Studies on the Structure and Properties of Human Interferon
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Anfinsen, Christian
In 1980, the biochemist Christian Anfinsen participated for the first time in a Lindau meeting. Listening to his introduction, one can hear that he liked the lecturing situation: A Nobel Laureate and a select audience of students and young researchers. As Anfinsen puts it, the main function could be to make it clear that people who win the Nobel Prize are not really different from other people (except, in his view, certain exceptions such as Albert Schweitzer, Linus Pauling and a few others!). Many Nobel Laureates coming to Lindau for the first time repeat (more or less) their Nobel lectures that, according to the Statutes of the Nobel Foundation, should be “on a subject relevant to the work for which the prize has been awarded”. But Anfinsen choose to talk on his work on the human interferon and what medical applications it could have. The idea was that interferon could be important in curing viral diseases (maybe even cancer) and the problem was to get enough interferon to be able to make large-scale medical studies. Anfinsen discusses two approaches, first the traditional one of a biochemist (with a lot of money): Use 1000 litres of human white blood cells, infect them with a virus, use biochemistry to produce about 100 micrograms of interferon (which only amounts to a limited amount of doses for a patient). The second approach discussed was the modern one: Produce the interferon protein molecule using genetic engineering, since it is probably easier to find the structure of the interferon gene than that of the protein it produces. He may not have known it, but at the same time as Anfinsen gave his talk, the Nobel Committee for Chemistry discussed the 1980 Nobel Prize. In the early autumn it was decided that one of the fathers of genetic engineering, Paul Berg, should receive one of the two prizes. Today his technique is in fact, as Anfinsen discussed, used to produce several kinds of interferon for medical purposes! Anders Bárány
 Thank you, Professor Ho. I would like first to take the opportunity to thank you, Kat and Katas Benadot and the Oba Bugamast of Lindau and everyone else who has been so gracious in making us feel so comfortable here in Lindau. It's a beautiful place and this afternoon when the sun comes out it will be even nicer. Before I begin formally speaking and since it's still early in the morning so I'm, everybody's a little bit sleepy. I thought I could just say two words about some thoughts I was having about the significance of these meetings. It's a tremendous effort to get 20 wonderful geniuses like myself here. Together with all these enthusiastic students and so on. I was thinking about the true value of this kind of meeting. If we had two months I think we could actually achieve some kind of education back and forth. But as it is the time is very short and I think perhaps one of the main functions is to make it clear to students who are beginning now in their careers in science, make it clear to them that people who win Nobel prizes are not very different than other people. They may have had mothers that made them work a little harder in school or they may be a little more aggressive but they're really quite ordinary except for a few unusual. Once in a while you get unusual types like Schweitzer and Linus Pauling and so on. But I think that's perhaps one of the main messages is to make it clear that we're all ordinary people together. Some people work perhaps a little more diligently, perhaps a little luckier, have better postdocs coming to the laboratory and so on. So that's, I think I'm now more awake than I was before. I would like to be able to begin this lecture with a sentence that I've often thought would be ideal at the beginning of the lecture. Cancer may be cured as follows. That would be a nice way to begin. And I think if you read Time Magazine and all the newspapers and listen to the television most people have the impression that this interferon molecule is the cure of everything. As a matter of fact it may very well turn out to be an extremely useful substance in the treatment of some viral diseases. The problem in the case of cancer for example is that we really don't know that cancers are generally viral diseases. There may be some cancers that are viral and some that are not. We know that interferon is definitely an antiviral substance. It's very difficult to get enough interferon to test on a large scale. And what I'd like to tell you about today is the current status of our own more chemical approach to the problem and a few things about the current status of the availability of interferon through genetic engineering and other techniques. It has been tested clinically now not in a very thorough way but sufficiently to make it almost certain that it will be useful as a drug against some viral diseases notably hepatitis B I think, herpes zoster which is a very serious neurological disease, juvenile papilloma has been some very nice work done in Sweden on this disease in the throat of young of juveniles. Possibly in some forms of cancer osteosarcoma has been under study in Sweden for a number of years by Strander and his colleagues. And in recent year, in the past year, enough money has become available from a number of governments to buy large quantities of interferon as much as possible from the group in Finland that is manufacturing interferon from the white cells that are obtained from blood bank contributions. So that perhaps in another year we will have some definite information on such serious large scale problems as breast cancer and other forms of cancer that are now being studied systematically. Another problem that I'll talk more about is the fact that the interferon that is available is generally only about one or two or three percent pure so that when we do these tests at the moment it's not entirely clear whether it's only the interferon that's doing whatever happened good or bad but possibly other small proteins that are mixed with the interferon. The substance was discovered by Isaacs and Lindemann in England in 1957. They did some experiments and biological experiments to examine an old well-known phenomenon in medicine known as interference. If you've been sick from a viral disease and you recover from this disease there's a sort of subjective impression that one is somewhat more resistant to other viral diseases. The body has somehow developed some resistance. They show that if they took chicken, chick cells in culture and exposed these cells to killed flu virus that the cell and then washed off the killed flu virus after a certain time that the cells had produced a substance which made them resistant to live flu virus. In other words the virus, the viral material had induced the production of an antiviral substance and they were able to show by indirect methods with these tiny, tiny amounts of material that it was indeed a protein of fairly small molecular weight and that it was species specific that is to say interferon produced in a mouse would not protect a horse or vice versa but that interferon from producing a human cell culture would protect human cells against many viruses. So it's species non-specific, species specific but virus non-specific. And the observation was very interesting and attracted some attention but not on a large scale. Only because there was not much available. Most of the early work was done with white cell interferon and as I mentioned before in Finland, Kerry Cantel arranged with the Red Cross in Finland to get almost all of the Buffy coat, the white cells from blood cell, from blood contributions and taking these white cells and adding to it Sendai virus, he was able to stimulate interferon production by these cells, remove the cells and then purify the interferon somewhat and distribute this for clinical trial. And most of that early material and indeed throughout the 60s and partially throughout the 70s, most of that was used in clinical trials on a rather small scale and mainly in Finland although some work was being done in other countries in England, France, Belgium, United States. So that the quantities were limited and there was not that much activity. Suddenly it became clear, I think partially through the press, that here was a possible substance that might be of value in viral diseases including cancer and immediately a lot of biochemists like myself and a lot of physicians got into the business and began to think about isolation and production and clinical trial. I'm not a physician myself so I don't want to show any clinical results. I have one slide on a clinical trial which was carried out by my friend Michelle Ravel in Israel together with some physicians in Tel Aviv on a disease known as shipyard disease. It's an adenovirus conjunctivitis which does cure itself after 35 or 40 days although it can leave serious permanent damage to the cornea. The first slide please is a summary of some experiments that can't reveal and his colleagues made where they took out