Development and characterization of a theranostic multimodal anti-PSMA targeting agent for imaging, surgical guidance, and targeted photodynamic therapy of PSMA-expressing tumors

Abstract

Rationale: Prostate cancer (PCa) recurrences after surgery frequently occur. To improve the outcome after surgical resection of the tumor, the theranostic multimodal anti-PSMA targeting agent ¹¹¹In-DTPA-D2B-IRDye700DX was developed and characterized for both pre- and intra-operative tumor localization and eradication of (residual) tumor tissue by PSMA-targeted photodynamic therapy (tPDT), which is a highly selective cancer treatment based on targeting molecules conjugated to photosensitizers that can induce cell destruction upon exposure to near-infrared (NIR) light. Methods: The anti-PSMA monoclonal antibody D2B was conjugated with IRDye700DX and DTPA and subsequently radiolabeled with ¹¹¹In. To determine the optimal dose and time point for tPDT, BALB/c nude mice with PSMA-expressing (PSMA⁺) s.c. LS174T-PSMA xenografts received the conjugate (24-240 µg/mouse) intravenously (8 MBq/mouse) followed by µSPECT/CT, near-infrared fluorescence imaging, and ex vivo biodistribution at 24, 48, 72 and 168 h p.i. Tumor growth of LS174T-PSMA xenografts and overall survival of mice treated with 1-3 times of NIR light irradiation (50, 100, 150 J/cm²) 24 h after injection of 80 µg of DTPA-D2B-IRDye700DX was compared to control conditions. Results: Highest specific tumor uptake was observed at conjugate doses of 80 µg/mouse. Biodistribution revealed no significant difference in tumor uptake in mice at 24, 48, 72 and 168 h p.i. PSMA⁺ tumors were clearly visualized with both µSPECT/CT and NIR fluorescence imaging. Overall survival in mice treated with 80 µg of DTPA-D2B-IRDye700DX and 1x 150 J/cm² of NIR light at 24 h p.i. was significantly improved compared to the control group receiving neither conjugate nor NIR light (73 days vs. 16 days, respectively, p=0.0453). Treatment with 3x 150 J/cm² resulted in significantly prolonged survival compared to treatment with 3x 100 J/cm² (p = 0.0067) and 3x 50 J/cm² (p = 0.0338). Principal conclusions:¹¹¹In-DTPA-D2B-IRDye700DX can be used for pre- and intra-operative detection of PSMA⁺ tumors with radionuclide and NIR fluorescence imaging and PSMA-targeted PDT. PSMA-tPDT using this multimodal agent resulted in significant prolongation of survival and shows great potential for treatment of (metastasized) prostate cancer.

Keywords: PSMA; intra-operative; near-infrared fluorescence; prostate cancer; targeted photodynamic therapy.

Figure 1

Internalization kinetics of ¹¹¹In‐DTPA-D2B (A) and ¹¹¹In‐DTPA‐D2B‐IRDye700DX (B) in LS174T‐PSMA cells. Binding and internalization is presented as the percentage of the added activity (mean ± SD). C) Cell viability of LS174T-PSMA cells in vitro following incubation with 10 µg/mL DTPA-D2B-IRDye700DX and NIR light irradiation with radiant exposures of 2, 5, 10, 30, 50, 100, and 150 J/cm².

Figure 2

A) Biodistribution of ¹¹¹In-DTPA-D2B-IRDye700DX (30 µg and 8 MBq per mouse, n=5 per group) in the PSMA-expressing tumors and several healthy tissues. B) NIRF (top) and µSPECT/CT (bottom) in mice (different mice per time point were used) with s.c. LS174T-PSMA tumors at 24, 48, 72, and 168h after injection of 30 µg of ¹¹¹In-DTPA-D2B-IRDye700DX.

Figure 3

Biodistribution of different doses of ¹¹¹In-DTPA-D2B-IRDye700DX (24, 80, or 240 µg/mouse, 8 MBq/mouse, n=5 mice per group) at 24 h after injection. One group of mice received 300 µg of D2B i.v. 2 days prior to i.v. application of 24 µg of ¹¹¹In-DTPA-D2B-IRDye700DX to block the uptake (Xs cold condition).

Figure 4

Kaplan-Meier plots of overall (A) and progression-free survival (B) (7 mice/group, excluded mice are indicated with apostrophes in the graphs) and tumor growth (C-F) of male BALB/c nude mice with s.c. PSMA⁺ LS174T-PSMA tumors after i.v. injection of 80 µg of DTPA-D2B-IRDye700DX, followed by exposure to different NIR light irradiation regimens. In panel C-F, tumor growth in mice that did not receive the conjugate but where irradiated with the highest NIR light dose (3x 150 J/cm²) (control group), was compared to that in mice treated with a single administration of the conjugate followed by NIR light exposure of 3x 150 J/cm² (C), 1x 150 J/cm² (D), 3x 100 J/cm² (E), and 3x 50 J/cm² (F).

Figure 5

A) HE, PSMA, caspase-3, ƴH2aX, and Ki-67 stainings of s.c. PSMA⁺ LS174T-PSMA tumors after i.v. administration of PBS (left) or 80 µg of ¹¹¹In-DTPA-D2B-IRDye700DX (middle (tumors dissected 1h after tPDT) and right (tumors dissected 24h after tPDT)). All scale bars indicate 500 µm. B) Staining intensity of immunohistochemical stainings of treated (n=5) and non-treated tumor.