Evaluation of (89)Zr-pertuzumab in Breast cancer xenografts

Abstract

Pertuzumab is a monoclonal antibody that binds to HER2 and is used in combination with another HER2-specific monoclonal antibody, trastuzumab, for the treatment of HER2+ metastatic breast cancer. Pertuzumab binds to an HER2 binding site distinct from that of trastuzumab, and its affinity is enhanced when trastuzumab is present. We aim to exploit this enhanced affinity of pertuzumab for its HER2 binding epitope and adapt this antibody as a PET imaging agent by radiolabeling with (89)Zr to increase the sensitivity of HER2 detection in vivo. Here, we investigate the biodistribution of (89)Zr-pertuzumab in HER2-expressing BT-474 and HER2-nonexpressing MDA-MB-231 xenografts to quantitatively assess HER2 expression in vivo. In vitro cell binding studies were performed resulting in retained immunoreactivity and specificity for HER2-expressing cells. In vivo evaluation of (89)Zr-pertuzumab was conducted in severely combined immunodeficient mice, subcutaneously inoculated with BT-474 and MDA-MB-231 cells. (89)Zr-pertuzumab was systemically administered and imaged at 7 days postinjection (p.i.) followed by terminal biodistribution studies. Higher tumor uptake was observed in BT-474 compared to MDA-MB-231 xenografts with 47.5 ± 32.9 and 9.5 ± 1.7% ID/g, respectively at 7 days p.i (P = 0.0009) and blocking studies with excess unlabeled pertuzumab showed a 5-fold decrease in BT-474 tumor uptake (P = 0.0006), confirming the in vivo specificity of this radiotracer. Importantly, we observed that the tumor accumulation of (89)Zr-pertuzumab was increased in the presence of unlabeled trastuzumab, at 173 ± 74.5% ID/g (P = 0.01). Biodistribution studies correlate with PET imaging quantification using max SUV (r = 0.98, P = 0.01). Collectively, these results illustrate that (89)Zr-pertuzumab as a PET imaging agent may be beneficial for the quantitative and noninvasive assessment of HER2 expression in vivo especially for patients undergoing trastuzumab therapy.

Keywords: 89Zr; HER2; NOG; PET; breast cancer xenograft; pertuzumab; trastuzumab.

Figure 1

ESI-MS quantified up to four DFO chelates per antibody. (A) Unconjugated pertuzumab depicted in +25, +24, and +23 charge states and (B) DFO-Bz-NCS-pertuzumab conjugate in the same charge states as the unconjugated control.

Figure 2

In vitro cell binding studies show ⁸⁹Zr-pertuzumab retains immunoreactivity and specifically binds to a different HER2 binding site than trastuzumab. Both antibodies internalize at the same rate. (A) Immunoreactivity of ⁸⁹Zr-pertuzumab in SKBR3 cells was determined with a y intercept of 1.39 ± 0.04 and calculated an immunoreactive fraction of 72.2%. (B) Cell uptake studies at 37 °C in BT-474 (HER2+), SKBR3 (HER2+), and MDA-MB-231 (HER2−) demonstrate significantly higher ⁸⁹Zr-pertuzumab uptake in the HER2+ cell lines compared to the HER2– control (P < 0.001). No statistical difference was observed between BT-474 and SKBR3 (P > 0.05). (C) Competitive binding studies of ⁸⁹Zr-pertuzumab with unlabeled pertuzumab in SKBR3 cells at 4 °C yields an EC₅₀ of 2.4 ± 0.11 nM, and no competition was observed with unlabeled trastuzumab. (D) Cell internalization studies in SKBR3 cells at 37 °C up to 48 h demonstrate 50% of ⁸⁹Zr-pertuzumab internalizes at about 20 h, similar to ⁸⁹Zr-trastuzumab. Control reactions when receptor internalization is reduced at 4 °C show minimal internalization of radiolabeled antibodies at 48 h.

Figure 3

In vivo studies show high and specific tumor uptake of ⁸⁹Zr-pertuzumab in HER2+ tumors, enhanced in the presence of trastuzumab. (A) Maximum intensity projection of PET/CT images of ⁸⁹Zr-pertuzumab in BT-474 (HER2+) and MDA-MB-231 (HER2−) breast cancer xenografts at 7 days p.i. The scale bar is from 0–600 kBq/mL. (B) Max SUV quantification of ⁸⁹Zr-pertuzumab tumor uptake in BT-474 was 10.5 ± 6.1, increased in the presence of unlabeled trastuzumab (19.0 ± 5.4), decreased in the presence of a blocking concentration of unlabeled pertuzumab (2.2 ± 2.5), and decreased in MDA-MB-231 xenografts (2.2 ± 0.8). (C) Biodistribution studies correlate with PET imaging quantification with 47.5 ± 32.9% ID/g for ⁸⁹Zr-pertuzumab in BT-474, 173 ± 74.5% ID/g in the presence of unlabeled trastuzumab (P = 0.01), 8.5 ± 6.6% ID/g in the presence of unlabeled pertuzumab (P = 0.0006), and 9.5 ± 1.7% ID/g in the HER2– MDA-MB-231.

Figure 4

Biodistribution of ⁸⁹Zr-pertuzumab in HER2+ BT-474 and HER2– MDA-MB-231 xenografts. All mice were injected with ⁸⁹Zr-pertuzumab. A cohort of BT-474 xenografts were coinjected with either unlabeled trastuzumab (checkered bars) or unlabeled pertuzumab (horizontal line bars). ⁸⁹Zr-pertuzumab alone with no other cotreatments added were studied in BT-474 (black bars) or MDA-MB-231 (unfilled bars).