of a group of patients they took about 17 controls who were given simply some human albumin eye drops in the eye five or six times a day and another 15 who were given interferon as eye drops in the eye. Then you will see down where it says average length of disease that the controls cured themselves mainly after about 27, 30 days whereas those who received interferon were free of the conjunctivitis after six and a half days on the average and if they selected among the experimental patients those with double with bilateral conjunctivitis and treated one eye and not the other so they had an internal control once again the treated eye is cured in seven days and treated eye in 25 days. Lights please. It's a trivial example but I show it only to indicate to you that interferon does indeed stop the course of a viral disease and we hope that much more dramatic results will be forthcoming in the next year. So what I want to do now is to tell you something about preparation, characterization and possibilities for the future. There are three possible ways one can think of making enough interferon. One is to grow large amounts of human cells in culture and then stimulate the cells with virus to make interferon and then purify the interferon from these cultures. That's what we've been doing for the last five or six years. It's a very slow, tedious, large scale project. A second way would be to hope that one could get enough interferon to chemically characterize the molecule and then knowing the structure, knowing the amino acid sequence of the chain of amino acids to be able to do the classical process of organic synthesis to make the molecule from individual amino acids. As you'll see, it's not a small molecule so this becomes a very large task, a very difficult task but it is a possibility and it's the one that happens at my own associates and I have selected. The third became possible only in the last few years and is now beginning to become quite exciting and this is the possibility of taking the human gene for interferon, putting it into a bacterial cell, an E. coli cell for example, and allowing the bacterium to make the interferon for us and then isolating the interferon either from the bacterial cell or from the medium in which the bacterium swims. As you all undoubtedly know from the press and elsewhere, within some hundred kilometers, Charles Weisman at the University of Zurich and his colleagues have already managed to make some white cell interferon in E. coli by genetic engineering and there's been a group in Japan, Taniguchi, who have done this with fibroblast interferon and there are two or three other groups now who have clones of bacterial cells that can make one of several different kinds of interferon and there seem to be a number of different, the human chromosome seems to have perhaps as much as 10 different interferon genes so that a number of different interferons are made in different cells. So this is the third possibility for making large amounts. Now I thought I might just tell you something about to give you some impression of the power, the powerfulness of this drug, of this protein and also about the minute amounts that one obtains to describe how you assay the interferon, how do you test for interferon. The standard technique is as follows. You take a porcelain plate with 8 by 12 holes, 96 depressions and in each of these depressions you grow fibroblasts, human cells that grow in sheets. You grow about a million cells in each of these 96 wells and then you expose each of these wells to an interferon solution, first the undiluted and then say 1 to 2, 1 to 4, 1 to 8, 1 to 16 and allow it to sit overnight and you shake out the interferon and then you put in a virus, standard virus suspension into each hole and then the next day you look at the plate under the microscope and determine the dilution at which half the cells have been protected against cell destruction by the interferon. On that point it's considered one unit. It's a very, very crude test. It's accurate only to about three tenths of a log, about plus or minus 50, 100%, 50%. So it's quite inaccurate and takes two to three days but at the moment that is the only relatively safe way of assaying for interferon. So namely one unit would be the amount to protect half of a million cells against the standard viral dose. Now when we grow a thousand liters of human white cells in culture and infect these cells when they're grown up to about three times ten to six, three million cells per milliliter with virus out of a thousand liters of cell culture we are very lucky if we can have a total amount of perhaps 20 milligrams and after isolation perhaps two milligrams, generally more like a tenth of it, like 100 micrograms so that the amounts what it gains are very disappointingly small. Furthermore it turns out that one milligram is equal to about two times ten to the eighth units. Now if you were treating patients you would like to give, it's become standard practice to administer not less than a million units per day to a patient. So if you had say one milligram of interferon you could give 20 days of treatment to one person and it works out that if you had one gram of interferon in pure form you would be able to treat a thousand patients for perhaps 200 days. Of course what we need is enough to treat 10 million patients for 100 days. So obviously the amounts required are much larger than that. I'd like to show some, a few slides of our own studies on the purification of interferon to give you some idea of the difficulties that are involved. The next slide please. This is it. What we did originally, this was seven or eight years ago, we decided that the perhaps the most efficient selective purification method would be to use immunological techniques. So we took some of the interferon that we obtained from Finland and purified it on a Cephodex gel filtration column. It's simply a column that separates on the basis of size. And if we look at this left, on the left side the dark circles represent the peak of activity, the interferon activity coming off this column. The white circles are total protein. So most of the impurities go out first and then comes the interferon with only a small amount of protein. It's still at this point only perhaps half of 1% pure. But it's good enough to give as an antigen to animals to prepare antibody. So we gave one or two micrograms every two weeks for 16 to 18 weeks to sheep. And it turns out that interferon is a very good antigen and made rather high titers of anti-interferon in three or four months time. So that we then had an antibody which would catch interferon quite efficiently. The antibodies produced by the sheep, of course, are not only against interferon but also all the other protein impurities that are in the material that was injected into the sheep. And we tried then to purify this antibody. The next slide is a slide showing that if you use the following technique you can make much better antibody. We prepared what we called a cocktail column. What we did was to take all of the protein impurities that we could think of that might be in the material that we gave to the sheep, serum proteins, egg proteins, virus proteins and so on, attach them to a column, and then we passed the antibody through that column. All the impurities caught the antibodies against the impurities. And what went through the column in the beginning contained the anti-interferon, and the anti-impurities could then be taken off with acid, and the column could be washed, and then you could repeat the process many times. And eventually one could get an antibody preparation which was mainly free of antibodies against the impurities. Now this purified antibody could then be attached to another column. We chemically attached the antibody to a Cepharose column, and passing crude interferon through the column then permits the interferon in that crude substance to be caught by the antibodies, and the impurities go through. You can wash the impurities off. The next slide, you see that most of the protein goes through the column without losing much interferon, and you wash and wash and wash, and finally you can take the interferon off the column at once again at a low pH, a higher acidity, and you get out some material which has now been purified between 500 and 5000 times in this one step. So this is a way of getting from very large volumes down to very small volumes fairly quickly. The only way we felt that we could make very large quantities eventually was to obtain some kind of human, was to use a human cell which would grow well in tissue culture. And fortunately, Strander and his colleagues in Sweden had examined many kinds of B lymphocytes for their capacity to produce interferon when stimulated with viruses. And one particular kind of B lymphocyte is known as Nimalva. That's a pet name for this cell. It was originally isolated from burgut lymphoma, a virus produced lymphoma. The next slide I think shows a picture of this cell. It's not terribly pretty. It grows very well in solution. You grow it in a rich salt solution