Camel single-domain antibodies as modular building units in bispecific andbivalent antibody constructs

Single-domain antibodies against various antigens are isolated from the uni que heavy-chain antibodies of immunized camels and llamas. These minimal si zed binders:are very robust and bind the antigen with high affinity in a mo nomeric state. We evaluated the feasibility to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-do main antibody fragments as building blocks. Two single-domain antibody: fra gments were tethered by the structural upper hinge of a natural antibody to generate bispecific molecules. This linker was chosen for its protease res istance in serum and its natural flexibility to reorient the upstream and d ownstream located domains. The expression levels, ease of purification, and the solubility of the recombinant proteins were comparable with those of t he constituent monomers. The individual moieties fully retain the binding c apacity and the binding characteristics within the recombinant bispecific c onstructs. The easy generation steps and the biophysical properties of thes e bispecific and bivalent constructs based on camel single-domain antibody fragments makes them particularly attractive for use in therapeutic dr diag nostic programs.