Tetravalent antibodies are more potent and efficacious erythropoiesis‐stimulating agents than erythropoietin in vivo

Recent studies have shown that tetravalent antibodies are potent and efficacious agonists of the erythropoietin (EPO) receptor (EPOR) both in vitro and in vivo. To identify antibody-based erythropoiesis-stimulating agents (ESAs) with therapeutic potential, we evaluated various tetravalent antibody formats for EPOR agonism and key biophysical properties necessary for biologic drug development. We identified two distinct tetravalent antibody formats that strongly stimulated the growth of UT7/Epo cells, which rely on EPOR signaling for proliferation. Moreover, one of these formats exhibited ideal biophysical characteristics for drug development. This format consisted of a diabody (Db) and two antigen-binding fragment (Fab) arms fused to the N- and C-termini of an Fc domain, respectively, to form a tetravalent Db-Fc-Fab (EPRA-0322). In a mouse model expressing the human EPOR, EPRA-0322 induced erythropoiesis with greater potency, efficacy, and duration than darbepoetin, a hyperglycosylated EPO currently used in clinical practice. These findings highlight tetravalent antibodies, and the Db-Fc-Fab format in particular, as promising next-generation ESAs suitable for large-scale production and clinical use.