Expression of antibodies using single-open reading frame vector design and polyprotein processing from mammalian cells.

We describe a novel polyprotein precursor-based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single-open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein-mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N-terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method.

Tumor necrosis factor antagonist mechanisms of action: a comprehensive review.

During the past 30 years, elucidation of the pathogenesis of rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis and ankylosing spondylitis at the cellular and molecular levels has revealed that these diseases share common mechanisms and are more closely related than was previously recognized. Research on the complex biology of tumor necrosis factor (TNF) has uncovered many mechanisms and pathways by which TNF may be involved in the pathogenesis of these diseases. There are 3 TNF antagonists currently available: adalimumab, a fully human monoclonal antibody; etanercept, a soluble receptor construct; and infliximab, a chimeric monoclonal antibody. Two other TNF antagonists, certolizumab and golimumab, are in clinical development. The remarkable efficacy of TNF antagonists in these diseases places TNF in the center of our understanding of the pathogenesis of many immune-mediated inflammatory diseases. The purpose of this review is to discuss the biology of TNF and related family members in the context of the potential mechanisms of action of TNF antagonists in a variety of immune-mediated inflammatory diseases. Possible mechanistic differences between TNF antagonists are addressed with regard to their efficacy and safety profiles.

The biotechnology era: has the promise been fulfilled?

Biotechnological advances have changed the traditional landscape of the drug discovery process. Mutually beneficial relationships between small, innovative and nimble biotechnology companies and experienced, well-funded pharmaceutical companies have resulted in a more rapid application of new technologies to drug development and diversification. Protein-based drugs, such as therapeutic monoclonal antibodies, exemplify the fulfillment of the promise of the biotechnology revolution.

Use of new biotechnology to design rational drugs against newly defined targets.

Success in drug discovery depends largely on the implementation of appropriate strategies that build on new technologies and the appropriate mix of drug-discovery platforms and research management procedures. Close collaboration between pharmaceutical companies, biotechnology companies and academic institutions during the many intricate phases of drug discovery is necessary to address the need to co-ordinate and streamline target discovery and validation activities, which typically take much longer than anticipated. Antibodies have become an important segment of newly developed therapeutics for a wide range of indications and offer the appropriate risk/benefit profile to balance drug-discovery and development portfolios for optimum success. However, as with other discovery activities, long-term commitment and experience are required to exploit these new techniques fully. Companies with experience in managing the appropriate mix of small-molecule and antibody discovery efforts while implementing novel techniques will remain at the forefront of drug development.