OXavidin for tissue targeting biotinylated therapeutics

Abstract

Avidin is a glycoprotein from hen egg white that binds biotin with very high affinity. Here we describe OXavidin, a product containing aldehyde groups, obtained by ligand-assisted sugar oxidation of avidin by sodium periodate. OXavidin chemically reacts with cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks while preserving the biotin binding capacity. The long tissue residence of OXavidin as well as that of OXavidin/biotinylated agent complex occurs in normal and neoplastic tissues and immunohistochemistry shows a strong and homogenous stromal localization. Once localized in tissue/tumor, OXavidin becomes an "artificial receptor" for intravenous injected biotin allowing tumor targeting with biotinylated therapeutics like radioisotopes or toxins. Moreover, present data also suggest that OXavidin might be useful for the homing of biotinylated cells. Overall, OXavidin exhibits a remarkable potential for many different therapeutic applications.

Figure 1

Diffusion kinetic of ¹²⁵I-labeled avidin or streptavidin injected in a limb and distribution in blood, liver, and kidney. At indicated time points, mice were sacrificed and tissue samples weighted and counted in a gamma-counter. Data are expressed as % ID/100 mg of tissue. Each point is the average of 5 mice. Bars represent standard deviation.

Figure 2

Tissue residence of avidin, PEGavidin, and oxidized avidin. ¹²⁵I-labeled avidin, PEGavidin, and oxidized avidin were formulated in 100 mM sodium acetate pH 5.5 and injected in the limb muscle at the dose of 50 μg in 15 μL/mouse. Mice were sacrificed 24 hours after injection and tissue samples weighted and counted in a gamma-counter. Data are expressed as % ID/100 mg of tissue. Each point is the average of 5 mice. Bars represent standard deviation. Student's t test was used for statistical analysis. NS: Not significant.

Figure 3

Oxidized avidin in vivo residence and biotin uptake. Diffusion kinetic of ¹²⁵I-labeled avidin and oxidized avidin injected in a limb of Balb/c mice (a)–(d) and uptake of intravenously administered ¹¹¹In-ST2210 (e)–(h), after 1, 24, or 48 hours. Mice were sacrificed 1 hour after ¹¹¹In-ST2210 injection and injected sites as well as samples of non target organs were weighted and counted in a gamma-counter. Each point is the average of 5 mice. Bars represent standard deviation. Student's t test was used for statistical analysis. NS: Not significant.

Figure 4

Immunohistochemistry of avidin (a,c) or OXavidin (b,d) injected normal muscle (a,b) or MDA-MB-468 human breast carcinoma xenotransplanted mass (c,d) 48 hours after injection.

Figure 5

Uptake of ¹¹¹In-ST2210, intravenously injected 48 hours after intramuscular administration of OXavidin or avidin in a limb. Groups of mice were sacrificed at the indicated time points after ¹¹¹In-ST2210 injection and samples of treated limb, spleen, liver, and kidney weighted and counted in a gamma counter. Data are expressed as % ID/g of tissue. Each point is the average of 5 mice. Bars represent standard deviation.

Figure 6

Tissue residence of OXavidin/biotin complex. ¹²⁵I-labeled avidin, avidin/ST2210, OXavidin, or OXavidin/ST2210 were injected in the limb of Balb/c mice. At the indicated time points after intramuscular injection mice were sacrificed and samples of treated limb weighted and counted in a gamma counter. Data are expressed as % ID/100 mg of tissue. Each point is the average of 5 mice. Bars represent standard deviation. Student's t test was used for statistical analysis. NS: Not significant.

Figure 7

OXavidin uptake of biotinylated cells. OXavidin shows highly efficient uptake of biotinylated GFP-expressing B16 cells compared to avidin. Black and white histograms indicate the average of nine fields of B16 and biotinylated B16 cells, respectively. Bars represent standard deviation. Student's t test was used for statistical analysis.