Photoimmunotherapy targeting prostate-specific membrane antigen: are antibody fragments as effective as antibodies?

Abstract

Photoimmunotherapy is a highly cell-selective cancer therapy based on an armed antibody conjugate with a phthalocyanine-based photosensitizer, IR700. Photoimmunotherapy induces rapid and highly specific necrosis in targeted cancer cells after exposure to near-infrared (NIR) light. Cells not expressing the antigen are not affected. To date, photoimmunotherapy has been demonstrated only with full antibody-IR700 conjugates. In this study, small and bivalent antibody fragments, including anti-prostate-specific membrane antigen (PSMA) diabody (Db) and minibody (Mb), were compared with intact IgG for their effectiveness as photoimmunotherapy agents.

Methods: Radioiodinated antibody and antibody fragments with (125)I were used to determine the timing of maximum binding of each anti-PSMA antibody fragment on the cell surface in vivo in mice bearing either PSMA-positive or -negative PC3 tumors. Then therapeutic efficacy of photoimmunotherapy was examined by exposing mice to NIR light at 2 time points based on the time of maximum cell surface binding at 6 h after injection for Db-IR700 and 24 h after injection for Mb-IR700 and IgG-IR700 as well as 24 h after the peak uptake times.

Results: Photoimmunotherapy with the same molar concentration of PSMA-Db-IR700, PSMA-Mb-IR700, and PSMA-IgG-IR700 conjugate showed similar therapeutic effects in vitro and in vivo on PSMA-positive PC3 tumor xenografts in cytotoxicity and survival curves (P > 0.05).

Conclusion: The use of PSMA-Db-IR700 conjugate results in the shortest time interval between injection and NIR exposure without compromising therapeutic effects of photoimmunotherapy.

Keywords: diabody; minibody; monoclonal antibody; pharmacokinetics; photoimmunotherapy; prostate specific membrane antigen.

FIGURE 1.

Differential interference contrast and fluorescence microscopy of photoimmunotherapy-treated mix of PC3pip (PSMA-positive) and flu (PSMA-negative) cells. Cells were incubated with anti-PSMA antibodies (IgG-IR700), Mbs (Mb-IR700), or Dbs (Db-IR700) at 10 μg/mL. Just after fluorescence imaging, only PSMA-positive cells demonstrate necrotic cell death (cell budding/rupture). Scale bar 5 50 μm. PIT 5 photoimmunotherapy.

FIGURE 2.

Target-specific cell death in response to IgG-, Mb-, or Db-IR700–mediated photoimmunotherapy in PSMA-positive (A) or -negative (B) cells. Target-specific cell death in response to photoimmunotherapy was dose-dependent to intensity of irradiation. Data are mean ± SEM (n ≥ 3).

FIGURE 3.

Analysis of in vivo biodistribution of radioactivity after injection of ¹²⁵I-PSMA-IgG (A), Mb (B), or Db (C) into mice bearing PSMA positive (PSMA1) and negative (PSMA−) tumors. Each value was calculated using percentage injected dose per gram of tissue (Supplemental Fig. 1) for each animal and represented as mean ± SEM (n = 3–5): ratios of each organ and tumor to blood (left), subtraction of PSMA-negative tumor from PSMA-positive tumor (center), and ratios of PSMA-positive tumor to PSMA-negative tumor (right). * = value cannot be calculated because some denominators are 0.

FIGURE 4.

Tumor volume and survival curves of PSMA-positive tumor-bearing mice. Mice were randomized to 1 of 3 groups at 3 d after tumor cell injection (n = 9–10). IgG-IR700 (A), Mb-IR700 (B), or Db-IR700 (C) was injected, and photoimmunotherapy was performed at 1 and 2 d after injection for IgG-IR700 or Mb-IR700 and at 6 h and 1 d after injection of Db-IR700. Mice were observed until tumor volume reached more than 500 mm³, at which time mice had to be euthanized. *P < 0.05 vs. control, Dunnett multiple-comparison test. #P < 0.05 vs. control, log-rank test. Ab = antibody.