The introduced mutations did not interfere with protein expression and localization to the tumor. competition ELISA. Biodistribution studies in BALB/c mice using 125I- and 131I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was ~12 days. Additionally, 124I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn conversation provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting. Introduction Monoclonal antibodies and antibody fragments are not new to the pharmacology industry in both cancer treatment and imaging. Although favorable in terms of stability, target affinity, and specificity, native antibodies are noted for their prolonged serum half-life. Thus, a problem arises when IRL-2500 intact antibodies are conjugated to radionuclides or other toxic brokers. Because immunoglobulin G (IgG) remains in circulation for extended periods of time, the conjugate can cause significant irradiation and toxicity to normal organs, such as the bone marrow, liver, and kidneys. This would translate to reduced therapeutic dose, less frequent administrations, and ultimately compromised pharmacologic feasibility. Currently, the most common approach for minimizing antibody circulation persistence is reduction in size by deletion of domains. Several laboratories, including our own, have generated recombinant domain-deleted antibodies. One example is the antiCcarcinoembryonic antigen (CEA) T84.66 minibody, a dimeric engineered antibody fragment assembled VL-linker-VH-hinge-CH3, where VL is the light chain variable region, VH is the heavy chain variable region, and CH3 is the human IgG1 third constant domain name (Fig. 1]. This antibody fragment, in contrast to the minibody, behaves similarly to intact antibodies specifically regarding serum persistence and tumor uptake (4, 5). The scFv-Fc antibody fragment includes an intact Fc region, which is crucial for prolonging the half-life of antibodies (6) and antibody fragments. Specific interactions between antibody Fc domain name amino acid residues and the protective neonatal Fc receptor (FcRn; Brambell receptor) essentially divert IgGs from the lysosomal degradative pathway compared with other serum proteins (7C10). Open in a separate window Physique 1 schematic representation of an intact chimeric antibody and designed fragments. structure of the Fc region of human IgG1 with residues selected for mutation. The physique was generated using the RASMOL program (Roger Sayle, Bioinformatics Research Institute, University of Edinburgh, GB). design of the anti-CEA scFv-Fc fragment. Gene assembly including the 18 amino acid linker sequence between the V regions and the restriction sites used in the cloning and subcloning actions. Rabbit Polyclonal to HSP60 The FcRn has long been known to control the transfer of immunity (IgGs) from the mother to the offspring (11C17). Recent studies have shown a more intricate function of the FcRn receptor in maintaining the levels of IgGs in the circulation (i.e., by favoring antibody recycling rather than catabolism; refs. 8C10). In vascular endothelial cells, IgGs are taken up from the serum by fluid-phase endocytosis and delivered to early endosomes where FcRn resides. IgG binds FcRn with high affinity at slightly acidic pH (<6.5) but with low affinity at neutral pH (18, 19). At modest levels of antibody (due to the saturable nature of the intracellular FcRn-IgG conversation; ref. 20), most of the ligand binds FcRn and it is either recycled back to the circulation or delivered by transcytosis from the apical to the basolateral side of the endothelial cell. In either case, the neutral pH of the serum or the interstitial fluid promotes dissociation from the FcRn receptor and release of immunoglobulins. Essential for the FcRn binding, in both humans and rodents, are the residues Ile253 and His310 in the CH2 domain name and His435 in the CH3 domain name (Kabat numbering system; Fig. 1; refs. 21C24). Ward IRL-2500 et al. IRL-2500 showed that mutation of these amino acid residues correlates with reduced antibody fragment half-life (22). Furthermore, the solution of the structure of co-crystallized FcRn and Fc supported these findings by delineating the protein interface in human FcRn-human Fc and rat FcRn-rat Fc complexes (25, 26). Mutation of.