At the recent Cambridge Healthtech “Peptalk Conference”, a range of topics related to protein expression and characterization were covered, but the event was notable for its discussion of various approaches to the optimization of biologic therapeutics.
Genes for potential therapeutic proteins can be easily cloned and expressed, but frequently the results are disappointing. Yields may be paltry or nonexistent and the molecules often fail to fulfill their earlier promises. To improve the efficacy and robustness of biological drugs, many companies are turning to sophisticated revamping and engineering technologies.
“DNA is extremely uniform, and DNA production projects are more than 99% successful,” said James L. Hartley, Ph.D., of SAIC (www.saic.com), a contractor for the NCI. “On the other hand, protein expression projects may have a success rate of less than 10%, due to their tremendous variability.”
Dr. Hartley’s protein expression group carries out about 100 projects/year for the NCI/NIH, mostly mammalian proteins. Starting with cDNAs from vendors, the team targets improvements and modifications in order to improve the quality and quantity of the proteins. In engineering their proteins, Dr. Hartley prefers the use of viral expression elements, arguing that because viruses are selected by evolution for protein overexpression, they are the most logical choice for high yields.
Dr. Hartley is the inventor of Invitrogen’s(www.invitrogen.com) Gateway cloning system and favors its use in recombinational cloning for reliability and speed. His experiences with E. coli demonstrate that while it can work well for smaller proteins, above 30–40 kd the success rate falls off rapidly. For this reason, mammalian cells are the obvious choice for obtaining the most native product.
“We try different kits for optimizing DNA purification and test many different culture media, which can yield big improvements in cell performance,” Dr. Hartley stated. “While mammalian cells are very successful for producing secreted proteins, the baculovirus and/or insect cell platform usually outproduces mammalian cells for cytoplasmic proteins.”
Proteolysis is a frequent issue during purification, and Dr. Hartley recommends speed and increasing the amount of protein per cell, assuming the amount of protease is unchanged, to maximize the yield of the protein of interest.
“We are able to address deficiencies in therapeutic products through our mutational strategies,” states Manuel Vega, Ph.D., CEO of Nautilus Biotech (www.nautilusbiotech.com), “which allows us to move toward the holy grail of oral protein therapeutics.”
Protein therapeutics are a gigantic market—$57 billion in 2005—so there is tremendous incentive to pursue improvement strategies. The lack of sophistication of products derived from native proteins means that ample opportunity exists for a range of promising reconfigurations.
Nautilus seeks to address various deficiencies, the most obvious being the short half-life of native proteins. A brief residency time in the circulation requires numerous administrations of the drug, which means that proteins must be introduced through the invasive and labor intensive processes of either subcutaneous or intravenous injection.
“The decreased half-life is the result of proteolysis in blood and tissues in addition to kidney filtration,” Dr. Vega explained, “so we identified the entry points for proteolysis in the proteins and then mutated one or more amino acids, thereby decreasing the sensitivity of the proteins to degradation and extending their half-life in the bloodstream.”
The Nautilus scientists used a proprietary technology to screen hundreds of different amino acid substitutions for the most optimal choices. These were conservative mutations that blocked proteolysis without introducing alterations into the biological activity of the protein. A single mutation in the Interferon Alpha molecule could increase the half-life from 2 to 40 or more hours, and additional mutations can have an additive effect.
Another benefit of these protease-resistant proteins is that, following subcutaneous injection, the molecules survive much longer in the skin, compared to the native counterparts, and only gradually enter the circulation. The skin compartment thus becomes a source of protein that is sustainably released into blood over time.
The Nautilus scientists are now evaluating oral dosing in which the engineered proteins are lyophilized and packed into coated capsules that resist the acidic environment of the stomach. Once in the intestine, the proteins are released from the capsule and remain available for absorption directly through the intestinal mucosa. This approach would increase patient compliance and lower the costs of administration, especially for drugs requiring multiple injections. Animal studies have shown no toxicity or serious side effects, the company reported, and Phase I clinical studies on these orally delivered proteins are scheduled to begin later this year.
The Nautilus approach has the added benefit of creating new intellectual property, as many of the older biologicals are coming off patent soon, if they have not already done so. Of course, the disadvantage is that a new series of clinical trials will be required in order to gain approval, but invariably a modification of an already approved product will have easier sailing through the evaluation process.
With a number of products in the pipeline, including alpha and beta interferon and human growth hormone, Dr. Vega and his colleagues are considering a range of other possibilities, including single chain antibodies. There clearly is no limit to this strategy, he said, which could revolutionize biological drug development.