SINCE life emerged about 3.7 billion years ago, biological evolution has ensured that the genes that make enzymes, which catalyse chemical reactions, and binding proteins, which assist in binding two or more molecules together, are optimised for specialised bodily functions and the sustenance of human life against hostile environments. Similarly, the genes of other organisms, too, have evolved to improve their fitness and survival. This is the Darwinian principle of evolution. Through evolution, nature has found solutions to life’s many complex chemical problems, and the diversity of life on earth is testimony to the power of natural evolution.
For thousands of years, humans have been breeding animals and plants with desirable traits using genetic selection, which actually is, in a sense, speeding up the process of Darwinian selection through human intervention, albeit by trial and error. Success is achieved over a few years or decades instead of the thousands of years the natural process takes.
This year’s Nobel Prize in Chemistry has been awarded to three scientists who developed laboratory methods of controlling evolution and harnessing its power to engineer enzymes and binding proteins—using the same tools as nature, of genetic mutation and selection—for the benefit of mankind. This is called “directed evolution”, a man-made procedure that compresses the evolution timescale by orders of magnitude down to a few years—much less than breeding timescales—to produce new enzymes and proteins in the laboratory that are useful for human survival. The notion of “survival of the fittest” in nature’s scheme of things is mimicked in the laboratory through directed evolution to create new useful entities.
One half of the nine million Swedish krona (about $1 million) goes to the 62-year-old Frances H. Arnold, a professor of chemical engineering, bioengineering and biochemistry at the California Institute of Technology, “for the directed evolution of enzymes”. She is only the fifth woman to get the Chemistry Nobel Prize.
The other half of the award is shared equally by the 77-year-old George P. Smith, professor emeritus at the University of Missouri, Columbia, United States, and the 67-year-old Sir Gregory P. Winter of the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, “for the phage display of peptides and antibodies”. By developing innovative methods to engineer new enzymes and new proteins through directed evolution, the laureates have revolutionised both chemistry and the development of new pharmaceuticals.
Back in 1979, as a graduate in mechanical and aerospace engineering, Frances Arnold had a clear vision that her efforts to develop new technology must be for the benefit of humanity. According to the background document put out by the Nobel Committee, as an engineer she initially started out to work on solar power. But, with the future of the industry moving radically towards the new DNA technology at that time, she shifted to biology. “It was clear that a whole new way of making materials and chemicals that we needed in our daily lives would be enabled by the ability to rewrite the code of life,” she has been quoted as saying in the Nobel Committee document. She turned her focus to using the tools of chemistry to engineer key enzymes of life. She thought if, learning from nature, she could design new enzymes with new properties, she could fundamentally change chemistry.
As she told the interviewer from nobelprize.org soon after the announcement, coming into biology from a different field, she was able to see things differently. “I was able to look at the problem with a totally fresh set of eyes,” she said. “A problem that had challenged people since the techniques of site-directed mutagenesis, for example—which won the Nobel Prize—were available. And I realised that the way that most people were going about protein engineering was doomed to failure.”
In the 1980s, Frances Arnold tried to redesign subtilisin, a bacterial enzyme, so that it could dissolve in organic solvents such as dimethylformamide (DMF) instead of only in water-based solutions, for the enzyme’s wider applicability and flexibility of use. Initially, however, she approached the problem in a rational, logical manner like all other researchers did. The basic building blocks of enzymes are the 20 amino acids that appear on the genetic code, which can be combined in all possible combinations, and a single enzyme can be made of thousands of amino acids linked in long chains forming complex special 3D structures. It is, therefore, not very surprising that she did not succeed in these early attempts.
Despite what was already known about life’s chemistry and the huge computing power that was available, Frances Arnold soon realised that remodelling nature’s elaborate architecture by logic to engineer an enzyme with new properties was a hopeless task, and as she has said, it was “a somewhat arrogant approach” in the face of nature’s superiority. So, she decided to follow nature’s own method, namely evolution. That is, to let evolution play out in a test tube as it were.
