title: Assessing combinatorial strategies to multimerize libraries of single-domain antibodies.
abstract: Single-domain antibodies (SDAs) are among the most studied and interesting antibody fragments. These molecules combine advantages of antibodies and small molecules. However, SDAs present a low efficiency of in vivo targeting because of their low binding avidity and fast clearance from blood circulation. Multimerization of SDA can overcome these drawbacks and increase their therapeutic potency. In this work, we developed and compared three strategies that allow construction of SDA dimers derived from rabbit light chains--PCR overlap, sticky PCR, and restriction/ligation. The restriction/ligation strategy proved to be the most efficient and feasible method to construct a successful library of SDA dimers. To further explore this technique, we constructed different libraries that differed in linker length between the two SDAs, and assessed its efficiency to deliver antigen-specific SDA dimers. We efficiently increased both the molecular size and avidity of antibody fragments, increasing the possibility of these molecules to bind to their antigen. Therefore, this work describes efficient tools for therapeutic development of SDA dimers.
authors.affiliation: URIA-Centro de Patogenese Molecular, Faculdade de Farmacia, da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Soraia S
authors.initials: SS
authors.lastname: Oliveira
authors.forename: Frederico
authors.initials: F
authors.lastname: Aires da Silva
authors.forename: Sara
authors.initials: S
authors.lastname: Lourenco
authors.forename: Acilino
authors.initials: A
authors.lastname: Freitas-Vieira
authors.forename: Ana Catarina
authors.initials: AC
authors.lastname: Cunha Santos
authors.forename: Joao
authors.initials: J
authors.lastname: Goncalves
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011817:Rabbits; D061905:Single-Domain Antibodies / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D019151:Peptide Library; D061905:Single-Domain Antibodies; D014409:Tumor Necrosis Factor-alpha