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Jun 15, 2007 (Vol. 27, No. 12)

Improving Drugs with Protein Engineering

How to Choose the Best Target, Optimize Delivery, and Reduce Immunogenicity

  • One challenge for protein engineers is to produce biopharmaceuticals that can withstand the proteolytic conditions of the gastrointestinal tract and serum. This was one of many challenges discussed at Cambridge Healthtech’s “PEGS Summit” held last month in Boston. To meet that demand, Manuel Vega, CEO of Nautilus Biotech (www.nautilusbiotech.com), described a 2-D scanning approach that identified a single residue change that could confer proteolysis resistance in plasma and tissues in Nautilus’ two lead products—interferon alpha (Belerofon) and human growth hormone (HGH; Vitatropin).

    The approach includes experimental scans of proteolytic sites across the entire linear peptide sequence. “In the end, we have been able to identify all of the sites for proteolysis that matter,” said Vega, “These are sites that have an impact on the in vivo half life.” Researchers at Nautilus validated Belerofon, Vitatropin, and a few other proteins in vivo in multiple animal models, including rat, monkey, and mouse for pharmacokinetics and biological activity.

    Though Nautilus reduced proteolysis of interferon alpha, HGH, and other proteins with single point mutations, Vega admitted that some proteins may need more than one mutation at proteolysis sites. Vega said Nautilus researchers successfully tested this approach on proteins that ranged in size from 80 amino acids up to 450. Proteolysis-resistant variants also showed improved PK profiles. “That means that proteolysis plays a role in the process of clearance of circulating proteins,” said Vega.

    Proteolysis-resistant proteins show extended half-lives in the gastrointestinal tract as well, thus favoring absorption through the blood barrier. This, Vega said, opens the door for oral administration of therapeutic proteins.

  • Therapeutic Protease Engineering

    Protease activity is not always a problem to be avoided. Human proteases themselves can be exploited as biologics. The major problems with many applications of so-called therapeutic proteases, however, are that they are not active enough, not specific enough, or are too rapidly inactivated by the highly active serine protease inhibitors, SERPINs, naturally present in human serum.

    Wayne M. Coco, vp of protein engineering at Direvo Biotech (www.direvo.com), presented a strategy that uses combinatorial libraries and confocal fluorimetry screening to engineer a nonspecific human protease, trypsin, for high specificity against anti-TNFalpha, a cytokine involved in systemic inflammation. The Direvo scientists accomplished this goal while creating a trypsin variant with an 80-fold increase in specific activity and with several thousand-fold less inhibition in 100% human serum (IC50), according to Coco.

    “Our unique combination of library strategies with confocal screening in 100% human serum represents a general platform for the comprehensive optimization of therapeutic proteases,” said Coco. “We can do highly miniaturized, high-throughput screens in formats that are predictive of ultimate application.”

    Combinatorial libraries for variant testing included whole gene random mutagenesis and libraries focused on either individual residues or protein domains. “From the improvements that we get in those initial libraries, we recombine the best mutations with proprietary DNA-shuffling techniques,” said Coco.

    The Direvo screening technique uses confocal laser technology developed by Evotec (www.evotec.com) and exclusively owned by Direvo for protein engineering. The confocal laser not only detects changes in fluorophore-labeled protein populations, but changes at the individual molecule level, thus contributing to miniaturization, throughput, and unique assay formats.

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