I-Labeled anti-prostate stem cell antigen affinity-matured A11 minibody

The use of intact antibodies as molecular imaging agents has some disadvantages, such as large size, poor tumor penetration, and immunogenicity (1-4). To minimize these problems, various formats of antibody fragments have been designed with the variable regions of the light chain (V) and the heavy chain (V) (1, 5, 6). Monovalent single-chain Fv (scFv) constructs (molecular weight, 25–30 kDa) represent the smallest antibody fragments, which are generated with V and V connected by a flexible linker. Although monovalent scFv fragments exhibit efficient tumor penetration, they are cleared rapidly from blood and can demonstrate poor antigen binding. For example, the β half-life of monovalent scFv in plasma is only 0.5–2.0 h (in contrast to 48–72 h for intact antibodies) (1). Bivalent antibodies such as diabodies (molecular weight, 55–60 kDa) and minibodies (molecular weight, ~80 kDa) are ideal for tumor targeting because of their compact sizes and better antigen binding compared to monovalent scFv fragments (1, 7). Diabodies are dimeric molecules consisting of two scFv fragments connected with a short linker. Minibodies are formed by the fusion of scFv fragments with the immunoglobulin G1 (IgG1) C3 domain. However, decreased antigen binding remains an issue for most antibody fragments compared with intact antibodies (1, 6, 8). In previous studies, Olafsen et al. produced a humanized anti–prostate stem cell antigen (PSCA) intact antibody (hu1G8) and evaluated the antibody as an imaging agent (3). I-Labeled hu1G8 has been shown to specifically accumulate in LAPC-9 prostate tumor xenografts; however, it takes 1 week to reach the maximal tumor/background signal. To improve the antibody pharmacokinetics for imaging use, Leyton et al. engineered several antibody fragments on the basis of the hu1G8 sequence (7). The hu1G8 minibody (scFv-C3 dimer; molecular weight, 80 kDa) is one of the fragments, and shows rapid blood clearance kinetics (MICAD chapter on I-anti-PSCA 2B3 minibody). However, the apparent affinity of the hu1G8 minibody is only 5 nmol, representing a nine-fold loss compared with the apparent affinity of the intact hu1G8 antibody (46 nmol) (7). To increase the hu1G8 minibody affinity, Lepin et al. generated three affinity-matured minibody variants (A11, A2, and C5) with molecular evolution associated with yeast display, a powerful strategy for antibody affinity maturation (1). This strategy enables selection of scFv fragments with higher affinity from a yeast library generated with error-prone polymerase chain reaction (PCR). Lepin et al. demonstrated that the A11 variant has a higher affinity to PSCA than the parental hu1G8 minibody and exhibits more favorable pharmacokinetics for tumor imaging than other variants (1).