Immunogenicity and kinetics of distribution and elimination of sheep digoxin-specific IgG and Fab fragments in the rabbit and baboon

Abstract

To evaluate the relative merits of purified IgG and Fab preparations of defined specificity for potential clinical use, immunogenicity studies were carried out in baboon and rabbit experimental models. Distribution and elimination kinetics of purified sheep digoxin-specific IgG and Fab fragments were also studied following intravenous administration to baboons. Serial plasma and urine Fab concentrations were determined from trichloroacetic acid-precipitable 125I counts from pre-labelled preparations and also by measurement of the antibody's functional 3H-digoxin binding capacity. Results were compared with data obtained from IgG by 3H-digoxin binding. Kinetic data analysed by computer-fitted functions demonstrated that plasma Fab disappearance was best described by a tri-exponential function, whereas a bi-exponential function best described the IgG data. Initial distribution half-life (t 1/2) of Fab (0.28-0.32 hr) was considerably shorter than that of IgG (4.0 hr) and contributed a greater proportion of the total fall in plasma level over 24 hr. Fab elimination t 1/2 (9-13 hr) was also shorter than IgG (61 hr), but appreciably longer than earlier estimates in rabbits, guinea-pigs, rats and mice. The total volume of distribution of Fab was 8.7 times greater than that of IgG measured by the same method. Over the first 24 hr after administration 30-45% of administered Fab was recoverable in active form in urine, while 93% of total administered 125I counts from 125I-Fab preparations (bound and free) could be recovered. Less than 1% of administered IgG binding activity was recovered in urine during the initial 24 hr. The relative immunogenicities of sheep digoxin-specific IgG and Fab fragments were studied in six baboons. Both IgG and Fab elicited prompt immune responses when injected intramuscularly with Freund's complete adjuvant. Intravenous injection of soluble sheep IgG resulted in a prompt immune response in one baboon while repeated injections caused only a late, weak response in a second animal. Soluble sheep Fab fragments elicited only delayed and weak responses in the two baboons thus challenged. Further immunogenicity studies in ninteen rabbits showed significantly earlier and greater antibody responses to intravenously administered sheep IgG antigen than to Fab fragments derived from the same IgG population. These studies demonstrate that digoxin-specific Fab fragments undergo more rapid and extensive distribution to the extra vascular compartment and also more rapid renal excretion than IgG. Furthermore, Fab fragments are significantly less immunogenic than the parent IgG population. These data indicate potentially important therapeutic advantages for digoxin-specific Fab compared with IgG when administered for the reversal of life-threatening digitlis toxicity.