Erythropoietin (Epo) is the naturally occurring hematopoietic growth factor required for the production of mature red blood cells. Epo has a molecular mass of 18.4 kD excluding carbohydrate, and when naturally glycosylated is 35 kD (Roberts, D. and Smith, D. J., J. Mol. Endocrinology 12, 131, 1994). The protein is encoded by only one gene (Youssoufian, H., Zon, L. I., Orkin, S. H., D'Andrea, A. D. & Lodish, H. F. Mol. Cell. Biol. 10, 3675–3682 (1990), Maouche, L., et al. Blood 78, 2557–2563 (1991). This growth factor stimulates the proliferation of early and late erythroid specific progenitor cells as well as the hemoglobination of proerythroblasts and their differentiation into mature red blood cells.
Recombinant human Epo (rEpo) has an established market and is routinely used in the care of patients with renal failure, where kidney damage results in anemia due to insufficient production of Epo (Foa, P. Acta Haematol 86, 162–168 (1991)). Furthermore, Epo has also been shown to be useful in specific clinical settings, such as autologous blood transfusion prior to elective surgery, prevention and/or treatment of anemia induced by cytoreductive drugs, and for the treatment of anemia patients receiving zidovudine for HIV infection (Ascensao, J. A., Bilgrami, S. & Zanjani, E. D. Am. J. Pediat. Hematol. 13, 376–387 (1991)). Therapy with rEpo is remarkably well tolerated by most patients with few, if any, major adverse reactions reported. Despite the success of rEpo in the clinic, its full potential has not been realized due to limitations imposed by the short half life of rEpo that require frequent dosing and the high cost of treatment. The current recommended dosing is 3× a week delivered subcutaneously.
Epo is a member of a family of structurally and genetically related ligands, which include IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, LIF, G-CSF, GM-CSF, M-CSF, Epo, growth hormone and PRL (see Young, P. R. Curr. Opin. Biotech. 3, 408, (1992) for review). The structures of several of the ligands have been determined by X-ray crystallography and/or NMR, and all have a basic core structure of a four α-helical bundle with an up—up-down—down connectivity. A similar fold is predicted for other members of the family based on modeling and gene structure.
Epo acts through a cell surface receptor which belongs to the hematopoietic cytokine receptor family. EpoR has been cloned from mouse and human and exists in both membrane-bound and secreted forms (D'Andrea, A. D., Lodish, H. F. & Wong, G. G. Cell 57, 277–285 (1989), Jones, S. S., D'Andrea, A. D., Haines, L. L. & Wong, G. G. Blood 76, 31–35 (1990), Nakamura, Y., Komatsu, N. & Nakauichi, H. Science 257, 1138–1141 (1992) and Todokoro, K., Kuramochi, T., Nagasawa, T., Abe, T. & Ikawa, Y. Gene 106, 283–284 (1991)). The extracellular domain of the receptors contains the “hematopoietic motif” which consists of two 100 amino acid long fibronectin-like domains and a conserved WSXWS sequence motif, while the intracellular domains contain several conserved regions but do not encode an endogenous kinase activity. Examination of the growth hormone ligand-receptor complex structure (De Vos, A. M., Ultsch, M. & Kossiakoff, A. A. Science 255, 306, (1992)) and extensive mutagenesis of the ligands (reviewed in Young, P. R., supra,) suggests that, in general, the interaction between receptor and ligand is similar for all members of the hematopoietic cytokine family, with the loops of the two fibronectin domains of each receptor subunit interacting with the amino and carboxy-terminal α-helices.
All ligands in this family stimulate biological activity by causing the aggregation of single or multiple receptor subunits in target cells. In the case of Epo, the critical event appears to be the dimerization of a single receptor subunit. Mutant cloned receptors which lead to constitutively active, ligand-independent growth in transfected cell lines, are constitutively dimeric (Watowich, S. S., et al. Proc. Natl. Acad. Sci. USA 89, 2140, (1992)). Furthermore, in vitro studies of complex formation between Epo and the extracellular domain of EpoR suggest a 1:2 ligand:receptor interaction (Harris, K. W., Mitchell, R. A. & Winkelmann, J. C. J. Biol. Chem. 267, 15205, (1992), Philo, J. S., Aoki, K. H., Arakawa, T., Narhi, L. O. & Wen, J. Biochemistry 35, 1681, (1996)). More recently a peptide with Epo minimetic activity was shown to dimerize the receptor (Wrighton, N. C. et al., Science 273, 458–463 (1996)).
The interaction of Epo with its receptor initiates a chain of events involving tyrosine and serine-threonine protein kinases which culminate in changes in the pattern of cellular gene expression, proliferation and differentiation. While there have been many advances in the understanding of the signal transduction pathways following Epo binding to its receptor (for a review see: Ihle, J. N. Nature 377, 591–594 (1995)), it is still not clear how progenitor cells decide between proliferation and differentiation.
The finding that dimerization of the receptor is a key step in the stimulation of mitogenesis by Epo suggests another approach to novel Epo-like agonists. In at least three examples of other receptors where homodimerization is induced by receptor binding, monoclonal antibodies have been developed which also had agonist properties. These include monoclonal antibodies to EGF, TNF and growth hormone receptors (Schreiber, A. B., Libermann, T. A., Lax, I., Yarden, Y. & Schlessinger, J. J. Biol. Chem. 258, 846–853 (1983), Defize, L. H. K., Moolenaar, W. H., van der Saag, P. T. & de Laat, S. W. EMBO J. 5, 1187–1192 (1986), Engelmann, H., et al. J. Biol. Chem. 265, 14497–14504 (1990), Fuh, G., et al. Science 256, 1677–1680 (1992)). In all three cases, the monoclonal antibody, by virtue of its two antigen recognition sites, was able to bring together two receptors and activate them. Fab fragments made from these mAbs were inactive. In some cases, the apparent affinity of the antibody for receptor was comparable to that of the ligand (e.g., growth hormone, Fuh, G., et al. Science 256, 1677–1680 (1992)). More recently, there were reports of monoclonal antibodies raised to the Epo receptor that have Epo-like activity (Schneider. H. et al., Blood 89, 473482 (1997) and Elliot, E. Et al., J. Biol. Chem. 271, 24691–24697 (1996)). However, these reports indicated that the frequency of obtaining agonist monoclonal antibodies to the Epo receptor was very low, and their potency was low and hence unsuitable for use therapeutically.
Clearly, there is a need to develop high affinity, potent agonist antibodies to the EpoR which will have sufficient activity to work in vivo at therapeutically acceptable concentrations.
There are available well known methods for humanization of non-human mAbs that result in less immunogenic antibodies for human therapy, yet retain full binding avidity. These methods can be applied to receptor agonist mAbs whose mode of action is the dimerization of receptors in a manner that mimics the action of the natural receptor ligand.
A humanized agonist mAb with equal or better affinity than rEpo for its receptor and an appropriate Fc region would be expected to have a longer in vivo half-life. This would be expected to produce an Epo-like protein with a lower frequency of dosing compared to rEpo, which is presently given three times a week by subcutaneous injection.