Antibodies as drug carriers. II. For proteins

Purpose. To evaluate the potential use of antibodies as a carrier for monov alent protein haptens. Methods. A single -SH functionality present in the human IgG light chain wa s 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 we re radiolabeled using [H-3]-propionic acid N-hydroxysuccinimide ester, and administered via tail vein to FL-immunized or mock-immunized mice. The bloo d radioactivity was measured over a 72-h period. Attempts were made to meas ure the affinity constant for the interaction between the conjugates and an ti-FL antibodies by fluorescence quenching, surface plasmon resonance spect roscopy, and competitive ELISA. Results. All of the three methods used produced supportive, if not conclusi ve, evidence of decreased binding affinity with increased conjugate size. S ubsequent to tail-vein injection to FL-immunized mice, FL-LC showed approxi mately 4-fold smaller volume of distribution than mock-immunized mice: 0.04 1 +/- 0.005 vs. 0.16 +/- 0.02 mL/g. Corresponding values for FL-(LC), 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 earl ier, the dose-normalized concentrations of the protein conjugates started a t a higher level but declined more rapidly with time. In mock-immunized mic e, the radioactivity disappeared very rapidly after administration, followe d by an extremely slow decline with half-life close to 60 h. Evidence is pr ovided to support that the radiolabel dissociated in the kidney, however, b inding to anti-FL antibodies greatly stabilized the conjugate. Conclusions. Based on an entropic principle alone the affinity of monovalen t hapten-antibody interaction is expected to diminish with increase in hapt en size. As such, the size of a hapten should be an important determinant o f its pharmacokinetics in animals harboring antibodies that recognize the h apten. Relative to what was observed with small MW FL-EA, the protein conju gates showed substantially sustained circulation as a result of antibody bi nding, but this effect was diminished at later time points. Both affinity a nd pharmacokinetic data are consistent with the hypothesis of reduced affin ity with increasing MW for monovalent hapten conjugates, but neither offere d overwhelming proof.