Tumor penetration and epidermal growth factor receptor saturation by panitumumab correlate with antitumor activity in a preclinical model of human cancer

Abstract

Background: Successful treatment of solid tumors relies on the ability of drugs to penetrate into the tumor tissue.

Methods: We examined the correlation of panitumumab (an anti-epidermal growth factor [EGFR] antibody) tumor penetration and EGFR saturation, a potential obstacle in large molecule drug delivery, using pharmacokinetics, pharmacodynamics, and tumor growth rate in an A431 epidermoid carcinoma xenograft model of human cancer. To determine receptor saturation, receptor occupancy, and levels of proliferation markers, immunohistochemical and flow cytometric methods were used. Pharmacokinetic data and modeling were used to calculate growth characteristics of panitumumab-treated tumors.

Results: Treatment with panitumumab in vivo inhibited pEGFR, Ki67 and pMAPK levels vs control. Tumor penetration and receptor saturation were dose- and time-dependent, reaching 100% and 78%, respectively. Significant tumor inhibition and eradication (p < 0.05) were observed; plasma concentration associated with tumor eradication was estimated to be 0.2 μg/ml. The tumor inhibition model was able to describe the mean tumor growth and death rates.

Conclusions: These data demonstrate that the antitumor activity of panitumumab correlates with its ability to penetrate into tumor tissue, occupy and inhibit activation of EGFR, and inhibit markers of proliferation and MAPK signaling.

Figure 1

Panitumumab inhibited ligand-induced pEGFR in vitro and in vivo. Immunoprecipitation of both phospho- and total- EGFR harvested from (A) panitumumab-treated A431 epidermoid carcinoma cells in vitro 15 minutes after a treatment with EGF and (B) panitumumab-treated A431 xenograft tumors 30 minutes after an intraperitoneal injection of EGF.

Figure 2

The observed and modeled panitumumab PK profiles in xenograft mice after intraperitoneal administration of panitumumab twice a week. Serum concentrations of panitumumab were assessed 1, 2, 3, 4, 7, and 14 days after the first dose (symbols, n = 5/time point); lines represent modeled pharmacokinetic profiles.

Figure 3

Penetration of panitumumab into tumor xenograft tissue is dose and time dependent. Panitumumab initially surrounded the afferent blood supply and then penetrated diffusely into the surrounding tumor tissue. A431 tumor xenograft samples of animals receiving 0, 20, 200, or 500 μg panitumumab were collected (A) 24 hours or (B) 96 hours after initiation of dosing and incubated with anti-idiotype IgG₂-bound panitumumab to detect administered panitumumab (blue chromagen) and DAKO anti-EGFR to detect EGFR (red chromagen). Micron bars = 10 μm all high magnification images and black arrows identify blood vessels. (C) IHC staining intensities for the anti-idiotype IgG₂-bound panitumumab were qualitatively graded and plotted as an estimated percent of total staining intensity.

Figure 4

Treatment with panitumumab resulted in the saturation of EGFR on A431 epidermoid carcinoma cells in vitro and in vivo in xenograft tumors as determined by flow cytometry. (A) A431 cells were incubated in vitro with increasing concentrations of unlabeled panitumumab and phycoerythrin (PE)-labeled panitumumab to determine lowest concentration required to achieve cell-surface binding saturation. (B, C [inset]) FACS dot plots of PE-panitumumab vs Alexa-EGFR of A431 cells treated with 17 nM of either B, control IgG or C, panitumumab for 1 hour. (D) The percent of EGFR saturation following treatment was determined in vivo by measuring the level of bound panitumumab on the dissociated tumor xenograft cells by flow cytometry and plotting test results against the standard curve generated in Figure 4A. (E, F [inset]) FACS dot plots of PE-panitumumab vs Alexa-EGFR of dissociated A431 xenograft cells treated with 500 μg of either E, control IgG or F, panitumumab; tumor cells were dissociated and assayed 7-days post-treatment.

Figure 5

Markers of proliferation (Ki67) and downstream kinase signaling (pMAPK) in A431 epidermoid carcinoma xenografts were decreased after treatment with panitumumab. Fixed tissue sections were prepared from A431 xenograft tumors from mice treated with either 500 μg IgG₂ control vehicle or 500 μg panitumumab twice a week for two weeks. Qualitative changes in Ki67 or pMAPK immunoreactivity were visualized with DAB and hematoxylin counterstain. Micron bars = 100 μm for low magnification images on the left and 10 μm for high magnification images on the right. Representative fields are shown.

Figure 6

Observed and model-fitted tumor growth curves in an A431 carcinoma xenograft model. (A) Mice with established A431 tumors received PBS (purple line), human IgG₂ 500 μg (blue line), panitumumab 5 μg (orange line), 20 μg (brown line), 200 μg (red line) or 500 μg (green line) for 52 days. Mice were monitored continuously for 300 days after the last dose of panitumumab was administered. (B) Tumor inhibition modeled data.