Isothiocyanate-trigalactose: application for antibody-targeted delivery of diagnostic and therapeutic agents.

Radiolabeled monoclonal antibodies (MAb) and MAb-streptavidin conjugates exhibit slow blood clearance which impedes radioimmunoimaging and radioimmunotherapy. To control blood clearance and lower background levels, lesion-specific targeting proteins can be modified with galactose derivatives for liver uptake via the hepatocyte galactose receptor. In this study, an isothiocyanate-trigalactose derivative (ITC-Tgal) designed for direct coupling to protein amino groups, was synthesized and characterized. In vitro experimentation demonstrated efficient conjugation of ITC-Tgal to streptavidin (SA) and MAb Fab fragment with a corresponding decrease in protein net charge. In vivo studies were conducted with radiolabeled ITC-Tgal modified and native SA and MAb Fab fragment. ITC-Tgal modified SA and Fab fragment exhibited increased blood clearance with the liver uptake and the rate of blood clearance controlled by the extent of ITC-Tgal modification.

Biochemical modification of streptavidin and avidin: in vitro and in vivo analysis.

UNLABELLED: The high affinity streptavidin (or avidin)/biotin system is being investigated for imaging and radiotherapy procedures. Streptavidin (SA) and avidin exhibit markedly different pharmacokinetics, with avidin clearing from the blood much faster than SA. To optimize blood clearance kinetics, SA and avidin were biochemically modified and analyzed in vitro and in vivo. METHODS: Galactose moieties were covalently attached to promote binding by hepatocyte galactose receptors and hasten SA clearance. To prolong avidin clearance, avidin was deglycosylated and/or neutralized by acetylation of its lysine amino acids. In vitro, the modified proteins were analyzed by isoelectric focusing, SDS polyacrylamide electrophoresis and a biotin binding saturation assay. The modified and native proteins were radiolabeled with 131I and injected into rabbits for pharmacokinetic, redistribution and imaging analysis. RESULTS: For SA, the resulting increase in blood clearance and liver accumulation was correlated to the amount of galactose bound to SA. For avidin, each type of modification increased its circulation time, with the slowest clearance resulting from a combination of deglycosylation and neutralization. CONCLUSION: Biochemical modification of SA and avidin resulted in altered pharmacokinetics compared to the native proteins. Modified SA or avidin, when cross-linked with a lesion-specific targeting agent, may be applicable for rapid two-step in vivo imaging techniques.

Fluence rate effects in photodynamic therapy of multicell tumor spheroids.

EMT6/Ro spheroids approximately 500 microns in diameter were subjected to photodynamic therapy administered at various incident radiation fluence rates. Following 24 h incubation with 10 micrograms/ml Photofrin, groups of spheroids were irradiated at 630 nm with an identical fluence of 60 J/cm2, delivered at fluence rates ranging from 25 to 200 mW/cm2. After treatment, spheroids were dissociated, cell yields were determined, and surviving cells were assayed for their colony-forming ability. A surviving fraction was calculated for each treatment group by computing the product of the fractional cell yield and the plating efficiency. The results exhibit a strong dependence on the fluence rate, with surviving fractions decreasing from approximately 0.5 to 0.07 as the incident fluence rate was lowered from 200 to 25 mW/cm2. These data were analyzed using a mathematical model of photochemical oxygen consumption in spheroids undergoing photodynamic therapy. Calculations showed that therapy-induced oxygen consumption creates hypoxic volumes within which cells would be protected from singlet oxygen-mediated damage and that the magnitude of these hypoxic volumes depends on the radiation fluence rate. The fluence rate dependence of the spheroid cell survival was consistent with such an interpretation.