Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies.

We have generated two distinct recombinant bispecific antibody molecules for the retargeting of adenoviral vectors to CEA-expressing tumour cells. These antibody molecules were produced by combining the antigen-binding sites of a neutralising anti-fibre knob scFv (S11) and an anti-CEA antibody either in a single-chain diabody format (scDb CEA-S11) or a tandem scFv format (taFv CEA-S11). In order to facilitate expression of taFv CEA-S11 in bacteria we selected from a phage display library taFv molecules with an optimised linker that connects the two scFv fragments. ScDb CEA-S11 and taFv CEA-S11 were expressed and purified in soluble form from the bacterial periplasm with yields of approximately 100 micro g per litre of bacterial culture. In vitro, both bispecific molecules mediated selective and enhanced transduction of CEA-expressing tumour cells by recombinant adenoviruses. These assays did not reveal any differences in efficiency of adenoviral transduction by the two antibody formats. However, compared with taFv CEA-S11, scDb CEA-S11 exhibited a 2- to 3-fold increased stability in human plasma at 37 degrees C. In summary, we could demonstrate that both formats are suitable for adenovirus targeting to tumour cells with similar biological activity in vitro.

We present a novel vascular tumor therapy approach based on lysing endothelial cells by cytotoxic T lymphocytes (CTLs). Retargeting of CTLs is achieved by a recombinant bispecific antibody molecule (bispecific single-chain diabody) directed against human endoglin (CD105, EDG) and the T-cell coreceptor CD3 (scDb EDGCD3). Bacterially expressed scDb EDGCD3 was able to bind to endoglin-expressing endothelial cells as well as CD3-expressing T lymphocytes. The single-chain diabody mediated killing of endothelial cells (HUVEC, HMEC) by activated cytotoxic T lymphocytes at picomolar concentrations, and cells not expressing endoglin were not affected. Because endoglin is up-regulated in the vasculature of many solid tumors, this antibody molecule should be capable of lysing tumor endothelial cells and thus destroying the vascular bed of the tumor.

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