Functional Antibody CDR3 Fusion Proteins with Enhanced Pharmacological Properties

摘要

Most mammalian antibodies have complementarity determining region (CDR) loops of 8-16 residues, while some human antibodies have longer, protruding CDR loops that play a role in virus neutralization. The bovine antibody repertoire features a subgroup of antibodies with exceptionally long heavy chain complementarity determining region 3 (CDR3H) loops. The lengths of CDR3H in these bovine antibodies range from 40 to 67 amino acid residues. We recently solved the X-ray crystal structure of bovine antibody BLV1H12 which has a CDR3H of 61 residues (Figure 1). The crystal structure revealed a novel CDR3H structure that forms a solvent-exposed two-stranded antiparallel sheet approximately 20 A (seven amino acids) in length (the stalk region). This |3-sheet terminates in a folded protein domain that is stabilized by three pairs of disulfide bonds (the knob region). This knob region was shown to play a critical role in recognition of a viral antigen in the case of one such bovine antibody. Herein, we asked whether the knob region of BLV1H12 can be substituted with other native protein and peptide ligands to create chimeric antibodies with new or enhanced properties. For example, such fusion proteins may have improved serum half-lives relative to the native polypeptide, improved binding affinity or specificity owing to interactions of the cognate receptor with the other bovine CDRs (or simply through bivalent binding), or improved expression and/or stability in the context of the antibody framework. To begin to explore these possibilities, we first attempted to substitute bovine granulocyte colony-stimulating factor (bGCSF) for the knob region in CDR3H of antibody BLV1H12. The resulting bovine antibody-bGCSF (Ab-bGCSF) fusion protein was stably expressed in mamma- lian cells, exhibited potency similar to bGCSF in proliferating GCSF-dependent cells, and significantly increased and sustained neutrophil populations for over three weeks in rodents.