title: Engineering and therapeutic application of single-chain bivalent TGF-β family traps.
abstract: Deregulation of TGF-β superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-β superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-β type II receptor-based, single-chain traps were designed, termed (TβRII)2 and (TβRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TβRII-ED), and a similar or slightly better potency than pan-TGF-β-neutralizing antibody 1D11 or an Fc-fused receptor trap (TβRII-Fc). Despite its short in vivo half-life (<1 hour), which is primarily due to kidney clearance, daily injections of the (TβRII)2 trap reduced the growth of 4T1 tumors in BALB/c mice by 50%, an efficacy that is comparable with 1D11 (dosed thrice weekly). In addition, (TβRII)2 treatment of mice with established 4T1 tumors (100 mm(3)) significantly inhibited further tumor growth, whereas the 1D11 antibody did not. Overall, our results indicate that our rationally designed bivalent, single-chain traps have promising therapeutic potential.
authors.affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada. john.zwaagstra@cnrc-nrc.gc.ca
authors.forename: John C
authors.initials: JC
authors.lastname: Zwaagstra
authors.forename: Traian
authors.initials: T
authors.lastname: Sulea
authors.forename: Jason
authors.initials: J
authors.lastname: Baardsnes
authors.forename: Anne E G
authors.initials: AE
authors.lastname: Lenferink
authors.forename: Cathy
authors.initials: C
authors.lastname: Collins
authors.forename: Christiane
authors.initials: C
authors.lastname: Cantin
authors.forename: Béatrice
authors.initials: B
authors.lastname: Paul-Roc
authors.forename: Suzanne
authors.initials: S
authors.lastname: Grothe
authors.forename: Sazzad
authors.initials: S
authors.lastname: Hossain
authors.forename: Louis-Philippe
authors.initials: LP
authors.lastname: Richer
authors.forename: Denis
authors.initials: D
authors.lastname: L'Abbé
authors.forename: Roseanne
authors.initials: R
authors.lastname: Tom
authors.forename: Brian
authors.initials: B
authors.lastname: Cass
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
authors.forename: Maureen D
authors.initials: MD
authors.lastname: O'Connor-McCourt
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D023061:Gene Order; D006801:Humans; D007165:Immunosuppression Therapy; D008024:Ligands; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D051381:Rats; D017207:Rats, Sprague-Dawley; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D008024:Ligands; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta