Generation and characterization of monospecific and bispecific hexavalent trimerbodies

• DOI: 10.4161/mabs.22698
• Publication Date: 2013-01-16T21:35:56Z
• Authors: Ana Blanco-Toribio; Noelia Sainz-Pastor; Ana Álvarez-Cienf...; Nekane Merino; Ángel M. Cuesta; David Sánchez-Martín; Jaume Bonet; Patricia Santos-V...; Laura Sanz; Baldo Oliva; Francisco J. Blanco; Luis Álvarez-Vallina
• Keywords: 0301 basic medicine; 577.2; 24 Ciencias de la Vida; CD3 Complex; Recombinant Fusion Proteins; T-Lymphocytes; Bioquímica (Farmacia); Antibody Affinity; Antibody engineering; Lymphocyte Activation; Transfection; Jurkat Cells; 03 medical and health sciences; Antibody Specificity; Report; Antibodies, Bispecific; Animals; Humans; 577.1; Trimerbody; Multivalent antibodies; Collagen Type XVIII; HEK293 Cells; Collagen; Laminin; Protein Multimerization; Bispecific antibodies; Biología molecular (Farmacia); Single-Chain Antibodies

Here, we describe a new class of multivalent and multispecific antibody-based reagents for therapy. The molecules, termed "trimerbodies," use a modified version of the N-terminal trimerization region of human collagen XVIII noncollagenous 1 domain flanked by two flexible linkers as trimerizing scaffold. By fusing single-chain variable fragments (scFv) with the same or different specificity to both N- and C-terminus of the trimerizing scaffold domain, we produced monospecific or bispecific hexavalent molecules that were efficiently secreted as soluble proteins by transfected mammalian cells. A bispecific anti-laminin x anti-CD3 N-/C-trimerbody was found to be trimeric in solution, very efficient at recognizing purified plastic-immobilized laminin and CD3 expressed at the surface of T cells, and remarkably stable in human serum. The bispecificity was further demonstrated in T cell activation studies. In the presence of laminin-rich substrate, the bispecific anti-laminin x anti-CD3 N-/C-trimerbody stimulated a high percentage of human T cells to express surface activation markers. These results suggest that the trimerbody platform offers promising opportunities for the development of the next-generation therapeutic antibodies, i.e., multivalent and bispecific molecules with a format optimized for the desired pharmacokinetics and adapted to the pathological context.