Pharmacokinetics and microdistribution of polyethylene glycol‐modified humanized A33 antibody targeting colon cancer xenografts

Abstract

Therapeutic proteins have been conjugated with polyeth-ylene glycol (PEGylation) to reduce immunogenicity and enhance circulating dose. Here we have investigated the effect of PEGylation on immunogenicity, pharmacokinetics, and histologic microdistribution of tumor-targeting antibodies with humanized A33 antibody (huA33) as a model system. Conjugation of huA33 with methoxy-PEG of M r 5,000 (32%-34% of primary amines modified) or M r 20,000 (16%-18% modification) preserved >50% of native huA33 binding to SW1222 colon cancer cells. In mice, both PEGylated forms cleared from serum moderately slower than native huA33. After repeated immunization with PEG-huA33, anti-antibody titers in immunocompetent mice were <5% of those in huA33-treated controls. Both PEG-huA33 forms reached approx. 75% of the maximum tumor dose of huA33 in SW1222-xenografted mice, but their tumor:blood ratios were considerably reduced. To demonstrate immunologic specificity of PEG-huA33 targeting in SW1222 tumor-bearing mice, antigenic sites were presaturated by injecting excess native huA33. This reduced subsequent uptake of PEG-huA33 by up to 80%, whereas presaturation with hu3S193 control antibody had no significant effect. To assess the mi-crodistribution of antibody uptake in the same xenograft model, tumor tissue resected at different time points after antibody administration was examined for human IgG by immunohistochemistry. Both PEG preparations achieved the same peak staining intensity and homogeneity as native huA33 with a delay of several hours. Given the measured reduction in immunoreactivity in vitro, these results demonstrate that the tumor targeting potential of huA33 in vivo is preserved at PEGylation levels sufficient to suppress immu-nogenicity. Int.

PEGylation Does Not Significantly Change the Initial Intravenous or Subcutaneous Pharmacokinetics or Lymphatic Exposure of Trastuzumab in Rats but Increases Plasma Clearance after Subcutaneous Administration

“…In this study, PEGylation of the A33 antibody resulted in an increase in the terminal half-life when compared to the native antibody. 30 This may have been due to the fact that PEGylation of the A33 antibody was performed by conjugating multiple (15 to 30) smaller molecular weight PEGs (5000−20000 Da) to a single antibody (final PEG to antibody ratios used were 15:1 PEG 20000 to antibody and 30:1 PEG 5000 to antibody). In addition, differential binding kinetics between PEGylated and native trastuzumab with soluble HER2 in plasma may play a role in the pharmacokinetic behavior of trastuzumab.…”

“…29 Furthermore, the PEGylation of antibodies has been shown in mouse tumor xenograft models to have the potential to increase disposition toward solid primary tumors and therefore improve chemotherapeutic activity. 30,31 This has been suggested to be via increases in molecular weight leading to more prolonged plasma exposure after iv administration and enhanced tumor uptake via the enhanced permeation and retention effect. 30,31 PEGylation therefore has the potential to improve the sc pharmacokinetic behavior of trastuzumab and increase lymphatic and solid tumor disposition.…”

“…30,31 This has been suggested to be via increases in molecular weight leading to more prolonged plasma exposure after iv administration and enhanced tumor uptake via the enhanced permeation and retention effect. 30,31 PEGylation therefore has the potential to improve the sc pharmacokinetic behavior of trastuzumab and increase lymphatic and solid tumor disposition. To this point, however, the impact of PEGylation on the sc pharmacokinetics and lymphatic disposition of very large proteins, such as antibodies, has not been explored.…”

“…Long-term control rats were dosed with 2 mg/kg PEG−trastuzumab as a bolus in sterile saline via a lateral tail vein (500 μL volume) or sc into the inner left heel (in a volume of 0.5 mL/kg) under isoflurane anesthesia. Blood samples (150 μL) were then collected from a lateral tail vein (alternate vein to the one receiving the iv dose for iv dosed rats) 30 Iv-dosed short-term control and thoracic lymph duct cannulated rats were administered PEG−trastuzumab at a dose of 2 mg/kg in 1 mL sterile saline via infusion over 2 min via the jugular vein. Sc-dosed short-term and thoracic lymph duct cannulated rats were administered PEG−trastuzumab as described above for sc dosed long-term rats.…”

“…Sc-dosed short-term and thoracic lymph duct cannulated rats were administered PEG−trastuzumab as described above for sc dosed long-term rats. Blood samples were collected via the carotid artery cannula in short-term control rats immediately after dosing (t 0 ) and at 5 min, 30 10 All blood and lymph samples were kept on ice until further processed. Blood samples were centrifuged at 3500g for 5 min to obtain plasma.…”