“Frances’ insight was to recognise that the most amazing molecules in the world weren’t created by chemists but rather by the biological world,” Jesse Bloom, a computational biologist and a former student of Frances Arnold’s, was quoted as saying by Quanta Magazine . “Most scientists were accustomed to thinking of evolution in terms of fossils of long-dead creatures, while chemical engineering was something that involved synthetic chemicals in the lab. The idea of applying evolutionary biology to problems in chemistry and biotechnology was a real leap,” Bloom said.
She created several mutated genes with random mutations in the enzyme’s genetic code. She engineered them into bacteria for them to produce thousands of variants of subtilisin. Her idea was to find out which of these worked best in DMF. First, on the basis of the knowledge that subtilisin breaks down the milk protein casein, she selected the variant that was most effective in breaking down casein in a solution with 35 per cent DMF. She then carried out one more round of mutations in this selected variant of subtilisin. This time she found a variant that worked even better in DMF. By the end of the third round, she had found a variant that worked 256 times better in DMF than the original enzyme. This third-generation subtilisin had a combination of 10 different mutations, and their effect on the enzyme could in no way have been predicted (Figure 1).
Thus, the power of allowing chance and directed selection, which is how nature works, in developing new enzymes, rather than depending solely on human logic and rationality, had been demonstrated. The work was published in Proceedings of the National Academy of Sciences in 1993. Not very long afterwards, the chemical revolution based on this ingenious idea followed.
Stemmer's 'DNA shuffling'
Willem P.C. Stemmer, a Dutch scientist and entrepreneur, would have probably shared the Nobel award with Frances Arnold if he had been alive. He died in 2013 when he was 56. “DNA shuffling” was one of the several biotechnology techniques he invented. An important element he had conceived and introduced in Frances Arnold’s directed evolution of enzymes was mating in a test tube. In natural evolution, a robust organism results when good genes from different individuals of the species get combined through mating or pollination and get selected, and less functional genes slowly disappear after a few generations. Stemmer’s DNA shuffling was essentially the laboratory equivalent of mating.
In 1994, Stemmer showed that if one took different variants of a gene, cut them into small pieces and shuffled them, with the tools of biotechnology one could piece together a complete gene, a new variant that would have a mosaic of the original versions. At the end of several cycles of DNA shuffling, Stemmer produced a variant of an enzyme that was much more efficient than the original improved enzyme.
With the enormous development that has occurred in DNA technology and the ever-increasing sophistication in the resulting tools, the technique of directed evolution is today considerably more advanced and more refined than the original, and Frances Arnold herself has been leading many of the developments in the field. Enzymes being produced in her laboratory can catalyse processes that do not exist in nature, which produce altogether new materials.
The pharmaceutical industry has benefitted from her specially tailored enzymes. Some of her enzymes have resulted in processes that obviate the use of heavy metals, which otherwise were used in the traditional chemical route to various substances, particularly pharmaceuticals, causing harm to the environment.
Frances Arnold has now returned to her original interest, working on renewable energy but with tools that have resulted from her Nobel Prize-winning work. Her group has developed enzymes that convert simple sugars into isobutanol, which can be used to produce biofuels and environment-friendly plastics. One of her long-term goals is to use the enzymes she has developed to produce sustainable fuels, which can be used in cars and aeroplanes, to create a greener transport sector.
Phage display technique The method of directed evolution in the hands of the other two laureates has resulted in the development of pharmaceuticals that can neutralise toxins, arrest the progression of autoimmune disorders and, in some cases, even cure metastatic cancer. The organism that the scientists used to implement directed evolution is a tiny virus that infects bacteria and replicates within bacteria, called bacteriophage or simply phage, and the method they developed is known as “phage display”.
