In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V-H) and variable light (V-L) chain domains but arranged in different orientations. V-H/V-L orientation, i.e., V-H-linker-V-L or V-L-linker-V-H, showed significant effects oil the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V-H/V-L orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degreesC in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, Our results underscore the importance of identifying/selecting optimal V-H/V-L orientation/arrangement for efficient production of active bDAb. (C) 2004 Elsevier Inc. All rights reserved.