Interferon: The Dawn of Recombinant Protein Drugs PDF
Preview Interferon: The Dawn of Recombinant Protein Drugs
Ernst Schering Research Foundation Workshop Supplement 5 Interferon: The Dawn of Recombinant Protein Drugs Springer-Verlag Berlin Heidelberg GmbH Ernst Schering Research Foundation Workshop Supplement 5 Interferon: The Dawn of Recombinant Protein Drugs J. Lindenmann, W.-D. Schleuning Editors With 7 Figures and 1 Table Springer Series Editors: G. Stock and M. Lessl ISSN 0947-6075 ISBN 978-3-662-03789-8 CIP data applied for Die Deutsche Bibliothek - CIP-Einheitsaufnahme Schering-Forschungsgesellschaft <Berlin>: [Ernst Schering Research Foundation Work shop / Supplement] Ernst Schering Research Foundation Workshop. Supplement. Reihe Supplemnt zu: Schering-Forschungsgesellschaft <Berlin>: Ernst Schering Re search Foundation Workshop 5. Interferon .. -1999 Interferon: the dawn of recombinant protein drugs / l.Lindemann ; W.-D. Schleuning ed. (Ernst Schering Research Foundation Workshop: Supplement; 5) ISBN 978-3-662-03789-8 ISBN 978-3-662-03787-4 (eBook) DOI 10.1007/978-3-662-03787-4 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illus trations, recitation, broadcasting, reproduction on microfilms or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag Berlin Heidelberg GmbH. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1999 Originally published by Springer-Verlag Berlin Heidelberg New York in 1999 Softcover reprint of the hardcover I st edition 1999 The use of general descriptive names, registered names, trademarks, etc. in this publica tion does not imply, even in the absence of a specific statement, that such names are ex empt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Typesetting: Data conversion by Springer-Verlag SPIN: 10730445 13/3134-543210-Printed on acid-free paper Preface Forty years of Interferon I wish to dedicate this short introduction to the memory of Alick Isaacs (1921-1967), and to that of Sir Christopher Andrewes (1896-1988). Let us go back more than 40 years. In 1956 Isaacs was in charge of the Wodd Influenza Centre. Andrewes was head of the division of bac teriology and virology, and deputy director of the National Institute for Medical Research in London. When researchers are faced with a seemingly new phenomenon, ex planations are easy to come by. These explanations fall into two broad categories: the phenomenon in question is either due to something or to the lack of something. I apologize for the primitive way in which I ex press this, but I am going to give three examples, scattered over 100 years, of what I mean. First example: in 1880 the great French microbiologist Louis Pas teur was involved in work on chicken cholera. He was struck by the following observation: if a suitable chicken broth was inoculated with the bacterium, the organism grew profusely and the liquid became tur bid. If he now freed the fluid, by sedimentation or filtration, from the bulk of the organisms and re-inoculated it with the same bacterium, no growth occurred. He immediately offered two possible explanations: either the bacterium, during its first round of growth, had used up some essential constituent of the broth or it had produced a substance which, VI Preface The participants of the workshop upon reaching a certain concentration threshold, inhibited further growth. This second alternative would have been analogous to alco holic fermentation, with which Pasteur was familiar. In the case of chicken cholera Pasteur attempted to but failed to reveal the existence of such a hypothetical inhibitor. He therefore settled for the first expla nation, the so-called exhaustion hypothesis, which he extended to the phenomenon of immunity in vivo. In the following years a lively de bate developed over the issue of whether immunity was caused by the lack of something or by the addition of something. Second example: 100 years later, in a paper in Lancet by Murray, Murray, Murray and Murray (1980) on the rarity of planar warts in Cushite nomads, the authors wrote: "There may be a factor ... which inhibits virus replication. Alternatively, there may be a deficiency ... of a nutrient essential for the replication of the virus." Third example, and now I am getting closer to my subject: almost halfway between the first two examples, in 1942, Christopher An drewes published an important paper on viral interference in tissue cul- Preface VII ture. In this he stated: "The virus first upon the scene uses up some es sential foodstuff in the cell. An alternative ... hypothesis ... would be, of course, the generation of some inhibitory substance." Notice the words "of course". They show Andrewes' modesty and generosity. To him it was clear that anybody who gave only 5 min thought to the problem would hit upon these two explanations. Like Pasteur 60 years earlier he attempted to show the existence of such an inhibitor, but without success. There the matter rested when Alick and I started experiments on vi ral interference, using an extraordinarily simple system which allowed repeated manipUlations of fragments of chick chorioallantoic mem branes suspended in a salt and glucose solution. We had one advantage over what Andrewes had done earlier: whereas he had used interfer ence between two strains of live virus, it had become clear in the meantime that interference also occurred between inactivated virus and live virus. The use of inactivated virus as the interfering agent made it from the start less likely that the explanation which Andrewes had fa vored, the exhaustion of some essential foodstuff, also applied in our case, although it was by no means excluded, since the inactivated vi rus, after all, did something. In fact, we entertained for a time the idea that some sort of abortive replication of the interfering virus occurred. Alick once told me that when trying to explain to one of his chil dren what he did, he had said: "Well, you see, we ask questions and then we try to answer them." Whereupon his son said: "And they pay you for that?" What were the questions that we asked? In fact, they had little to do with interference per se. Before coming to Mill Hill I had done a few experiments on viral interference in which I had used inactivated influ enza virus firmly attached to red cells as the interfering agent. This technique appealed to Alick because he thought that it might be used to answer the following question: does the virus, as was known at that time from bacteriophages, inject its nucleic