Bacteriophages consist of a small loop of genetic material encapsulated in a protective coating of proteins. Their genomes can contain as few as four genes or as many as up to 100 genes. They replicate by injecting their genome into bacteria and integrating with the host DNA, thus hijacking their host’s metabolism. The bacteria then produce new copies of the phage’s genetic material and the proteins on the protective coat, which will then form new phages. In the 1980s, when Smith started using bacteriophages in his research, it was mainly with the idea of using them as hosts to clone genes. The idea was to insert a gene into bacteria, which would then mass produce the protein to be studied. Deciphering the human genome was still about 20 years away, and scientists had no straightforward way of identifying the specific gene for a given protein: it was like looking for the proverbial needle in the haystack.
Smith had this brilliant idea that researchers should be able to find the gene for a known protein by exploiting the simple structure of a phage. Molecular libraries containing large numbers of fragments of various unknown genes were available to scientists at that time. Smith’s idea was that these unknown gene fragments could be assembled together with the gene for one of the proteins in the phage capsule. When new phages were produced, the proteins from the unknown genes would also end up on the surfaces of the phages as part of the capsule protein (Figure 2). That is, one would have a medley of phages carrying a multitude of proteins on their surfaces.
Smith further argued that it should be possible to use antibodies as bait to fish out the correct phages carrying known proteins from this random mixture. Antibodies have the ability to home in on a single protein from among thousands of others with great precision and bind to it. Smith reasoned that if one identified a phage by the antibody to a known protein attached on its surface, the unknown gene for the known protein could now be deduced. In 1985, Smith demonstrated that his idea actually works with the following experiment.
He engineered a phage that carried a part of a protein, a peptide, on its surface. Using an antibody to the protein, he could identify the phage that he had constructed out of the soup of phages. This laid the foundation for what is known today as phage display. The idea is brilliant; the method, where the phage functions as the link between the protein and the gene, at once simple and elegant. When asked during the telephonic interview after the prize announcement whether the idea had come to him suddenly or whether it was a long process, Smith told the interviewer from nobelprize.org: “Oh no… certainly not suddenly, no. It was an idea from… many sources because I had all these streams that were very much in my background at the time. So it was definitely not something that just popped into my head…. It is very much [a case of] the right person and the right time. I mean I was trained in immunology, but I also knew a lot about this phage, and classical, you know, molecular biology, that’s what my basic training was in college.”
However, it was not in the identification of genes that the technique had its major impact. This happened when others started using phage display for biomolecules in the 1990s. Winter was one of the people who adopted this technique for his research.
Therapeutic antibodies
Antibodies are large Y-shaped proteins produced by the human lymphatic system and come in hundreds of thousands of different types. They are designed such that the far ends of the two arms can attach on to specific proteins the way a key fits into a lock. The evolution of the human immune system has ensured that they do not attach themselves to the body’s own biomolecules but selectively to those of an external pathogen, such as virus or bacteria, which is a signal to the immune system to mount a response and destroy the foreign body. (Autoimmune disorders occur when the immune system misfires and begins to attack the body’s healthy tissues and organs by mistake. Scientists do not yet understand what causes the immune system to (mis)behave thus.) Antibody therapeutics is an idea that goes back to the 1980s. In the hope of generating antibodies to specific targets, mice were injected with antigenic proteins, such as from cancer cells, so that the resultant antibodies could be harvested and turned into drugs. But the idea did not work for various reasons, chief among them being the extreme selectivity of antibodies. Patients’ immune systems recognised these mice-generated antibodies as foreign and destroyed them.
It was this hurdle that led Winter to apply Smith’s phage display technique to his research. His aim now was to develop drugs based on human antibodies so that the patient’s immune system would tolerate the drugs without any side effects. He inserted the genetic information for the end part of the antibody to the gene of a phage’s capsule protein and demonstrated in 1990 that the antibody-binding site was expressed on its coat. The antibody site he used was designed to latch on to a known molecule called phOx. With this molecule as the bait, Winter succeeded in pulling out the phage carrying the genetic information he had inserted into the original phage’s genome out of four million phages.
