Immunoconjugates deliver beneficial effects; further peptide modifications may increase cytotoxicity.

Single-chain variable fragments (scFvs) of anti-Lewis y hu3S193 humanized antibody were constructed by joining the V H and V L domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V H domain were removed (-1 residue, -2 residue) and then joined directly to the V L domain. An scFv construct in the reverse orientation with the V L joined directly to the V H domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.Keywords: scFv multimers; diabody; triabody; trimer; tetrabody; anti-Lewis y antibody; hu3S193Single-chain variable fragments (scFvs) were designed to link the variable heavy-chain domain (V H ) and variable light-chain domain (V L ) via a flexible peptide linker to stabilize and enhance their interaction with each other to form an antigen-binding site (Huston et al. 1991;Malby et al. 1993). Peptide linkers >12 amino-acid residues provide sufficient spatial flexibility for the V H and V L domains to associate predominantly as scFv monomers with only small amounts of higher molecular mass multimers evident (Griffiths et al. 1993;Kortt et al. 1994;Whitlow et al. 1994). Decreasing the linker length to <10 residues prevents the V domain from interacting with its attached partner and promotes the interaction of complementary V H /V L pairs from two scFv molecules to associate to form noncovalent multivalent multimers Iliades et al. 1997;Kortt et al. 1997;Pei et al. 1997;Atwell et al. 1999;Le Gall et al. 1999;Dolezal et al. 2000). The formation of scFv dimers, trimers, and tetramers by noncovalent association of tethered V domains is dependent on the length of the linker joining the domains and the orientation of the V domains in the scFv construct . In general, with linkers of 3 to 10 residues, scFv dimers (diabodies) are predominantly formed whereas trimers (triabodies) and tetramers (tetrabodies) are formed with linkers of 2 residues or less.

A single-chain antibody fragment (scFv) of the humanised monoclonal antibody, hu3S193, that reacts specifically with Le(y) antigen expressed in numerous human epithelial carcinomas was constructed. A five-residue linker joined the C-terminus of the V(H) and the N-terminus of the V(L), which prevented V-domain association into a monomeric scFv and instead directed non-covalent association of two scFvs into a dimer or diabody. The diabody was secreted into the E. coli periplasm using a heat-inducible vector, pPOW3, and recovered as a soluble, correctly processed protein, following osmotic shock or solubilised with 4 M urea from the insoluble fraction. The diabody from both fractions was isolated by a rapid batch affinity chromatography procedure, using the FLAG affinity tag to minimise degradation and aggregation. The purified diabody has an Mr of approximately 54 kDa, was stable and demonstrated similar binding activity as the parent monoclonal antibody, as measured by FACS and BIAcore analyses. The radiolabelled diabody showed a rapid tumour uptake, with fast blood clearance, proving it to be an excellent potential candidate as a tumour-imaging agent.

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BLCA-38 can target prostate cancer in vivo almost as effectively as J591. Given that J591 is used clinically, BLCA-38, which targets a different antigen, has potential for radioimmunoscintigraphy and for therapeutic targeting of prostate cancer.

A panel of monoclonal antibodies (MAbs) directed to outer membrane protein I were generated with the ultimate aim of detecting Neisseria gonorrhoeae in patient samples by a direct immunofluorescence (IF) test. In an initial evaluation of the sensitivity of these reagents, a cocktail of six IF MAbs recognized 491 (91%) of 540 gonococci isolates from several centers in Sydney, Australia. IF MAbs designated 185 and 228 recognized serovars of WI serogroup and IF MAbs 208, 210, and 312 recognized serovars of WII/III serogroup. IF MAb 198 recognized serovars within both serogroups. Three additional IF MAbs, designated 322, 323, and 330, were then generated by using strains which failed to react with the original MAb cocktail and which belonged to particular serovars. The new cocktail of nine IF MAbs recognized 96% of the gonococcal isolates, which incidentally contained representatives of serovars shown to have a worldwide distribution in previous studies. Although subtle differences were apparent in the reaction patterns found with coagglutination (serotyping) and IF, there nonetheless seems to be merit in the approach of continually evaluating the sensitivity of diagnostic reagents such as MAbs. This is especially true with an organism such as N. gonorrhoeae, which has the capacity to regularly alter the antigenic structure of its outer membrane proteins.