Targeted, activatable, in vivo fluorescence imaging of prostate-specific membrane antigen (PSMA) positive tumors using the quenched humanized J591 antibody-indocyanine green (ICG) conjugate

Abstract

In patients with prostate cancer, a positive surgical margin is associated with an increased risk of cancer recurrence and poorer outcome, yet margin status cannot be determined during the surgery. An in vivo optical imaging probe that could identify the tumor margins during surgery could result in improved outcomes. The design of such a probe focuses on a highly specific targeting moiety and a near-infrared (NIR) fluorophore that is activated only when bound to the tumor. In this study, we successfully synthesized an activatable monoclonal antibody-fluorophore conjugate consisting of a humanized anti-Prostate-Specific Membrane Antigen (PSMA) antibody (J591) linked to an indocyanine green (ICG) derivative. Prior to binding to PSMA and cellular internalization, the conjugate yielded little light; however, after binding an 18-fold activation was observed permitting the specific detection of PSMA+ tumors up to 10 days after injection of a low dose (0.25 mg/kg) of the reagent. This agent demonstrates promise as a method to image the extent of prostate cancer in vivo and could assist with real-time resection of extracapsular extension of tumor and positive lymph nodes.

Figure 1

A. Quenched (left) and chemically dequenched (right) J591-ICG and Tra-ICG. Dequenched conjugates show increased fluorescence signal shown in green. In vitro cell experiment with flow cytometry (B-D) and microscopy (E) showing specific binding of J591 to PSMA and activation of fluorescence signal after binding. Scale bar indicates 20 μm.

Figure 2

A. In vivo serial fluorescence signals of PC3-PSMA+ and PC3-PSMA- tumor as well as background in the body of mice injected with 50, 25, 12.5, and 6.25 μg of J591-ICG. B. In vivo serial fluorescence images of a PC3-PSMA+ and PC3-PSMA- tumor bearing mouse injected with 6.25 μg of J591-ICG. Only PC3-PSMA+ yields ICG fluorescence signal 3 days after injection or later.

Figure 3

A. In vivo fluorescence images of a PC3-PSMA+ and PC3-PSMA- tumor bearing mice 10 days after injection of 50, 25, 12.5, or 6.25 μg of J591-ICG are shown. Although ICG signal from PC3-PSMA+ tumors is stronger in mice injected with a larger dose, all PC3-PSMA+ tumors show ICG fluorescence signal. B. Ex vivo fluorescence images of five PC3-PSMA+ and PC3-PSMA- tumors obtained 10 days after injection of 6.25 μg J591-ICG are shown. PC3-PSMA+ tumors clearly show specific fluorescence signal of ICG. C. PC3-PSMA+ tumor-to-PC3-PSMA- tumor signal intensity ratio is plotted. All doses show a similar trend of changes on PC3-PSMA+ tumor-to-PC3-PSMA- tumor signal intensity ratio. Scattered PC3-PSMA- tumors, which showed signal below background, are eliminated from the calculation.

Figure 4

Side-by-side comparison of images (A) and serial fluorescence intensity curves (B) for PC3-PSMA+ and PC3-PSMA- tumor bearing mouse injected with 12.5 μg of J591-ICG versus 3T3HER2+ and 3T3HER2- tumor bearing mouse injected with 12.5 μg of Tra-ICG. Images (A) are obtained 3 days after injection of antibodies. The anti-PSMA conjugate yields higher signal than the anti-HER2 conjugate suggesting better delivery and/or more efficient internalization.