with vitamins and amino acids and so on. And unfortunately, you get the best growth when you add 10% fetal cast serum which is now becoming extremely expensive. And we have to develop some new techniques for growing on a large scale that will be a lot less expensive. The next slide shows some properties of this lymphoblastoid interferon that we are working with. This shows you that it's very stable, both to temperature and to acidity. For example, at almost boiling temperature at 97 degrees centigrade, one can allow the material to sit for 10 minutes and still have essentially the same activity so that it's extremely stable to heat and low pH. Next slide. It's however quite heterogeneous in the sense of its electrical properties. It turns out that interferon is a glycoprotein. It's a protein molecule, globular protein molecule, on which carbohydrate is also attached. And these carbohydrate side chains contain acidic residues, sialic acid residues, which are present in different numbers on different molecules, sometimes there are three, sometimes four, sometimes two. So that if you do an isoelectric focusing experiment, you get quite a large heterogeneity in the isoelectric points. These peaks that you see are different forms of interferon differing by their charge due to the difference in sugar. However, if you remove the sugar, thereby removing the negative charge from all of these side chains of carbohydrate, the material becomes much more homogeneous. This shows first of all that the heterogeneity is due to the carbohydrate side chains. And secondly of all, and secondly, that the carbohydrate can be removed without losing activity because it's still active in tests. The next slide indicates the results of treating partially purified lipoblastoid interferon with a mixture of enzymes which choose off carbohydrate, which we obtained from a bacterium dipococcus pneumoniae. The molecular weight of the interferon molecule shifts to a smaller weight when you take off carbohydrate. And if as shown in the next slide, you plot the results on a standard type of figure showing the relationship between weight and behavior on a column against some standard proteins of known molecular weight, you can see that the interferon after treatment with enzymes has shifted to a lower molecular weight. As a matter of fact, one can chop off about 4,000 Dalton's of molecular weight, and the weight goes from about 22,000 to about 18,500 having taken off most of the carbohydrate. It's still active and upon injection into, let's say, a rat, you can show that it is maintained in the circulation at least as long as the normal untreated interferon so that it's not destroyed quickly. Consequently, we feel that it is realistic to think of synthesizing the protein part of the interferon and not having to worry about the carbohydrate part because that would be essentially impossible. There's no organic chemistry at the moment that permits the systematic synthesis of carbohydrate side chains on proteins, but apparently we don't need that, so that's a very lucky thing. The problem now is to make large amounts. The next slide is the kinetics of growth, kinetics of production of interferon by these lymphoblastoid cells. What we do is to grow the cells up in large tanks until they reach about 2 million per milliliter and they're infected with the new castle disease virus which induces the synthesis of interferon by these cells and they secrete the interferon out into the medium that they're growing in. There's a slight lag at the beginning, at the end of about 20 hours you have reached the maximum production of interferon. Then the cells are removed by taking the whole contents of the tank through a large cream separator, actually a cream separator like you use on a farm turns out to be the most convenient way of removing the cells. The fluid comes out and we precipitate all of the protein including the interferon and that's the starting crude material. The next slide shows that one can actually save a little money and it is important in these experiments to do that. Fetal calf serum at the moment costs I think on the order of $200 per liter. It's extremely expensive. We use 10% fetal calf serum in growing these cells. In 1000 liters we have 100 liters at $200 per liter. It's a little bit rich. However, it turns out that the lymphoblastoid cell produces interferon much more efficiently at a lower cell concentration than it does at a higher cell concentration. You see if you infect with Newcastle disease virus at 2 times 10 of the 6 cells per mil, you get in this case 1800 units per mil. If you dilute down to 0.4 times 10 of the 6, we get 7,000. Simply by adding salt solution, you increase the ability of the efficiency of production of interferon. What we do routinely is grow up, instead of 1000, we grow up 250 liters and then dilute to 1000 with salt solution. Next slide shows here the same thing. At the top is the undiluted cells and their efficiency in producing interferon. At the bottom is the efficiency of the cells. They're diluting with salt solution containing glutamine, which is an essential component during the production. Basically we get 4 times as much interferon as we would otherwise, simply by diluting the cells. The next slide. This is a typical kind of slide that you can't read. Simply summarizing all of the steps that we go through. We start, as I mentioned before, we take off the cells in the cream separator. Then we precipitate all the proteins in that clear fluid with trichloroacetic acid. Then we go through a number of steps. First to remove the trichloroacetic acid, then this antibody column that I spoke of, which catches the interferon. A number of other steps including sizing on columns, ion exchange separations, and finally a polyacrylamide gel separation in SDS, sodium nodesisulfate slab gel purification. By this time we're down to very small amounts. In this particular slide the total recovery was only 6%, which is very bad. We now have this up to about 15%, and I hope we'll soon have perhaps 50% with some new tricks that we are beginning to use. But it's still a very slow and tedious process. As you can see, at this point we have on the order of 2-tenths of a milligram of protein from 200 liters of cells. The specific activity of the pure material, this is the 18,500 k, 18,500 molecular weight, is about 2.2 times 10 to the eighth units, 2 to 4 times 10 to the eighth units per milligram. This is the purity of such material. There is one other component, the 21.5, which is the same as the 18.5, very likely, with carbohydrates still attached, which makes it heavier by that many units. The next slide, this is a polyacrylamide analytical slab gel with the interferon activity plotted as black points at the bottom, and at the top the pattern of the gel stained with comassie blue to indicate the position of the components. You see the dark band corresponding to the main interferon peak, and another smaller band with activity to the left. If you then cut out this main band and rerun it on another slab, the next slide, you see on the right a pure sample of human lymphoblastoid interferon against some known proteins as markers on the left to give some idea of the molecular weight. The first pure material was produced by Ernest Knight at DuPont about two years ago from fibroblast interferon. This is our lymphoblastoid interferon. Both varieties have about the same molecular weight and the same specific activity. That is to say, they both, one milligram is approximately two or three times 10 to the eighth units. The next slide. Once again, protein chemists always show amino acid analysis. Not terribly useful, but this is simply the amino acid analysis done with the micro method for amino acid analysis on some pure interferon. It's perhaps only interesting to point out that there is quite a high amount of hydrophobic amino acids like leucine, phenylalanine, valine, and a few others. The next slide is more of the same, but here what we've done is to take the amino acid analysis from fibroblast lymphoblastoid, leukocyte, and mouse, mouse interferon. Amino acid analyses have been done on pure specimens of all of these. This is simply to show that as you go from a mouse to a leukocyte to a fibroblast to a lymphoblastoid cell, the amino acid composition is about the same. Each type has about the same amount of lysine or histidine or arginine and so on. That last fall in November, we felt we had done enough on purification to accumulate material that could be put into the amino acid sequinator, the machine that has been designed over the last years by Edmund and his followers that will tell you something about the structure of a protein, one by one. Next slide shows results which were produced actually by Hood and Hunkapiller, two scientists at Caltech in California who have one of these very sensitive machines for determining sequence. We sent to him, together with Pete Knight at DuPont with his fibroblast interferon, and Leng Yell and his group at Yale who had mouse interferon samples that these people could degrade. It turned out