New Strategy for the Extension of the Serum Half-Life of Antibody Fragments

Abstract

Antibody fragments can recognize their cognate antigen with high affinity and can be produced at high yields, but generally display rapid blood clearance profiles. For pharmaceutical applications, the serum half-life of antibody fragments is often extended by chemical modification with polymers or by genetic fusion to albumin or albumin-binding polypeptides. Here, we report that the site-specific chemical modification of a C-terminal cysteine residue in scFv antibody fragments with a small organic molecule capable of high-affinity binding to serum albumin substantially extends serum half-life in rodents. The strategy was implemented using the antibody fragment F8, specific to the alternatively spliced EDA domain of fibronectin, a tumor-associated antigen. The unmodified and chemically modified scFv-F8 antibody fragments were studied by biodistribution analysis in tumor-bearing mice, exhibiting a dramatic increase in tumor uptake for the albumin-binding antibody derivative. The data presented in this paper indicate that the chemical modification of the antibody fragment with the 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate albumin-binding moiety may represent a general strategy for the extension of the serum half-life of antibody fragments and for the improvement of their in vivo targeting performance.

Bicyclic Peptides Conjugated to an Albumin-Binding Tag Diffuse Efficiently into Solid Tumors

“…This was expected because the binding affinity for uPA is weak and/or the concentration of uPA in tumor relatively low. This is in contrast with the studies of Dennis and colleagues and Neri and colleagues (13,16), in which antibody fragments were strongly enriched in tumors. The low affinity for the target protein allowed in our work studying the delivery of bicyclic peptide into tissues of tumor and organs solely based on diffusion as free or albumin-bound peptide.…”

“…The ligands can dissociate from the large albumin (66.5 kDa) and may diffuse into spaces such as cavities that are not accessible to large protein-based ligands. This strategy has been applied to enhance the accumulation of antibody fragments such as Fab, scFv, or single-chain diabodies in tumors (12)(13)(14)(15)(16). The much prolonged half-life resulted in a greatly improved targeting.…”

A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

“…The in vivo targeting performance of anti-EDA antibody fragments was evaluated by quantitative biodistribution analysis as previously described by Tarli et al 18 Briefly, antibodies were first loaded on Superdex 75, and the peaks corresponding to the desired molecular weight (according to the equation reported above) were collected. Purified antibody preparations were then radiolabeled with 125 I using the chloramineT method 62 and injected into the tail vein of immunocompetent 129SvEv mice bearing s.c. implanted F9 murine teratocarcinoma (8-10 μg per mouse). Mice were sacrificed 24 and 48 h after injection.…”

DOTA Conjugate with an Albumin-Binding Entity Enables the First Folic Acid–Targeted 177Lu-Radionuclide Tumor Therapy in Mice

“…These derivatives display a stable noncovalent binding interaction of variable affinity with both mouse and human serum albumin (27). The conjugation of the highaffinity albumin binder 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate (dissociation constant 3 mM) to antibody fragments resulted in a significantly increased tumor uptake (28).…”

“…Using this strategy, we synthesized a novel folic acid conjugate with both a DOTA chelator for coordination of a therapeutic radioisotope such as 177 Lu ( 177 Lu: half-life, 6.7 d; average b 2 energy, 134 keV; g-energies, 113 keV, 208 keV) and the low-molecular-weight albumin-binding entity (27,28) as an additional functionality (Fig. 1).…”