Ab-hEPO proliferated TF-1 cells with a potency comparable to that of hEPO (EC50 ~ 0.03 nM) and exhibits a significantly extended plasma half-life (>6 days) in mice relative to hEPO (~4 hours). show sustained elevated hematocrit for more than two weeks. This work demonstrates the utility of BLV1H12 CDR3 fusions as a novel approach for generating potent polypeptides with enhanced pharmacological properties. Introduction The ability to incorporate biologically active proteins and peptides directly into the hypervariable loops of antibodies may provide a general approach for modifying or enhancing the pharmacological properties of various cytokines, growth factors, peptide hormones and ion channel blockers. For example, the resulting fusion proteins are likely to have increased serum half-lives due Zfp264 to their size and conversation with the neonatal Fc receptors (FcRn). They may also express at higher levels in mammalian cells, be more easily purified, or have enhanced solubility and proteolytic stability. Moreover, antibody chimeras will have increased avidity due to the bivalent nature of the antibody molecule; additional binding interactions between the antibody CDR loops and the target receptor may also lead to increased potency or specificity. Finally, it may be possible to graft two or more Picroside III distinct proteins or peptides into the CDRs to afford fusion proteins with dual activities. Recently, we identified a bovine antibody (BLV1H12) with an ultralong heavy chain CDR3 (CDR3H) region that facilitates Picroside III engineering of such CDR fusions. The Xray crystal structure revealed an unusual CDR3H region that folds as a disulfide-bonded knob domain name fused to a solvent accessible, antiparallel -stranded stalk that protrudes from the antibody surface (Physique 1).(1) Unlike conventional antibodies with CDR loops of 10C15 residues in length, the novel architecture of this ultralong CDR3H provides an attractive platform for the creation of antibody chimeras with novel pharmacological activities.(1C5) Indeed, Picroside III we recently showed that bovine granulocyte colony-stimulating factor (bGCSF), when substituted into this ultralong CDR3H region, exhibits enhanced serum half-life in mice.(6) Open in a separate window Physique 1 Grafting of human erythropoietin (hEPO) onto the stalk region of bovine antibody BLV1H12. (A) X-ray crystal structures of bovine antibody BLV1H12 Fab fragment (PDB ID: 4K3D) and hEPO (PDB ID: 1EER). (B) Scheme for generation of antibody-hEPO fusion protein. (C) SDS-PAGE gel of purified BLV1H12 full-length IgG (Ab), hEPO, and Ab-hEPO. Erythropoietin (EPO), a cytokine mainly produced by kidney in adult, is usually a 34-kDa glycoprotein which stimulates erythroid progenitor cell differentiation and maturation and thus increases the erythrocyte population.(7, 8) Recombinant human EPO (hEPO) and its mimetics have been used clinically to treat anemia associated with chronic kidney disease and cancer chemotherapy.(8C12) However, their short circulating half-lives, which necessitate frequent subcutaneous (s.c.) administrations, have led to the development of second generation modified EPOs (e.g., darbepoetin alfa, methoxy polyethylene glycol-epoetin, etc.) with improved serum half-lives.(9) To further explore the generality of the BLV1H12 Picroside III antibody scaffold as a platform for generating biologically active fusion proteins, we asked whether grafting hEPO into the ultralong CDR3H region would afford an antibody-hEPO chimera with high potency and long serum half-life. Here we show that immediate grafting of hEPO in to the ultralong CDR3H area of the bovine antibody outcomes in an effectively expressed fusion proteins that stimulates TF-1 cell proliferation inside a dose-dependent way. Incredibly, this antibody-hEPO fusion proteins (Ab-hEPO) potently stimulates erythropoiesis in mice and sustains high degrees of hematocrit for a lot more than two weeks. Dialogue and Outcomes The folded, disulfide-bonded knob site from the bovine antibody BLV1H12 can be separated through the immunoglobulin site with a 20 ? solvent subjected, antiparallel -stranded stalk (Shape 1A). Thus, chances are that fusion from the N- and C-termini of hEPO using the related -stranded stalk won’t hinder folding of either the antibody or hEPO. Furthermore, as the receptor binding surface area of EPO can be on the contrary face from the molecule towards the string termini, the fusion protein should retain its erythropoietic activity. To create the Ab-hEPO fusion proteins, a artificial hEPO.