Tpo Af13948

Cwirla and coworkers (46) used several peptide library construction methods and covalent dimerization to produce AF13948, a 28-amino-acid peptide that was equipo-tent to the natural growth factor TPO in cell-based assays. Peptides that bind to TPOR were identified using both phage display and lac repressor display of random peptide libraries. Libraries were screened against the immobilized ECD of TPOR, and the resulting clones were categorized into two families and two subfamilies based on amino acid sequence homology.

Scientists constructed and screened several mutagenesis libraries to optimize the higher affinity sequences further. These libraries were constructed in the headpiece dimer and polysome vectors to generate even higher affinity ligands than those that were obtained using phage or lac repressor display. A 19-residue peptide (AF12434) obtained from these screens was then used as a template for additional mutagenesis to generate another library of peptides. This library produced a high-affinity peptide (AF12505, IC50 = 2 nM) that was subsequently covalently dimerized using the same branched-lysine strategy (chemical dimer) applied to the EMP series to generate AF13948 (33). Dimer AF13948 was 4000 times as potent as the monomer AF12505 and equipotent to rHuTPO in the cell proliferation assay (Fig. 6). The peptide dimer also stimulated in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and increased platelets by 80% in vivo (46).

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