that the mouse came in two varieties, or three varieties, A, B, and C. We show here some results on A and C. Simply to indicate, particularly here at the bottom, that the mouse aeroleicosides interferon called band C is rather similar to the material that we obtained from lymphoblastoid cells. There's quite a high degree of homology in structure. This is only the first 20 amino acids, but it is a beginning, and from the amino terminus it's the first 20 residues, and there's quite a high degree of similarity. It does at least tell us that the gene for interferon in the human and in the mouse, those two genes must be quite similar, at least for the first 20 amino acids. It means that this is probably quite an old gene. It can be found in animals as far as fish and turtles and frogs, so that the interferon gene has been around for quite a while. We were very proud of ourselves at this point, because after six or seven years we had finally achieved some beginnings of sequence. We really had a protein that was pure and it was characterizable. So we were working along, making more material to have the rest of the sequence finished, when suddenly the genetic revolution occurred in Zurich and elsewhere. One of the nice things about genetic engineering and producing material in E. coli cells is that you can obtain the DNA from the chromosome of the E. coli cell, and it's much easier to determine the structure of a DNA fragment than it is to determine the structure of a protein, so that when Charles Weisman, for example, obtained a clone of E. coli that produced interferon, he was able to very quickly take this material and buy some techniques, details which I won't go into, because first of all, I'm not terribly familiar with the field and it's quite complicated to explain. He was able to work out the total sequence of the gene that determined the interferon that was made by those cells for RNA message, RNA messenger RNA isolated from white cells. He used leukocyte message to prepare the original plasmid, which he put into the E. coli cell for translation. The next slide shows a very confusing slide, it's not as confusing as it looks actually. On this slide, what I've done is to put down Charles Weisman and his colleagues' sequence for one of the leukocyte interferons as a protein, that is to say, that's the center of the lines, the one that begins with the CYS and the box at the upper left. He goes along the center one all the way through is Weisman's leukocyte interferon, knowing the DNA strand sequence and knowing the genetic code, you can simply read off the amino acid that corresponds to the triplets of nucleotide bases in that DNA sequence and translate the DNA structure into a protein structure. The top line represents a similar translation of the DNA structure that was determined by Teneguchi and his colleagues' Japanese group from fibroblast DNA. This is another interferon gene. In their case, they selected the gene from fibroblast. So that's on the top line and on the bottom line is the sequence that we now have from our own material on lymphoblastoid. As you see, there are some empty spaces that are not finished, but we hope in the next months to complete this. The important thing is that when you go from fibroblast to leukocyte to a specific white cell like lymphoblastoid, there is a large amount of similarity. I have enclosed those areas where the same sequence occurs in all, in the three species. And there's obviously a great deal of homology down from one cell type to another. I've also cross-hatched the areas which include the cysteine residues, the amino acids that are responsible for making cross-links through SS bridges in the protein when it folds up. And there's one that I forgot to cross-hatch down at 140. There's a CYSALATRP cysteine alanine tryptophan, which occurs both in the fibroblast sequence and the leukocyte sequence. We unfortunately do not have it yet in our structure, although we know that we have one tryptophan in the molecule and we hope to have that section soon. The fact that that section and the section at the upper right has been so carefully maintained in these three varieties suggests that these may be particularly important cysteines that perhaps form an SS bridge. But this kind of detailed chemical consideration will have to really await the isolation of larger amounts. So what to do if you have this? For Taniguchi and Weisman, the future is very clear. They have to produce E. coli systems, E. coli cells themselves, plasmids to insert into these that are better and better and better and can produce more and more interferon. And then they have to purify the interferon away from the proteins of the bacterium sufficiently that it becomes pure enough to be able to give to human beings. You can't give human patients impure proteins because one develops antibodies against them and you have to avoid such phenomena as anaphylaxis and so on. So although it sounds phenomenal in time magazine, and I think these people have done a marvelous job and will probably eventually produce the interferon that we need, they still have a way to go in terms of purification and preparing better vectors for their work. In our own case, since we're not quite finished with our own sequence and since we're interested in synthesis, we have started to synthesize slowly the sequence from Weisman's leukocyte interferon structure, the middle line here. And we're doing this both by the new solid phase synthetic techniques developed by Merrifield and his colleagues and also by classical peptide synthesis fragment condensation, which is an enormous job and may take too many years to think about unless we can think of some tricks or perhaps some combination of both techniques. The other thing we would like to do is to grow enough material or to get enough material from Weisman or Taniguchi to do some careful structure function work. That is to say to try to determine whether one can chew away parts of the molecule and still keep the activity because if one could cut away half the molecule and make it 80 amino acids instead of 160, then it would become synthetically not so difficult. So that's our own hope at the moment. And perhaps we can even use the E. coli interferon for our structure function work. I think it would be interesting, I'm sure, to someone like Dorothy Hartzke and Bill Lipscomb sitting here to think that if the crystallography three-dimensional structure of interferon has ever worked out, it will probably be on synthetic material because we'll probably never have enough of the other kind to do it, but perhaps we will. It would be a nice first to try. One final point that might give some interest to some of your... We've known for some time that interferon and cholerotoxin compete for the same receptors on the surface of cells. We can add interferon and compete for the recognition by adding cholerotoxin simultaneously and vice versa. So I asked the people who put out this dictionary of protein sequences, Margaret Dayhoff and her colleagues who have a computer full of all the known sequences, to compare the leukocyte sequence of Weismann with all other known protein sequences of which there must be hundreds of them. And she called back and said in a very unbelieving voice that there seemed to be very little similarities that there was one rather interesting one that there was... Next slide, please. Rather interesting similarity between the interferon structure and the sequence of cholerotoxin, which I thought was very nice, because I didn't tell her in advance. It was out of the computer without any hinting. It turns out that at the very bottom here, there's a number called an alignment score, a score of 4.1 approximately, which is considered rather good. The alignment score for, say, myoglobin and beta chain of hemoglobin in man is something like nine. So this is really a rather high level of homology in sequence. And you can see that in this section here, there's quite a large number of identical amino acids. Could I go back to the last slide, please? The previous slide. So that as a beginning, we have started at the bottom right, and we are working backwards, and it turns out that this section that resembles cholerotoxin runs from about 150 back to about 120. In that region are these similarities. It'll be very interesting to see whether this peptide, which we now have essentially finished, will be competitive with cholerotoxin, and perhaps may serve, for example, as an antigenic site that we can use for further purification of our anti-interferon antibodies. And it might also give us some approach to the process by which, understanding of the process by which interferon recognizes the cell surface. I mentioned in an abstract that I sent here before we came that I would say something about the mechanism of action. I'm afraid that's very difficult to do. All one can say at the moment is that if you give interferon to cells, it is recognized by the cell and then induces the cell to make two or three enzymes, which either are absent or present in very small amounts before the interferon appears. The enzymes that are produced are all involved in the control of message translation, that is to say in protein synthesis. The translation of RNA message into protein. And interferon appears, therefore, at the moment from the work of a number of laboratories, to be involved in the inhibition of the translation of the viral message that comes into the cell. That's about all we know about the mechanism of action. Most of the rest is phenomenological, and I hope that we'll be able to say something specific about not only how it works, but against which diseases it is helpful if at all. And I feel quite confident myself that it will be very helpful against certainly some viral diseases. Cancer is still a knock on the wood question. Thank you. Thank you.
