RESUMO
PURPOSE: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. EXPERIMENTAL DESIGN: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. RESULTS: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CONCLUSION: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Quimiocina CXCL12/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Cricetinae , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Mapeamento de Epitopos , Feminino , Humanos , Camundongos , Modelos Moleculares , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Conformação Proteica , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4-61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.