Approach for Half-Life Extension of Small Antibody Fragments That Does Not Affect Tissue Uptake.

The utility of antigen-binding antibody fragments is often limited by their short half-lives. Half-life extension of such fragments is usually accomplished by attachment or binding to high-molecular-weight carriers that reduce the renal elimination rate. However, the higher hydrodynamic radius results in greater confinement in the vascular compartment and, thus, lower tissue distribution. We have developed a chemically controlled drug delivery system in which the drug is covalently attached to hydrogel microspheres by a self-cleaving β-eliminative linker; upon subcutaneous injection, the t1/2,β of the released drug acquires the t1/2 of linker cleavage. In the present work, we compared the pharmacokinetics of an anti-TNFα scFv, the same scFv attached to 40 kDa PEG by a stable linker, and the scFv attached to hydrogel microspheres by a self-cleaving linker. We also developed a general approach for the selective attachment of β-eliminative linkers to the N-termini of proteins. In rats, the scFv had a t1/2,β of 4 h and a high volume of distribution at steady state (Vd,SS), suggesting extensive tissue distribution. The PEG-scFv conjugate had an increased t1/2,β of about 2 days but showed a reduced Vd,SS that was similar to the plasma volume. In contrast, the tissue-penetrable scFv released from the hydrogel system had a t1/2,β of about 2 weeks. Thus, the cleavable microsphere-scFv conjugate releases its protein cargo with a prolonged half-life comparable to that of most full-length mAbs and in a form that has the high tissue distribution characteristic of smaller mAb fragments. Other antigen-binding antibody fragments should be amenable to the half-life extension approach described here.