 Thank you, Professor Ho.  I would like first to take the opportunity to thank you, Kat and Katas Benadot and the  Oba Bugamast of Lindau and everyone else who has been so gracious in making us feel  so comfortable here in Lindau.  It's a beautiful place and this afternoon when the sun comes out it will be even nicer.  Before I begin formally speaking and since it's still early in the morning so I'm, everybody's  a little bit sleepy.  I thought I could just say two words about some thoughts I was having about the significance  of these meetings.  It's a tremendous effort to get 20 wonderful geniuses like myself here.  Together with all these enthusiastic students and so on.  I was thinking about the true value of this kind of meeting.  If we had two months I think we could actually achieve some kind of education back and forth.  But as it is the time is very short and I think perhaps one of the main functions is  to make it clear to students who are beginning now in their careers in science, make it clear  to them that people who win Nobel prizes are not very different than other people.  They may have had mothers that made them work a little harder in school or they may be a  little more aggressive but they're really quite ordinary except for a few unusual.  Once in a while you get unusual types like Schweitzer and Linus Pauling and so on.  But I think that's perhaps one of the main messages is to make it clear that we're all  ordinary people together.  Some people work perhaps a little more diligently, perhaps a little luckier, have better postdocs  coming to the laboratory and so on.  So that's, I think I'm now more awake than I was before.  I would like to be able to begin this lecture with a sentence that I've often thought would  be ideal at the beginning of the lecture.  Cancer may be cured as follows.  That would be a nice way to begin.  And I think if you read Time Magazine and all the newspapers and listen to the television  most people have the impression that this interferon molecule is the cure of everything.  As a matter of fact it may very well turn out to be an extremely useful substance in  the treatment of some viral diseases.  The problem in the case of cancer for example is that we really don't know that cancers  are generally viral diseases.  There may be some cancers that are viral and some that are not.  We know that interferon is definitely an antiviral substance.  It's very difficult to get enough interferon to test on a large scale.  And what I'd like to tell you about today is the current status of our own more chemical  approach to the problem and a few things about the current status of the availability of  interferon through genetic engineering and other techniques.  It has been tested clinically now not in a very thorough way but sufficiently to make  it almost certain that it will be useful as a drug against some viral diseases notably  hepatitis B I think, herpes zoster which is a very serious neurological disease, juvenile  papilloma has been some very nice work done in Sweden on this disease in the throat of  young of juveniles.  Possibly in some forms of cancer osteosarcoma has been under study in Sweden for a number  of years by Strander and his colleagues.  And in recent year, in the past year, enough money has become available from a number of  governments to buy large quantities of interferon as much as possible from the group in Finland  that is manufacturing interferon from the white cells that are obtained from blood bank  contributions.  So that perhaps in another year we will have some definite information on such serious large  scale problems as breast cancer and other forms of cancer that are now being studied  systematically.  Another problem that I'll talk more about is the fact that the interferon that is available  is generally only about one or two or three percent pure so that when we do these tests  at the moment it's not entirely clear whether it's only the interferon that's doing whatever  happened good or bad but possibly other small proteins that are mixed with the interferon.  The substance was discovered by Isaacs and Lindemann in England in 1957.  They did some experiments and biological experiments to examine an old well-known phenomenon in  medicine known as interference.  If you've been sick from a viral disease and you recover from this disease there's a sort  of subjective impression that one is somewhat more resistant to other viral diseases.  The body has somehow developed some resistance.  They show that if they took chicken, chick cells in culture and exposed these cells to  killed flu virus that the cell and then washed off the killed flu virus after a certain time  that the cells had produced a substance which made them resistant to live flu virus.  In other words the virus, the viral material had induced the production of an antiviral  substance and they were able to show by indirect methods with these tiny, tiny amounts of material  that it was indeed a protein of fairly small molecular weight and that it was species specific  that is to say interferon produced in a mouse would not protect a horse or vice versa but  that interferon from producing a human cell culture would protect human cells against  many viruses.  So it's species non-specific, species specific but virus non-specific.  And the observation was very interesting and attracted some attention but not on a large  scale.  Only because there was not much available.  Most of the early work was done with white cell interferon and as I mentioned before  in Finland, Kerry Cantel arranged with the Red Cross in Finland to get almost all of  the Buffy coat, the white cells from blood cell, from blood contributions and taking  these white cells and adding to it Sendai virus, he was able to stimulate interferon  production by these cells, remove the cells and then purify the interferon somewhat and  distribute this for clinical trial.  And most of that early material and indeed throughout the 60s and partially throughout  the 70s, most of that was used in clinical trials on a rather small scale and mainly  in Finland although some work was being done in other countries in England, France, Belgium,  United States.  So that the quantities were limited and there was not that much activity.  Suddenly it became clear, I think partially through the press, that here was a possible  substance that might be of value in viral diseases including cancer and immediately  a lot of biochemists like myself and a lot of physicians got into the business and began  to think about isolation and production and clinical trial.  I'm not a physician myself so I don't want to show any clinical results.  I have one slide on a clinical trial which was carried out by my friend Michelle Ravel  in Israel together with some physicians in Tel Aviv on a disease known as shipyard disease.  It's an adenovirus conjunctivitis which does cure itself after 35 or 40 days although it  can leave serious permanent damage to the cornea.  The first slide please is a summary of some experiments that can't reveal and his colleagues  made where they took out of a group of patients they took about 17 controls who were given  simply some human albumin eye drops in the eye five or six times a day and another 15  who were given interferon as eye drops in the eye.  Then you will see down where it says average length of disease that the controls cured  themselves mainly after about 27, 30 days whereas those who received interferon were  free of the conjunctivitis after six and a half days on the average and if they selected  among the experimental patients those with double with bilateral conjunctivitis and treated  one eye and not the other so they had an internal control once again the treated eye is cured  in seven days and treated eye in 25 days.  Lights please.  It's a trivial example but I show it only to indicate to you that interferon does indeed  stop the course of a viral disease and we hope that much more dramatic results will be forthcoming  in the next year.  So what I want to do now is to tell you something about preparation, characterization and possibilities  for the future.  There are three possible ways one can think of making enough interferon.  One is to grow large amounts of human cells in culture and then stimulate the cells with  virus to make interferon and then purify the interferon from these cultures.  