Engineering and therapeutic application of single-chain bivalent TGF-β family traps.

Deregulation of TGF-beta 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-b 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-beta type II receptor-based, single-chain traps were designed, termed (T beta RII) 2 and (T beta RIIb) 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 (T beta RII-ED), and a similar or slightly better potency than pan-TGF-beta-neutralizing antibody 1D11 or an Fc-fused receptor trap (T beta RII-Fc). Despite its short in vivo half-life (<1 hour), which is primarily due to kidney clearance, daily injections of the (T beta RII) 2 trap reduced the growth of 4T1 tumors inBALB/c mice by 50%, an efficacy that is comparable with 1D11 (dosed thrice weekly). In addition, (T beta RII) 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. Mol Cancer Ther; 11(7); 1477-87. (c) 2012 AACR.