acid into the host cell, the viral envelope remaining outside? Or does the whole virus enter the cell? He proposed to use red cell ghosts (I had used intact red cells) coated with virus particles and to look at them with the electron micro scope before and after they had induced interference. To do this he en listed the help of an excellent electron microscopist, Robin Valentine. VIII Preface This approach proved impracticable. The virus particles were clearly visible at the beginning, but after the coated ghosts had induced interference, the picture became blurred by cellular debris and could not be interpreted. I believe this is a clear example of how experimental science is be ing done. Philosophers of science seem to believe that scientists first ask questions and then look around for experimental techniques that might answer them. I suspect it is the other way round: Scientists hit upon some nice technique and then they shop around for questions that could be answered. To put it aphoristically: Scientists don't do experi ments in order to answer questions, they ask questions that allow them to do experiments. In the course of this work we made an unexpected observation: when we tried to exhaust the interfering power of virus-coated ghosts by repeated rounds of interference, it looked as if new interfering activ ity was being generated. I called this hypothetical "something", jok ingly at first, interferon, and the first experiment that addressed this new question is labeled in Alick's lab notebook, now at the National Library of Medicine in Bethesda, "In search of an interferon". In search of an interferon does not mean that we were expecting several interferons, but rather, that we were looking for anything that would fit the notion that something was there and, of course, as An drewes might have said, we had to exclude the possibility that the phe nomenon was caused by the absence of something Figure I shows Alick in happy days with his twins. Somebody quipped: Alick always does his experiments in duplicate. This may have been a good joke, but it was not true. In fact, Alick told me he had learned from Sir Macfarlane Burnet that one should never repeat an experiment in exactly the same manner. Results of experiments should always be taken seriously. If you repeat an experiment pains takingly and then get the same result, you are no wiser than you were before. And should you get a different result, then both experiments cancel each other. So we did a series of experiments, the last one al ways overlapping the precedent one to some extent, but always with something new added. For instance, we titrated the activity, we at tempted to pass it through filters, to centrifuge it, to neutralize it with antiviral antibodies etc. At the beginning of 1957 we were convinced Preface IX Fig. I. Alick Isaacs with his twins that something was there, not the lack of something. People who were skeptical, as of course one should be, called it a misinterpreton. I left Mill Hill in 1957, my fellowship having expired. I was anx ious to find a research object that would be independent of interferon. With many others I shared the optimism that within a short time the purification of interferon, a biochemical business in which I did not want to engage, would be achieved, and that then biological questions could be asked more meaningfully. In the meantime I wanted to do ex periments. An opportunity presented itself when by chance I observed an inbred mouse strain that was highly resistant to orthomyxoviruses. I wrote about this to Alick, and in his reply he thought it unlikely that this had anything to do with interferon. This was perfectly reasonable advice, because interferon was thought to inhibit many different viruses, whereas the resistance phe nomenon I had observed was limited to a few related viruses. Many experiments suggested themselves that were easy to perform, and again the question had to be asked: is it something or the lack of something? During the following 18 years several collaborators and myself looked at the presence of inhibitors, at the absence of receptors, at powerful immune responses or the lack of immunopathology. All these experi- x Preface ments were fun to do, but gave negative results. The resistance phe nomenon remained as unexplained as ever. The resistance proved to be caused by a single dominant gene, which we called Mx. In the course of the years we had developed, again one of those experiments that suggested themselves, so-called congenic lines of mice. By systematic back-crosses over some 20 gen erations we had obtained two lines of inbred mice which were geneti cally identical except for a small segment of DNA containing the Mx locus. We argued that this locus yielded a gene product, a protein, which should be present in the resistant line but absent in the suscepti ble line. Because the two lines were congenic, this was expected to be the only substantial difference, and hence it should be possible, by im munizing susceptible mice with tissue extracts from resistant mice, to get an antiserum capable of recognizing the Mx gene product. We did this carefully, and tested the putative antiserum by the meth ods then available, by precipitation, gel diffusion, immunofluores cence, complement fixation, neutralisation, later by Western blotting, all without success. And then there was a sudden burst of light. My collaborator Otto Haller obtained from Ion Gresser a small sample of an extraordinarily potent anti-interferon serum, and a new experiment became possible: when resistant animals were treated with this antis erum, they became fully susceptible. Now it dawned upon us: the Mx gene product is normally switched off, and interferon switches it on. It was interferon after all, but acting indirectly through the Mx gene. We could now repeat the immuniza tion experiment more intelligently, by injecting, into susceptible mice, not simply organ extracts of resistant animals, but of interferon-treated resistant animals. Using this approach, highly specific antibodies were readily obtained and proved useful for the cloning of Mx. So resistance is caused by something, the presence of an interferon inducible gene. But we could have asked the other way round: what is the cause of susceptibility? And here the answer would have been: it is the lack of something, the lack of a functional Mx gene. Of course, as Sir Christopher would have said. 1. Lindenmann
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