That's what we've been doing for the last five or six years.  It's a very slow, tedious, large scale project.  A second way would be to hope that one could get enough interferon to chemically characterize  the molecule and then knowing the structure, knowing the amino acid sequence of the chain  of amino acids to be able to do the classical process of organic synthesis to make the molecule  from individual amino acids.  As you'll see, it's not a small molecule so this becomes a very large task, a very difficult  task but it is a possibility and it's the one that happens at my own associates and  I have selected.  The third became possible only in the last few years and is now beginning to become quite  exciting and this is the possibility of taking the human gene for interferon, putting it into  a bacterial cell, an E. coli cell for example, and allowing the bacterium to make the interferon  for us and then isolating the interferon either from the bacterial cell or from the medium  in which the bacterium swims.  As you all undoubtedly know from the press and elsewhere, within some hundred kilometers,  Charles Weisman at the University of Zurich and his colleagues have already managed to  make some white cell interferon in E. coli by genetic engineering and there's been a  group in Japan, Taniguchi, who have done this with fibroblast interferon and there are two  or three other groups now who have clones of bacterial cells that can make one of several  different kinds of interferon and there seem to be a number of different, the human chromosome  seems to have perhaps as much as 10 different interferon genes so that a number of different  interferons are made in different cells.  So this is the third possibility for making large amounts.  Now I thought I might just tell you something about to give you some impression of the power,  the powerfulness of this drug, of this protein and also about the minute amounts that one  obtains to describe how you assay the interferon, how do you test for interferon.  The standard technique is as follows.  You take a porcelain plate with 8 by 12 holes, 96 depressions and in each of these depressions  you grow fibroblasts, human cells that grow in sheets.  You grow about a million cells in each of these 96 wells and then you expose each of  these wells to an interferon solution, first the undiluted and then say 1 to 2, 1 to 4,  1 to 8, 1 to 16 and allow it to sit overnight and you shake out the interferon and then  you put in a virus, standard virus suspension into each hole and then the next day you look  at the plate under the microscope and determine the dilution at which half the cells have  been protected against cell destruction by the interferon.  On that point it's considered one unit.  It's a very, very crude test.  It's accurate only to about three tenths of a log, about plus or minus 50, 100%, 50%.  So it's quite inaccurate and takes two to three days but at the moment that is the only  relatively safe way of assaying for interferon.  So namely one unit would be the amount to protect half of a million cells against the  standard viral dose.  Now when we grow a thousand liters of human white cells in culture and infect these cells  when they're grown up to about three times ten to six, three million cells per milliliter  with virus out of a thousand liters of cell culture we are very lucky if we can have a  total amount of perhaps 20 milligrams and after isolation perhaps two milligrams, generally  more like a tenth of it, like 100 micrograms so that the amounts what it gains are very  disappointingly small.  Furthermore it turns out that one milligram is equal to about two times ten to the eighth  units.  Now if you were treating patients you would like to give, it's become standard practice  to administer not less than a million units per day to a patient.  So if you had say one milligram of interferon you could give 20 days of treatment to one  person and it works out that if you had one gram of interferon in pure form you would  be able to treat a thousand patients for perhaps 200 days.  Of course what we need is enough to treat 10 million patients for 100 days.  So obviously the amounts required are much larger than that.  I'd like to show some, a few slides of our own studies on the purification of interferon  to give you some idea of the difficulties that are involved.  The next slide please.  This is it.  What we did originally, this was seven or eight years ago, we decided that the perhaps  the most efficient selective purification method would be to use immunological techniques.  So we took some of the interferon that we obtained from Finland and purified it on a  Cephodex gel filtration column.  It's simply a column that separates on the basis of size.  And if we look at this left, on the left side the dark circles represent the peak of activity,  the interferon activity coming off this column.  The white circles are total protein.  So most of the impurities go out first and then comes the interferon with only a small  amount of protein.  It's still at this point only perhaps half of 1% pure.  But it's good enough to give as an antigen to animals to prepare antibody.  So we gave one or two micrograms every two weeks for 16 to 18 weeks to sheep.  And it turns out that interferon is a very good antigen and made rather high titers of  anti-interferon in three or four months time.  So that we then had an antibody which would catch interferon quite efficiently.  The antibodies produced by the sheep, of course, are not only against interferon but also all  the other protein impurities that are in the material that was injected into the sheep.  And we tried then to purify this antibody.  The next slide is a slide showing that if you use the following technique you can make  much better antibody.  We prepared what we called a cocktail column.  What we did was to take all of the protein impurities that we could think of that might  be in the material that we gave to the sheep, serum proteins, egg proteins, virus proteins  and so on, attach them to a column, and then we passed the antibody through that column.  All the impurities caught the antibodies against the impurities.  And what went through the column in the beginning contained the anti-interferon, and the anti-impurities  could then be taken off with acid, and the column could be washed, and then you could  repeat the process many times.  And eventually one could get an antibody preparation which was mainly free of antibodies against  the impurities.  Now this purified antibody could then be attached to another column.  We chemically attached the antibody to a Cepharose column, and passing crude interferon through  the column then permits the interferon in that crude substance to be caught by the antibodies,  and the impurities go through.  You can wash the impurities off.  The next slide, you see that most of the protein goes through the column without losing much  interferon, and you wash and wash and wash, and finally you can take the interferon off  the column at once again at a low pH, a higher acidity, and you get out some material which  has now been purified between 500 and 5000 times in this one step.  So this is a way of getting from very large volumes down to very small volumes fairly  quickly.  The only way we felt that we could make very large quantities eventually was to obtain  some kind of human, was to use a human cell which would grow well in tissue culture.  And fortunately, Strander and his colleagues in Sweden had examined many kinds of B lymphocytes  for their capacity to produce interferon when stimulated with viruses.  And one particular kind of B lymphocyte is known as Nimalva.  That's a pet name for this cell.  It was originally isolated from burgut lymphoma, a virus produced lymphoma.  The next slide I think shows a picture of this cell.  It's not terribly pretty.  It grows very well in solution.  You grow it in a rich salt solution with vitamins and amino acids and so on.  And unfortunately, you get the best growth when you add 10% fetal cast serum which is  now becoming extremely expensive.  And we have to develop some new techniques for growing on a large scale that will be  a lot less expensive.  The next slide shows some properties of this lymphoblastoid interferon that we are working  with.  This shows you that it's very stable, both to temperature and to acidity.  For example, at almost boiling temperature at 97 degrees centigrade, one can allow the  material to sit for 10 minutes and still have essentially the same activity so that it's  extremely stable to heat and low pH.  Next slide.  It's however quite heterogeneous in the sense of its electrical properties.  It turns out that interferon is a glycoprotein.  It's a protein molecule, globular protein molecule, on which carbohydrate is also attached.  And these carbohydrate side chains contain acidic residues, sialic acid residues, which  are present in different numbers on different molecules, sometimes there are three, sometimes  four, sometimes two.  