title: Generation and characterization of monospecific and bispecific hexavalent trimerbodies.
abstract: 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.
authors.affiliation: Leadartis SL, Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Blanco-Toribio
authors.forename: Noelia
authors.initials: N
authors.lastname: Sainz-Pastor
authors.forename: Ana
authors.initials: A
authors.lastname: Álvarez-Cienfuegos
authors.forename: Nekane
authors.initials: N
authors.lastname: Merino
authors.forename: Ángel M
authors.initials: ÁM
authors.lastname: Cuesta
authors.forename: David
authors.initials: D
authors.lastname: Sánchez-Martín
authors.forename: Jaume
authors.initials: J
authors.lastname: Bonet
authors.forename: Patricia
authors.initials: P
authors.lastname: Santos-Valle
authors.forename: Laura
authors.initials: L
authors.lastname: Sanz
authors.forename: Baldo
authors.initials: B
authors.lastname: Oliva
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.forename: Luis
authors.initials: L
authors.lastname: Álvarez-Vallina
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D043170:Collagen Type XVIII / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D007797:Laminin / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
chemical_list: D018033:Antibodies, Bispecific; D017252:CD3 Complex; D043170:Collagen Type XVIII; D007797:Laminin; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies