Use of SpyTag/SpyCatcher to construct bispecific antibodies that target two epitopes of a single antigen

Abstract

Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis.

Development and activities, including immunocomplex formation, of biparatopic antibodies and alternative scaffold proteins

“…For monomeric alternative scaffold proteins and fragment antibodies, generation of biparatopic proteins is an extremely efficient way of increasing avidity. Thus, a large number of these proteins have been developed as biparatopic agents [12,[16][17][18][19][20][21][22][23][24][25][26][27][28][29], which is not as important with conventional antibodies because they are already bivalent [5,7,8,10,[30][31][32][33][34][35][36][37].…”

“…Biparatopic antibodies and scaffold proteins have improved avidity compared with their monoparatopic analog because they usually contain binding units that do not compete with each other. Previously, our group reported the development and characterization of monoparatopic and biparatopic dimeric single-chain variable fragments (scFvs) against the tetrameric membrane antigen roundabout homolog 1 (ROBO1) [36]. Interaction of ROBO1 with the different domains of the antigen was strongest for the biparatopic scFvs ( Table 1).…”

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

“…Labeling the SpyTag or SpyCatcher prior to conjugation allows repetitive labeling of different batches of antibody or different antibodies with the same batch of labeled SpyTag or SpyCatcher. The SpyTag/SpyCatcher can be placed at any site within the antibody [17,41] without compromising expression yield and binding properties. In addition, this system provides control over the labeling ratio of antibodies and fragments by either modulating the labeling ratio of the SpyTag or SpyCatcher or by Bdoping^in unlabeled SpyTag or SpyCatcher into the ligation reaction.…”

Post‐translational Assembly of Protein Parts into Complex Devices by Using SpyTag/SpyCatcher Protein Ligase

“…Papers published in recent years have focused on a variety of methods of BsAb generation by chemical conjugation, hybridoma technology, and protein engineering involving recombinant DNA technology . Recently, the SpyTag/SpyCatcher system was used to construct a bispecific scFv that simultaneously interacted with two different epitopes of an antigen associated with cancer progression, roundabout homologue 1 (ROBO1) . The resulting bispecific antibody fragment showed simultaneous binding of both scFvs to their respective targets and, using the SpyTag/SpyCatcher platform, allowed the creation of antibody fragments that differed in valency and specificity.…”