001 vs. other control groups). More than two thirds of tumors were cured with NIR-PIT. In conclusion, the anti-PSMA antibody is suitable as an APC for NIR-PIT. Furthermore, NIR-PIT with the anti-PSMA-IR700 antibody is a promising candidate of the treatment of PSMA-expressing tumors and could be readily translated to humans.Implications: NIR-infrared photoimmunotherapy (NIR-PIT) using a fully human anti-PSMA-IR700 conjugate showed potential therapeutic effects against a PSMA-expressing prostate cancer that is readily translated to humans. Mol Cancer Res; 15(9); 1153-62. ©2017 AACR.

Figure 1. Confirmation of PSMA expression as a target for NIR-PIT in PC3 cells, and evaluation of in vitro NIR-PIT

(A) Validation of anti-PSMA-IR700 by SDS-PAGE (left: Colloidal Blue staining, right: fluorescence). Diluted anti-PSMA mAb was used as a control. (B) Expression of PSMA in PC3flu and PC3pip-luc cells was evaluated by FACS. After 6 h of anti-PSMA-IR700 incubation, PC3pip-luc cells showed high fluorescence signal. Fluorescence in PC3pip-luc cells was completely blocked by adding excess PSMA. On the other hand, PC3flu cells showed no fluorescence signal. (C) Differential interference contrast (DIC) and fluorescence microscopy images of PC3flu and PC3pip-luc cells after incubation with anti-PSMA-IR700 for 6 h. High fluorescence intensities were shown in only PC3pip-luc cells. Necrotic cell death was observed upon excitation with NIR light (after 15min) in PC3pip-luc cells. Scale bars = 20 μm. (D) Bioluminescence imaging (BLI) of a 10 cm dish demonstrated that luciferase activity in PC3pip-luc cells decreased in a NIR-light dose-dependent manner. (E) Luciferase activity in PC3pip-luc cells was measured, which also decreased in a NIR-light dose-dependent manner (n = 5, *p < 0.05 vs. untreated control, **p < 0.01 vs. untreated control, by Student’s t test). (F) Membrane damage of PC3flu cells induced by NIR light exposure was measured with the dead cell count using propidium iodide (PI) staining. No membrane damage was observed in PC3flu cells after NIR light exposure. (G) Membrane damage of PC3pip-luc cells induced by NIR-PIT was measured with the dead cell count using PI staining, which increased in a light dose dependent manner (n = 5, **p < 0.01, vs. untreated control, by Student’s t test). There was no significant cytotoxicity associated with NIR light exposure alone in the absence of anti-PSMA-IR700 and with anti-PSMA-IR700 alone without NIR light exposure.

Figure 2. In vivo fluorescence imaging of PC3pip-luc tumor

(A) In vivo anti-PSMA-IR700 fluorescence real-time imaging of tumor-bearing mice (right dorsum). The tumor showed high fluorescence intensity after injection and the intensity was gradually decreased over days. Most of the excess agent was excreted to the urine immediately after injection. ROIs were placed on the tumor and liver, then ROIs were also placed in the adjacent non-tumor region as background (left dorsum; blue circle and lower abdomen; green circle). (B) Time course of NIR fluorescence signal of IR700 in tumors and livers (n = 10). The IR700 fluorescence intensity of tumor and liver showed high intensities within 1 day after anti-PSMA-IR700 injection but this decreased gradually over days. (C) Time course of NIR fluorescence signal of TBR in tumors and livers (n = 10). TBR of tumor and liver showed high within four days after anti-PSMA-IR700 injection, then the TBR was gradually decreased over the following days.

Figure 3. In vivo effect of NIR-PIT for PC3pip-luc tumor

(A) NIR-PIT regimen. Fluorescence and bioluminescence images were obtained at each time point as indicated. (B) In vivo fluorescence real-time imaging of tumor-bearing mice in response to NIR-PIT. The tumor treated by NIR-PIT showed decreasing IR700 fluorescence after NIR-PIT. (C) In vivo BLI of tumor bearing mice in response to NIR-PIT. Before NIR-PIT, tumors were approximately the same size and exhibited similar BLI signals. The tumor treated by NIR-PIT showed decreasing luciferase activity after NIR-PIT. (D) Quantitative luciferase activity (before NIR-PIT is set to 100) showed a significant decrease in NIR-PIT tumors (n ≧ 10, **p < 0.001 vs. other groups, by Tukey’s test with ANOVA). Luciferase activity of tumor in other control groups showed an increase due to rapid tumor growth. (E) Tumor growth was significantly inhibited in the NIR-PIT treatment group with anti-PSMA-IR700 (n ≧ 10, **p < 0.001 vs other control groups, Tukey’s test with ANOVA). (F) Significantly prolonged survival was observed in the NIR-PIT treatment group with anti-PSMA-IR700 (n ≧ 10, **p < 0.0001 vs other control groups, by Log-rank test).

Figure 4. In vivo histological fluorescence distribution and histological NIR-PIT effect

(A) The regimen of NIR-PIT and imaging is shown. (B) Fluorescence images of resected PC3pip-luc tumors. White light images (upper) and IR700 fluorescence image (lower). High fluorescence intensity was shown in PC3pip-luc tumor 24 h after injection of anti-PSMA-IR700, but the fluorescence decreased 24 h after NIR-PIT. (C) Differential interference contrast (DIC) and fluorescence microscopy images of PC3pip-luc tumor xenografts. High fluorescence intensity was shown in PC3pip-luc tumor 24 h after injection of anti-PSMA-IR700, but the fluorescence decreased 24 h after NIR-PIT. Scale bars = 100 μm. (D) Resected tumor stained with hematoxylin and eosin (H&E). A few scattered clusters of damaged tumor cells were seen within a background of diffuse cellular necrosis and micro-hemorrhage after NIR-PIT, while no obvious damage was observed after anti-PSMA-IR700 alone with NIR light exposure. White scale bars = 100 μm. Black scale bars = 20 μm.