Generation of single-domain antibody multimers with three different self-associating peptides

Conventional Y-shaped antibodies have been widely used in research, diagnostics and therapeutics. Their large size result in disadvantages in certain applications, which makes production difficult. Different parts of an antibody have been used to replace the whole antibody to make it smaller. Single-domain antibodies (sdAbs) derived from the camelid heavy chain antibodies are among the smallest antibody fragments engineered for various applications. To improve the affinity of these single- sdAbs for correspondent antigens and provide suitable size of reagents for various applications, we fused an anti-epidermal growth factor receptor sdAb EG2 to self-associating peptides, RHCC derived from a right-handed coiled-coil peptide of an archaebacterium, COMPcc from human cartilage oligomeric matrix protein and C4bp derived from human plasma C4-binding protein -chain, respectively, to make multimeric antibodies. The multimeric antibodies were expressed as soluble cytoplasmic proteins to spontaneously form tetramers, pentamers or heptamers and were purified by affinity chromatography. The avidity of multimeric forms of sdAbs compared with that of the monomeric form of sdAbs was increased without altering binding specificity.