RESUMO
Impaired matrix metalloproteinase 1 (MMP-1) function, as result of the expression of increased levels of tissue inhibitor of metalloproteinase 1 (TIMP-1), plays an important role in the pathopysiolgical mechanism of fibrosis. In a recently performed clinically relevant rat animal model of established liver fibrosis, it could be shown, that blocking the interaction between the metalloproteinase and its inhibitor has beneficial effects in vivo. The rat TIMP-1 specific antagonistic antibody used in this study was derived from a human combinatorial antibody library (HuCAL) and blocks the interaction between rat TIMP-1 and MMP-13, the rat homologue of human MMP-1. We here describe the utilization of the same antibody source to generate fully human antibodies against human TIMP-1 which could be potential candidates for a therapy of fibrosis in man. In order to develop a highly potent antagonist of TIMP-1 action, antibodies isolated from the library were subjected to a number of different in vitro affinity maturation strategies. By these means, affinity and potency were improved by a factor of 87 and 65 fold, respectively, resulting in a valuable human therapeutic antibody candidate with a monovalent affinity of 150 pM and a potency for in vitro inhibition of TIMP-1/MMP-1 interaction of 200 pM.
Assuntos
Anticorpos/uso terapêutico , Neoplasias da Mama/terapia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Cirrose Hepática/terapia , Inibidor Tecidual de Metaloproteinase-1/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/imunologia , Afinidade de Anticorpos , Biotecnologia/métodos , Carcinoma/terapia , Técnicas de Química Combinatória , Regiões Determinantes de Complementaridade , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , RatosRESUMO
Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), which blocks matrix metalloproteinase (MMP) activity. TIMP-1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP-1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl(4)), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti-TIMP-1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl(4)-treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle alpha-actin (alpha-SMA). Compared to controls, rats administered anti-TIMP-1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti-TIMP-1 resulted in a marked decrease in alpha-SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP-2. In conclusion, administration of a TIMP-1 antibody attenuated CCl(4)-induced liver fibrosis and decreased HSC activation and MMP-2 activity.