Measuring the pharmacodynamic effects of a novel Hsp90 inhibitor on HER2/neu expression in mice using Zr-DFO-trastuzumab

Abstract

Background: The positron-emitting radionuclide (89)Zr (t(1/2) = 3.17 days) was used to prepare (89)Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.

Methodology/principal findings: Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [(89)Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in (89)Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. (89)Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3+/-2.1 MBq/mg (2.82+/-0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87+/-0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68+/-13.06%ID/g; 71.71+/-10.35%ID/g and 85.18+/-11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of (89)Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75+/-4.43%ID/g and 41.42+/-3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64+/-12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.

Conclusions/significance: The results indicate that (89)Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles.

Figure 1. Plot of the (total/bound) activity versus (1/[normalized cell concentration]), used to calculate the immunoreactivity fraction of ⁸⁹Zr-DFO-trastuzumab in BT-474 (HER2/neu positive) cells by extrapolation to infinite antigen excess (1/y-intercept).

Figure 2. In vitro Western blots showing the change in protein expression levels in response to treatment with PU-H71 at 0.01–1 µM concentrations.

DMSO vehicle treated experiments and β-actin are include as controls. Proteins investigated include: HER2/neu; Akt; phosphorylated Akt (P-Akt); the apoptosis marker, cleaved poly (ADP-ribose) polymerase (cPARP); RAF1; Hsp90; Hsp70; and β-actin as a protein loading control.

Figure 3. Bar charts showing selected tissue biodistribution data (%ID/g) for (A) uptake of high and low specific-activity formulations of ⁸⁹Zr-DFO-trastuzumab in BT-474 tumor-bearing mice, and (B) ⁸⁹Zr-DFO-trastuzumab uptake in control (vehicle-treated) and PU-H71 treated animals at 12, 24, 48 and 72 h post-i.v. administration of ⁸⁹Zr-DFO-trastuzumab (0.55–0.74 MBq, 5–7 µg of mAb, in 200 µL 0.9% sterile saline).

Figure 4. Pharmacodynamic studies on protein expression levels in BT-474 tumor tissue samples obtained at 12, 24, 48, 72 and 96 h after PU-H71 treatment.

Figure 5. ImmunoPET images of ⁸⁹Zr-DFO-trastuzumab (8.50–9.25 MBq, 80–90 µg of mAb, in 200 µL 0.9% sterile saline) recorded in (A) BT-474 (left shoulder) and (B) MDA-MB-468 (right flank) tumor-bearing mice between 1–120 h post-injection.

The transverse (top) and coronal (bottom) planar images intersect the center of the tumors.

Figure 6. Time-activity curves derived by region-of-interest analysis of the immunoPET images showing the mean %ID/g tissue uptake versus time/h, for control and PU-H71-treated mice bearing both BT-474 and MDA-MB-468 tumors.

Figure 7. Coronal immunoPET images of control (left) and PU-H71 (right) treated mice bearing s.c. BT-474 and MDA-MB-468 tumors recorded at 24 h post-i.v. administration of ⁸⁹Zr-DFO-trastuzumab.

Mean and maximum T/M ratios obtained from VOI analysis are presented (Table S3). For the control animal, the two coronal PET slices lie through the center of the HER2/neu positive and negative tumors.