The adenovirus E1A proteins form complexes with a group of cellular proteins, including a protein of 130 kD. EIA-associated p130 was purified through coimmunoprecipitation with EIA, and sequence data from four tryptic peptides was obtained. Oligonucleotides derived from the peptide sequences were used to clone a 4.85-kb eDNA. The eDNA contained an 1139-amino-acid open reading frame with homology to the retinoblastoma protein and E1A-associated p107 but was more closely related to p107. In vitro-translated p130 bound to E1A, and anti-p130 antibodies detected p130 in immunoblots of EIA immunoprecipitates, p130 was also detected in immunoprecipitates of cyclins A and E and was an efficient substrate in vitro for kinase activities associated with these eyclins. The p130 gene mapped to chromosome 16q12.2-13, a region that undergoes allelic loss in several types of tumors, including hepatocellular, prostate, and breast carcinomas.

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Inhibition of Cdk2 activity in mitogen-starved fibroblasts is usually performed by the CKI p27, and to a minor extent by p21. Remarkably p130, a protein in the Rb family that is not related to either p21 or p27, will directly substitute for the CKIs and restore normal CDK regulation by mitogens in cells lacking both p27 and p21. This compensatory pathway may be important in settings in which CKIs are not expressed at standard levels, as is the case in many human tumors.

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Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer halflife. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. IntroductionEPO, a naturally occurring hematopoietic growth factor produced by the kidney, is the primary regulator of erythropoiesis. 1 Recombinant human EPO (rHu-EPO) has important clinical uses in patients with anemia associated with renal disease and cancer. Analogs rHu-EPO with extended serum half-lives have been developed and of shown to provide a clinical advantage by allowing maintenance of stable hemoglobin levels with less frequent dosing. 2,3 A fulllength human agonistic antibody targeting the EPO receptor (EPOR) would offer a longer serum half-life and may support even less frequent dosing regimens that could better match with many chemotherapy regimens and may provide better convenience for both predialysis and peritoneal dialysis patients who need to attend the clinic only infrequently. In addition, an antibody EPO mimic is unlikely to induce pure red cell aplasia, a condition associated with some forms of rHu-EPO due to the formation of rHu-EPO-induced neutralizing antibodies. 4 Mouse monoclonal antibodies (mAbs) that are raised to the soluble extracellular domain (ECD) of the human EPOR and that mimic EPO activation by inducing ligand-dependent cell proliferation and differentiation have been described. 5,6 These mAbs, however, activate the EPOR less efficiently than the natural hormone does and consequently are less potent agonists and unsuitable for clinical use. Crystal structure of the EPO-(EPOR)2 complex reveals that EPO binds 2 distinct sites of the 2 cell-surface EPORs and that asymmetric molecules may therefore be required for optimal signaling. 7 This report describes a fully human agonistic antibody, ABT-007, that effectively stimulates both proliferation and erythroid differentiation. Since ABT007 exhibits a high degree of selectivity and does not recognize rodent EPOR, mice expressing the human EPOR transgene were used to establish its in vivo efficacy. Surprisingly, the activation signal achieved with the symmetric molecule ABT007 is sufficient to support potent and more sustained erythropoiesis in animal models compared with standard doses of rHu-EPO. We examined the crystal structure of human monomeric EPOR ECD complexed with a single antibody fragment of ABT007 (Fab-EPOR) to better understand the molecular basis for the erythropoietic potency of ABT007. Resolution of the resulting crystal structure identified a unique EPOR nonlinear epitope distinct from the EPO binding site, resulting in a receptor conformation that supports activation. In vivo properties of ABT007 may b...