Biodistribution of HuCC49DeltaCH2-beta-galactosidase in colorectal cancer xenograft model

Abstract

Antibody-enzyme conjugate (AbE) has been widely studied for site-specific prodrug activation in tumors. The purpose of this study is to characterize the pharmacokinetics and tissue distribution of HuCC49DeltaCH2-beta-galactosidase conjugate. HuCC49DeltaCH2 and beta-galactosidase were chemically conjugated and injected into a LS 174T colon cancer xenograft model. A colorimetric assay was developed to quantify the HuCC49DeltaCH2-beta-galactosidase levels in plasma and tissues. The HuCC49DeltaCH2-beta-galactosidase conjugate distributed into tumor tissue as early as 6h with the tumor/blood ratio of 5. This favored distribution of conjugate activity in the tumor tissue which was maintained up to 4 days post conjugate injection, while the conjugate was cleared rapidly from blood and other normal tissues (heart, spleen, lung, liver, kidney and stomach). At a high dose of 3000 U/kg, HuCC49DeltaCH2-beta-galactosidase conjugate saturated the antigen binding sites and yielded decreased tumor/normal tissue ratios compared to 1500 U/kg. These data suggest that HuCC49DeltaCH2-beta-galactosidase specifically target to the tumors to increase tumor selectivity.

Fig 1

A, The TAG-72 expression was detected using western blot in colorectal cancer cells (LS174T) in vitro, normal tissues and xenograft tumors in vivo. B, Immunostaining of HuCC49ΔCH2-β-galactosidase conjugates in the LS174T xenograft tumors 3 days post antibody-enzyme administration. 1, 20X; 2, 40X.

Figure 2

Standard Curve of colorimetric assay of enzymatic activity of β-galactosidase.

Figure 3

Plasma concentration vs. time of HuCC49ΔCH2-β-galactosidase conjugate activity after i.v. bolus administration (1000 units/kg) in the LS174T xenograft.

Figure 4

HuCC49ΔCH2-β-galactosidase conjugate activity (U/gram or U/mL) in tumor, plasma and normal tissues at various time points post injection of the conjugate (1000 units/kg)

Figure 5

Tumor: normal tissue ratios of HuCC49ΔCH2-β-galactosidase conjugate at various time points post conjugate injection (1000 units/kg).

Fig 6

Biodistribution of HuCC49ΔCH2-β-galactosidase activity in xenograft mice. Groups of 4–5 mice received 1500 or 3000 units/kg of conjugates. At 48 and 72 hours, the animals were sacrificed and the enzymatic activity of conjugates in tumors and normal organs was determined as described in method section (A and B). The tumor: tissue ratios are presented in C and D.