So that if you do an isoelectric focusing experiment, you get quite a large heterogeneity  in the isoelectric points.  These peaks that you see are different forms of interferon differing by their charge due  to the difference in sugar.  However, if you remove the sugar, thereby removing the negative charge from all of these side  chains of carbohydrate, the material becomes much more homogeneous.  This shows first of all that the heterogeneity is due to the carbohydrate side chains.  And secondly of all, and secondly, that the carbohydrate can be removed without losing  activity because it's still active in tests.  The next slide indicates the results of treating partially purified lipoblastoid interferon  with a mixture of enzymes which choose off carbohydrate, which we obtained from a bacterium  dipococcus pneumoniae.  The molecular weight of the interferon molecule shifts to a smaller weight when you take off  carbohydrate.  And if as shown in the next slide, you plot the results on a standard type of figure showing  the relationship between weight and behavior on a column against some standard proteins  of known molecular weight, you can see that the interferon after treatment with enzymes  has shifted to a lower molecular weight.  As a matter of fact, one can chop off about 4,000 Dalton's of molecular weight, and the  weight goes from about 22,000 to about 18,500 having taken off most of the carbohydrate.  It's still active and upon injection into, let's say, a rat, you can show that it is  maintained in the circulation at least as long as the normal untreated interferon so  that it's not destroyed quickly.  Consequently, we feel that it is realistic to think of synthesizing the protein part  of the interferon and not having to worry about the carbohydrate part because that would  be essentially impossible.  There's no organic chemistry at the moment that permits the systematic synthesis of  carbohydrate side chains on proteins, but apparently we don't need that, so that's a  very lucky thing.  The problem now is to make large amounts.  The next slide is the kinetics of growth, kinetics of production of interferon by these  lymphoblastoid cells.  What we do is to grow the cells up in large tanks until they reach about 2 million per  milliliter and they're infected with the new castle disease virus which induces the synthesis  of interferon by these cells and they secrete the interferon out into the medium that they're  growing in.  There's a slight lag at the beginning, at the end of about 20 hours you have reached  the maximum production of interferon.  Then the cells are removed by taking the whole contents of the tank through a large cream  separator, actually a cream separator like you use on a farm turns out to be the most  convenient way of removing the cells.  The fluid comes out and we precipitate all of the protein including the interferon and  that's the starting crude material.  The next slide shows that one can actually save a little money and it is important in  these experiments to do that.  Fetal calf serum at the moment costs I think on the order of $200 per liter.  It's extremely expensive.  We use 10% fetal calf serum in growing these cells.  In 1000 liters we have 100 liters at $200 per liter.  It's a little bit rich.  However, it turns out that the lymphoblastoid cell produces interferon much more efficiently  at a lower cell concentration than it does at a higher cell concentration.  You see if you infect with Newcastle disease virus at 2 times 10 of the 6 cells per mil,  you get in this case 1800 units per mil.  If you dilute down to 0.4 times 10 of the 6, we get 7,000.  Simply by adding salt solution, you increase the ability of the efficiency of production  of interferon.  What we do routinely is grow up, instead of 1000, we grow up 250 liters and then dilute  to 1000 with salt solution.  Next slide shows here the same thing.  At the top is the undiluted cells and their efficiency in producing interferon.  At the bottom is the efficiency of the cells.  They're diluting with salt solution containing glutamine, which is an essential component  during the production.  Basically we get 4 times as much interferon as we would otherwise, simply by diluting  the cells.  The next slide.  This is a typical kind of slide that you can't read.  Simply summarizing all of the steps that we go through.  We start, as I mentioned before, we take off the cells in the cream separator.  Then we precipitate all the proteins in that clear fluid with trichloroacetic acid.  Then we go through a number of steps.  First to remove the trichloroacetic acid, then this antibody column that I spoke of,  which catches the interferon.  A number of other steps including sizing on columns, ion exchange separations, and finally  a polyacrylamide gel separation in SDS, sodium nodesisulfate slab gel purification.  By this time we're down to very small amounts.  In this particular slide the total recovery was only 6%, which is very bad.  We now have this up to about 15%, and I hope we'll soon have perhaps 50% with some new  tricks that we are beginning to use.  But it's still a very slow and tedious process.  As you can see, at this point we have on the order of 2-tenths of a milligram of protein  from 200 liters of cells.  The specific activity of the pure material, this is the 18,500 k, 18,500 molecular weight,  is about 2.2 times 10 to the eighth units, 2 to 4 times 10 to the eighth units per milligram.  This is the purity of such material.  There is one other component, the 21.5, which is the same as the 18.5, very likely, with  carbohydrates still attached, which makes it heavier by that many units.  The next slide, this is a polyacrylamide analytical slab gel with the interferon activity plotted  as black points at the bottom, and at the top the pattern of the gel stained with comassie  blue to indicate the position of the components.  You see the dark band corresponding to the main interferon peak, and another smaller band  with activity to the left.  If you then cut out this main band and rerun it on another slab, the next slide, you see  on the right a pure sample of human lymphoblastoid interferon against some known proteins as  markers on the left to give some idea of the molecular weight.  The first pure material was produced by Ernest Knight at DuPont about two years ago from  fibroblast interferon.  This is our lymphoblastoid interferon.  Both varieties have about the same molecular weight and the same specific activity.  That is to say, they both, one milligram is approximately two or three times 10 to the  eighth units.  The next slide.  Once again, protein chemists always show amino acid analysis.  Not terribly useful, but this is simply the amino acid analysis done with the micro method  for amino acid analysis on some pure interferon.  It's perhaps only interesting to point out that there is quite a high amount of hydrophobic  amino acids like leucine, phenylalanine, valine, and a few others.  The next slide is more of the same, but here what we've done is to take the amino acid  analysis from fibroblast lymphoblastoid, leukocyte, and mouse, mouse interferon.  Amino acid analyses have been done on pure specimens of all of these.  This is simply to show that as you go from a mouse to a leukocyte to a fibroblast to  a lymphoblastoid cell, the amino acid composition is about the same.  Each type has about the same amount of lysine or histidine or arginine and so on.  That last fall in November, we felt we had done enough on purification to accumulate  material that could be put into the amino acid sequinator, the machine that has been  designed over the last years by Edmund and his followers that will tell you something  about the structure of a protein, one by one.  Next slide shows results which were produced actually by Hood and Hunkapiller, two scientists  at Caltech in California who have one of these very sensitive machines for determining sequence.  We sent to him, together with Pete Knight at DuPont with his fibroblast interferon,  and Leng Yell and his group at Yale who had mouse interferon samples that these people  could degrade.  It turned out that the mouse came in two varieties, or three varieties, A, B, and C.  We show here some results on A and C. Simply to indicate, particularly here at the bottom,  that the mouse aeroleicosides interferon called band C is rather similar to the material that  we obtained from lymphoblastoid cells.  There's quite a high degree of homology in structure.  This is only the first 20 amino acids, but it is a beginning, and from the amino terminus  it's the first 20 residues, and there's quite a high degree of similarity.  It does at least tell us that the gene for interferon in the human and in the mouse,  those two genes must be quite similar, at least for the first 20 amino acids.  It means that this is probably quite an old gene.  It can be found in animals as far as fish and turtles and frogs, so that the interferon  gene has been around for quite a while.  We were very proud of ourselves at this point, because after six or seven years we had finally  achieved some beginnings of sequence.  