After this success, Winter realised that he could apply directed evolution to antibodies using the phage display technique and produce antibodies to combat diseases. He then built up a huge library of phages that express billions of varieties of antibodies on their surfaces. This became the repository from which he pulled out antibodies specific to different, especially pathogenic, target proteins. Using this first generation library, he then built up a second generation by randomly mutating the antibody genes in which he found antibodies with even greater specificity (Figure 3). In 1994, for example, he used his directed evolution-generated antibodies that attached to cancer cells with a high degree of specificity.
When asked whether he was surprised by the swiftness with which his idea was picked up and whether he ever thought it would lead to therapeutic antibodies that came out of Winter’s work, Smith told nobelprize.org: “As I say, all those precedents that were in the air, so no, I wasn’t that surprised actually… I’m sharing… our half [of the award] with Greg Winter… he came out of this Cambridge group that had been, as they were calling… cloning the immune system at the time. So that was very similar, very allied, line of reasoning, line of research. And I was very aware of that too. So I actually wasn’t surprised that people would catch onto it because it was something that was a way of thinking very much in the air at the time.
“I don’t think that, certainly in [19]85, that I thought in those terms [of therapeutic antibodies], although I was very much interested in antibodies and very much aware of the work in the Cambridge group. But the first publication of single-chain antibodies…single-chain antibodies are sort of paired down antibodies that have the central feature of binding specifically to an antigen that are missing a whole bunch of other things and are single polypeptide chains. At that point it became quite obvious that, well I won’t say quite obvious, but it seemed very plausible that not just small peptides but larger folded domains like single-chain antibodies could be displayed on phage just like small peptides. And, of course, the Cambridge group realised that at the same time, and independently.”
Realising the impact of the success of his research work, Winter and his colleagues soon formed a company that would develop drugs using the phage display of antibodies. Its first drug, called adalimumab, was an antibody-therapeutic entity that neutralises a protein, TNF-alpha, that drives inflammation in many autoimmune disorders. This drug was approved in 2002 for the treatment of rheumatoid arthritis and is used for different types of psoriasis and inflammatory bowel disease.
Asked during his post-announcement interview by nobelprize.org, whether he thought his work to engineer antibodies by the phage display technique would actually translate into therapeutics, Winter’s response captures in a nutshell the whole thing about research in basic sciences—how much of it is driven by curiosity to understand nature, how it builds on the work of many others, how some of it translates into applications, that eventually lead to immense benefits to mankind: “I wasn’t actually thinking about doing translational work at the time I started the work… it was much more an interest in how one might create new molecules in general…. In my earlier work with protein engineering, I’d just been more interested in understanding how enzymes and things work, and then I started moving to antibodies again to try to understand how antibodies worked.
“And then I realised the power of evolutionary technologies to create large repertoires of them and to select them, and of course, I think there are two components to the work that were really very important. The first was the generation of repertoires, and making sure those repertoires were efficient, fully folded proteins. And then, secondly, the way of displaying them on the use of the phage which George Smith had provided pointers to… so it wasn’t as if I’d thought at the very beginning ‘right, I need to create pharmaceutical antibodies’.
“You kind of start working along a different route and then you find yourself gradually being, you know, seizing opportunities as they come up, and those opportunities were opportunities to actually overcome a really difficult problem which was how to make human antibodies against human self-antigens. And so I realised that we could create this by using evolutionary technology. In some sense, it’s a bit like how the immune system works… I mean, you could think in terms of early evolution, but if you think about the way in which the immune system works, that is an evolutionary system. So… effectively… what we did is… to rationalise it in terms of how we can mimic the immune system… to make human antibodies, but without all the checks and controls the immune system would have that prevent you from making anti-self antibodies.”
Thus, the methods developed by the Nobel Prize-winning trio of Frances Arnold, Smith and Winter have laid the foundations for the beginning of a new world in chemistry that can be tailored to benefit humanity.
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