Quantitating antibody uptake in vivo: conditional dependence on antigen expression levels

Abstract

Purpose: Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation.

Procedures: Using a cell line with high epithelial cell adhesion molecule expression and moderate epidermal growth factor receptor expression, fluorescent antibodies with similar plasma clearance were imaged in vivo. A mathematical model and mouse xenograft experiments were used to describe the effect of antigen expression on uptake of these high-affinity antibodies.

Results: As predicted by the theoretical model, under subsaturating conditions, uptake of the antibodies in such tumors is similar because localization of both probes is limited by delivery from the vasculature. In a separate experiment, when the tumor is saturated, the uptake becomes dependent on the number of available binding sites. In addition, targeting of small micrometastases is shown to be higher than larger vascularized tumors.

Conclusions: These results are consistent with the prediction that high affinity antibody uptake is dependent on antigen expression levels for saturating doses and delivery for subsaturating doses. It is imperative for any probe to understand whether quantitative uptake is a measure of biomarker expression or transport to the region of interest. The data provide support for a predictive theoretical model of antibody uptake, enabling it to be used as a starting point for the design of more efficacious therapies and timely quantitative imaging probes.

Figure 1

Effect of antigen expression on uptake. When the Clearance modulus and Thiele modulus are less than 1, the tumor concentration is a function of the antigen expression level. At subsaturating concentrations, the uptake is a function of transport to the tumor (A). Targeting of antibodies is similar between moderately expressed EGFR and highly expressed EpCAM at subsaturating concentrations (B). Image of a tumor targeted by anti-EpCAM and anti-EGFR antibodies showing qualitatively similar uptake (C). Scale bar = 5 mm

Figure 2

Quantifying antibody uptake. The fluorescence intensity between anti-EpCAM and anti-EGFR antibodies is similar with the effects of the fluorophore accounting for most of the difference.

Figure 3

Effect of saturating antigen with a non-fluorescent blocking antibody. A blocking dose that saturates EGFR but not EpCAM will lower uptake of anti-EGFR antibodies but not the anti-EpCAM antibodies (A). Mice given 150 μg doses of anti-EGFR and anti-EpCAM antibodies before administration of fluorescent antibodies had little effect on targeting of the EpCAM antibody but significantly lowered the uptake of anti-EGFR antibodies (B). Scale bar = 5 mm

Figure 4

Quantifying antibody uptake after preblocking the antigen. The targeting of the EpCAM antibody was not affected by the blocking dose, but the EGFR antibody had significantly lower uptake.

Figure 5

Targeting micrometastases and small tumors. Antibody diffusing inward from the surrounding normal tissue targets a larger fraction of a small tumor (A). Significant uptake of anti-EpCAM antibody and saturation of the EGFR in a small metastasis (B). Scale bar = 5 mm. Mathematical model of uptake in a small prevascular metastasis. The large number of binding sites immobilizes the EpCAM antibody in the periphery, while the few EGFR binding sites are saturated at a low level (C, top). The overall concentration of EpCAM antibody in the small metastasis is higher than EpCAM antibody in the large tumor due to the high surface area to tumor volume of the metastasis (C, bottom).