Abstract
There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed “trimerbody”, comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting.
Highlights
An optimized antibody fragment designed for targeting cancer in vivo should fulfill several requirements: high specificity and affinity for the target antigen, low immunogenicity; and be ready available form expression to purified protein [1]
Design and Expression of Trimerbody Constructs Structural analysis of the NC1 domain of collagen XVIII suggests that it consists of three segments, an N-terminal trimerization domain implicated in self-assembly of homotrimers; a central protease-sensitive hinge region; and the compact C-terminal endostatin domain [30]
We have previously shown that an engineered antibody containing the anti-laminin L36 scFv and the N-terminal trimerization domain of murine collagen XVIII NC1 was produced in human 293 cells in a functional active form [15]
Summary
An optimized antibody fragment designed for targeting cancer in vivo should fulfill several requirements: high specificity and affinity for the target antigen, low immunogenicity; and be ready available form expression to purified protein [1]. The pharmacokinetic properties of the antibody should be adjusted depending on the intended use. Intact IgG molecules (150 kDa) display low blood clearance and incomplete tumor penetration. Small monovalent single-chain variable fragments (scFv) (25–30 kDa) are more effective in tumor penetration but they are cleared too rapidly and have poor tumor retention because of their binding properties [2]. The ideal tumor-targeting antibodies are intermediate-sized multivalent molecules, which provide rapid tissue penetration, high target retention and rapid blood clearance. Recent biodistribution studies [3] indicate that bivalent antibodies such as diabodies (60 kDa), and minibodies (80 kDa) may be best suited for tumor imaging and therapy due to a higher total tumor uptake and better tumor-to-blood ratios than intact IgG molecules
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