Antibodies as Drug Carriers. II. For Proteins

Abstract

Purpose. To evaluate the potential use of antibodies as a carrier for monovalent protein haptens.

Methods. A single −SH functionality present in the human IgG light chain was fluoresceinated. This conjugate, FL-LC, was treated with pepsin to obtain FL conjugate of half light chain, FL-(LC)_1/2, of MW 11 kDa. These two were radiolabeled using [³H]-propionic acid N-hydroxysuccinimide ester, and administered via tail vein to FL-immunized or mock-immunized mice. The blood radioactivity was measured over a 72-h period. Attempts were made to measure the affinity constant for the interaction between the conjugates and anti-FL antibodies by fluorescence quenching, surface plasmon resonance spectroscopy, and competitive ELISA.

Results. All of the three methods used produced supportive, if not conclusive, evidence of decreased binding affinity with increased conjugate size. Subsequent to tail-vein injection to FL-immunized mice, FL-LC showed approximately 4-fold smaller volume of distribution than mock-immunized mice: 0.041 ± 0.005 vs. 0.16 ± 0.02 mL/g. Corresponding values for FL-(LC)_1/2 were significantly larger: 0.070 ± 0.013 and 0.30 ± 0.02 mL/g, respectively. Compared with a small FL conjugate of ethanolamine, FL-EA, we studied earlier, the dose-normalized concentrations of the protein conjugates started at a higher level but declined more rapidly with time. In mock-immunized mice, the radioactivity disappeared very rapidly after administration, followed by an extremely slow decline with half-life close to 60 h. Evidence is provided to support that the radiolabel dissociated in the kidney, however, binding to anti-FL antibodies greatly stabilized the conjugate.

Conclusions. Based on an entropic principle alone the affinity of monovalent hapten-antibody interaction is expected to diminish with increase in hapten size. As such, the size of a hapten should be an important determinant of its pharmacokinetics in animals harboring antibodies that recognize the hapten. Relative to what was observed with small MW FL-EA, the protein conjugates showed substantially sustained circulation as a result of antibody binding, but this effect was diminished at later time points. Both affinity and pharmacokinetic data are consistent with the hypothesis of reduced affinity with increasing MW for monovalent hapten conjugates, but neither offered overwhelming proof.