RESUMO
There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.
Assuntos
Angiopoietina-1/antagonistas & inibidores , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/imunologia , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Angiopoietina-1/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Córnea/efeitos dos fármacos , Córnea/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microvasos/efeitos dos fármacos , Microvasos/imunologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Fosforilação , Distribuição Aleatória , Receptor TIE-2/antagonistas & inibidores , Receptor TIE-2/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.