We really had a protein that was pure and it was characterizable.  So we were working along, making more material to have the rest of the sequence finished,  when suddenly the genetic revolution occurred in Zurich and elsewhere.  One of the nice things about genetic engineering and producing material in E. coli cells is  that you can obtain the DNA from the chromosome of the E. coli cell, and it's much easier  to determine the structure of a DNA fragment than it is to determine the structure of a  protein, so that when Charles Weisman, for example, obtained a clone of E. coli that  produced interferon, he was able to very quickly take this material and buy some techniques,  details which I won't go into, because first of all, I'm not terribly familiar with the  field and it's quite complicated to explain.  He was able to work out the total sequence of the gene that determined the interferon  that was made by those cells for RNA message, RNA messenger RNA isolated from white cells.  He used leukocyte message to prepare the original plasmid, which he put into the E. coli cell  for translation.  The next slide shows a very confusing slide, it's not as confusing as it looks actually.  On this slide, what I've done is to put down Charles Weisman and his colleagues' sequence  for one of the leukocyte interferons as a protein, that is to say, that's the center  of the lines, the one that begins with the CYS and the box at the upper left.  He goes along the center one all the way through is Weisman's leukocyte interferon, knowing  the DNA strand sequence and knowing the genetic code, you can simply read off the amino acid  that corresponds to the triplets of nucleotide bases in that DNA sequence and translate the  DNA structure into a protein structure.  The top line represents a similar translation of the DNA structure that was determined by  Teneguchi and his colleagues' Japanese group from fibroblast DNA.  This is another interferon gene.  In their case, they selected the gene from fibroblast.  So that's on the top line and on the bottom line is the sequence that we now have from  our own material on lymphoblastoid.  As you see, there are some empty spaces that are not finished, but we hope in the next  months to complete this.  The important thing is that when you go from fibroblast to leukocyte to a specific white  cell like lymphoblastoid, there is a large amount of similarity.  I have enclosed those areas where the same sequence occurs in all, in the three species.  And there's obviously a great deal of homology down from one cell type to another.  I've also cross-hatched the areas which include the cysteine residues, the amino acids that  are responsible for making cross-links through SS bridges in the protein when it folds up.  And there's one that I forgot to cross-hatch down at 140.  There's a CYSALATRP cysteine alanine tryptophan, which occurs both in the fibroblast sequence  and the leukocyte sequence.  We unfortunately do not have it yet in our structure, although we know that we have one  tryptophan in the molecule and we hope to have that section soon.  The fact that that section and the section at the upper right has been so carefully maintained  in these three varieties suggests that these may be particularly important cysteines that  perhaps form an SS bridge.  But this kind of detailed chemical consideration will have to really await the isolation of  larger amounts.  So what to do if you have this?  For Taniguchi and Weisman, the future is very clear.  They have to produce E. coli systems, E. coli cells themselves, plasmids to insert into  these that are better and better and better and can produce more and more interferon.  And then they have to purify the interferon away from the proteins of the bacterium sufficiently  that it becomes pure enough to be able to give to human beings.  You can't give human patients impure proteins because one develops antibodies against them  and you have to avoid such phenomena as anaphylaxis and so on.  So although it sounds phenomenal in time magazine, and I think these people have done a marvelous  job and will probably eventually produce the interferon that we need, they still have a  way to go in terms of purification and preparing better vectors for their work.  In our own case, since we're not quite finished with our own sequence and since we're interested  in synthesis, we have started to synthesize slowly the sequence from Weisman's leukocyte  interferon structure, the middle line here.  And we're doing this both by the new solid phase synthetic techniques developed by Merrifield  and his colleagues and also by classical peptide synthesis fragment condensation, which is  an enormous job and may take too many years to think about unless we can think of some  tricks or perhaps some combination of both techniques.  The other thing we would like to do is to grow enough material or to get enough material  from Weisman or Taniguchi to do some careful structure function work.  That is to say to try to determine whether one can chew away parts of the molecule and  still keep the activity because if one could cut away half the molecule and make it 80  amino acids instead of 160, then it would become synthetically not so difficult.  So that's our own hope at the moment.  And perhaps we can even use the E. coli interferon for our structure function work.  I think it would be interesting, I'm sure, to someone like Dorothy Hartzke and Bill  Lipscomb sitting here to think that if the crystallography three-dimensional structure  of interferon has ever worked out, it will probably be on synthetic material because  we'll probably never have enough of the other kind to do it, but perhaps we will.  It would be a nice first to try.  One final point that might give some interest to some of your...  We've known for some time that interferon and cholerotoxin compete for the same receptors  on the surface of cells.  We can add interferon and compete for the recognition by adding cholerotoxin simultaneously  and vice versa.  So I asked the people who put out this dictionary of protein sequences, Margaret Dayhoff and  her colleagues who have a computer full of all the known sequences, to compare the leukocyte  sequence of Weismann with all other known protein sequences of which there must be hundreds  of them.  And she called back and said in a very unbelieving voice that there seemed to be very little  similarities that there was one rather interesting one that there was...  Next slide, please.  Rather interesting similarity between the interferon structure and the sequence of cholerotoxin,  which I thought was very nice, because I didn't tell her in advance.  It was out of the computer without any hinting.  It turns out that at the very bottom here, there's a number called an alignment score,  a score of 4.1 approximately, which is considered rather good.  The alignment score for, say, myoglobin and beta chain of hemoglobin in man is something  like nine.  So this is really a rather high level of homology in sequence.  And you can see that in this section here, there's quite a large number of identical  amino acids.  Could I go back to the last slide, please?  The previous slide.  So that as a beginning, we have started at the bottom right, and we are working backwards,  and it turns out that this section that resembles cholerotoxin runs from about 150 back to about  120.  In that region are these similarities.  It'll be very interesting to see whether this peptide, which we now have essentially finished,  will be competitive with cholerotoxin, and perhaps may serve, for example, as an antigenic  site that we can use for further purification of our anti-interferon antibodies.  And it might also give us some approach to the process by which, understanding of the  process by which interferon recognizes the cell surface.  I mentioned in an abstract that I sent here before we came that I would say something  about the mechanism of action.  I'm afraid that's very difficult to do.  All one can say at the moment is that if you give interferon to cells, it is recognized  by the cell and then induces the cell to make two or three enzymes, which either are absent  or present in very small amounts before the interferon appears.  The enzymes that are produced are all involved in the control of message translation, that  is to say in protein synthesis.  The translation of RNA message into protein.  And interferon appears, therefore, at the moment from the work of a number of laboratories,  to be involved in the inhibition of the translation of the viral message that comes into the cell.  That's about all we know about the mechanism of action.  Most of the rest is phenomenological, and I hope that we'll be able to say something  specific about not only how it works, but against which diseases it is helpful if at  all.  And I feel quite confident myself that it will be very helpful against certainly some viral  diseases.  Cancer is still a knock on the wood question.  Thank you.  Thank you.
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