RESUMEN
Antibody fragments can be isolated rapidly using techniques such as phage display and can be expressed to high levels in microbial systems. However, to date such antibody fragments have been of limited use for many therapeutic applications because they are rapidly cleared from the body. We present a strategy for the site-specific chemical modification of antibody fragments with polyethylene glycol, which results in the production of antibody fragments with long in vivo half-lives and full retention of antigen-binding properties. This technology should allow more rapid and economical production of therapeutic antibodies for chronic disease therapy.
Asunto(s)
Fragmentos de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/uso terapéutico , Animales , Área Bajo la Curva , Semivida , Inmunoterapia , Macaca fascicularis , Masculino , Polietilenglicoles , Ratas , Ratas WistarRESUMEN
A novel group of mixed mode adsorbents has been developed for purification of monoclonal and polyclonal antibodies from a broad range of raw materials such as hybridoma cell culture, ascites fluid, animal sera, milk, whey and egg yolk. The aim of this study was to determine whether such mixed mode adsorbents were also useful for the recovery of recombinant proteins from microbial feedstocks. This paper describes the performance of one of these adsorbents for expanded bed capture of a human Fab fragment from recombinant E. Coli cell extracts. It is concluded that the mixed mode adsorbent binds the Fab fragment efficiently from crude extracts without any requirement for preconditioning the extract by for example de-salting or dilution. The capacity of the mixed mode adsorbent is approx. 12 mg Fab/ml matrix. The novel mixed mode adsorbent can be useful during production of highly purified Fab fragments as the first step in a purification scheme. In this respect the mixed mode adsorbent is advantageous over alternative commercially available ion-exchange materials which require pre-conditioning of cell extract for Fab' capture. Together with the concentration and clarification effect a significant enrichment of the Fab fragment is obtained in one single high yield operation.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Adsorción , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
A series of fusion proteins have been generated between human and mouse CD18. These proteins have been used to carry out preliminary mapping studies on a number of anti-CD18 antibodies including KIM127 an antibody that promotes CD18-dependent adhesion. This antibody maps to a region of the CD18 molecule between amino acids 406 and 570 in a region containing cysteine-rich repeats.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD18/inmunología , Adhesión Celular/inmunología , Cisteína/genética , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Western Blotting , Antígenos CD18/química , Antígenos CD18/genética , Línea Celular/fisiología , Cisteína/análisis , Mapeo Epitopo , Expresión Génica/genética , Expresión Génica/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de TiempoRESUMEN
The envelope glycoprotein, gp160, of human (HIV) and simian (SIV) immunodeficiency viruses mediates virus-host cell binding followed by fusion of the viral and plasma membranes. The envelope proteins are known to exist as non-covalently associated oligomers on the virus surface. The production of permanent mammalian cell lines that constitutively secrete relatively high levels of soluble forms of SIV gp160 is described and we show that these proteins are secreted predominantly as tetramers with lower levels of dimer forms. Oligomeric forms were purified to greater than 90% purity using a simple gel filtration method. The purified proteins bind CD4 suggesting that they remain in their native conformation. The purified oligomeric proteins provide the basis for more relevant structural, functional and immunological studies than recombinant gp120 as they more closely resemble the envelope protein oligomer.