The Story of César Milstein and Monoclonal Antibodies: Introduction
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`A HEALTHCARE REVOLUTION IN THE MAKING
`The Story of César Milstein and Monoclonal Antibodies
`Collated and written by Dr Lara Marks
`Today six out of ten of the best selling drugs in the world are monoclonal antibody therapeutics. One of these, Humira®, which is a
`treatment for rheumatoid arthritis and other autoimmune conditions, was listed as the top selling drug across the globe in 2012 with a
`revenue of US$9.3 billion. Based on its current performance many predict the annual sales of the drug will surpass the peak sales of Lipitor,
`a treatment for lowering cholesterol, that is the best selling therapeutic of all time. Currently monoclonal antibody drugs make up a third
`of all new medicines introduced worldwide.
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`The Story of César Milstein and Monoclonal Antibodies: Introduction
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`Portrait of César Milstein.
`Photo credit: Godfrey Argent Studio
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`Monoclonal antibodies are not only successful drugs, but are powerful tools for a wide range of medical applications. On the research front
`they are essential probes for determining the pathological pathway and cause of diseases like cancer and autoimmune and neurological
`disorders. They are also used for typing blood and tissue, a process that is vital to blood transfusion and organ transplants. In addition,
`monoclonal antibodies are critical components in diagnostics, having increased the speed and accuracy of tests. Today the antibodies are
`used for the detection of multiple conditions, ranging from pregnancy and heart attacks, to pandemic flu, AIDS and diseases like anthrax
`and smallpox released by biological weapons. Beyond human healthcare, monoclonal antibodies help detect viruses in animal livestock or
`plants, prevent food poisoning and investigations into environmental pollution.
`
`Monoclonal antibodies are indispensable in so many walks of daily life thanks to their ability to target a single type of cell. Produced in the
`laboratory, these antibodies are derived from naturally occurring proteins made by the body's immune system to recognise and fight foreign
`invaders, such as bacteria and viruses. The antibodies are generated through the fusion of a myeloma cancer cell with spleen cells taken
`from an immunised animal.
`
`Yet the story of how these unsung microscopic heroes came into the world and helped change healthcare remains largely untold. Moreover,
`their significance was largely overlooked at the time of their creation. The journey of monoclonal antibodies all started when an
`Argentinian émigré called César Milstein arrived at the Laboratory of Molecular Biology in Cambridge, the same laboratory where Francis
`Crick and James Watson discovered the structure of DNA in 1953. It was to be here that Milstein, together with Georges Köhler, pioneered
`the seminal technique for the production of monoclonal antibodies in 1975 and showed their clinical application for the first time.
`
`This exhibition of the life and work of César Milstein provides a window into the world where monoclonal antibodies were first developed.
`Showing Milstein's notebooks and writings for the first time, this exhibition provides first-hand the complexities that were involved in the
`creation of monoclonal antibodies and brings to life the many challenges scientists face in devising a viable biotechnological tool and its
`application in healthcare. Transforming monoclonal antibodies, which started life as a laboratory tool into something that could be of use in
`the outside world was neither straightforward nor inevitable.
`
`From Milstein's papers we learn first-hand how the newly-created monoclonal antibodies spread from the confines of Milstein's laboratory in
`Cambridge to scientists across the world and were then adapted for clinical applications. They highlight the logistical difficulties Milstein
`and his team faced in transporting monoclonal antibodies to other laboratories, and the fact that other scientists initially had little idea
`about how to grow and maintain the antibodies, let alone any idea what purpose they might serve.
`
`Strikingly, initially Milstein had very few requests for monoclonal antibodies. By 1977, however, he was being inundated with requests for
`samples and had to search for outside support in the distribution process. This was to pave the way to the earliest commercialisation of the
`technology with the help of Sera-Lab, a small British company set up to supply animal serum reagents to the scientific community. The
`relationship between Milstein and Sera-Lab illustrates the process of technology transfer in biotechnology during its formative years. All of
`this was done with little public fanfare and no venture capital or government support. Yet, the collaboration between Milstein and Sera-Lab
`laid the foundation for the wide-scale commercialisation of monoclonal antibodies.
`
`The exhibition also offers a way of understanding why the original technology developed by Milstein and Köhler was not patented in Britain
`and instead formed the basis of patents taken out by the Polish-American virologist, Hilary Koprowski, and his team based at the Wistar
`Institute in Philadelphia. The latter were thus the first scientists to be granted patents for monoclonal antibodies. Generating major
`controversy in the late 1970s, the patent story told in this exhibition reveals some of messy business of patenting research science and the
`implications this holds for those working in both the laboratory and the commercial world.
`
`It also provides some insight into Milstein's very early efforts to demonstrate the practical application of monoclonal antibodies. He and his
`colleagues paved the way for the use of monoclonal antibodies as tools for the purification of natural compounds for drugs and as reagents
`for blood typing. Their work also demonstrated the use of monoclonal antibodies as probes to investigate the pathological pathway of
`neurological conditions and a wide range of other diseases. This paved the way to the adoption of monoclonal antibodies as diagnostic tools
`and an invaluable platform in the move towards personalised medicine. The final part of the exhibition shows how Milstein encouraged the
`use of genetic engineering to improve the safety and efficacy of monoclonal antibodies thereby enabling their use as therapeutics on a large
`scale.
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`Milstein's early life and work >>
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`The Story of César Milstein and Monoclonal Antibodies: Introduction
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`Page 3 of 3
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`Sponsored by the Medical Research Council as part of its Centenary Programme.
`
`Supported by the Department of Social Science,
`Health & Medicine, King's College, London
`
`About What is Biotechnology | Advisory Board | Contact us | Terms and Conditions
`
`Website design by Silico Research the creative minds behind BioPartnering.
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`The Story of César Milstein and Monoclonal Antibodies:Milstein's early life
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`Milstein's early life
`
`The journey from Argentina
`
`The son of Jewish im m igrants, César Milstein grew up in Bahía Blanca, a port town located by the Atlantic ocean som e 500 m iles south of
`Buenos Aires. Jews had begun to settle in Bahía Blanca from around 1900, m any of them com ing from central and eastern Europe.
`
`Family background
`
`Milstein's father, Lazaro, was born in a village in the Ukraine and m igrated
`to Argentina in 1913 at the age of 14 with his aged aunt and uncle. For m any
`years he lived in Jewish settlem ents near Bahía Blanca trying his hand at
`different trades, including farm labour, carpentry and railway work. During
`this tim e he taught him self Spanish and was an enthusiastic reader. He was
`also active in social and cultural activities, helping to preserve Yiddish
`literature and working for non-religious Jewish organisations, som e with
`anarcho-syndicalist connections.
`
`Lazaro m et Maxim a, his wife-to be, in Bahía Blanca. Maxim a was born in
`Argentina. She was the daughter of poor Ukrainian im m igrants who m ade
`great sacrifices to ensure she had a secondary school education and went to
`college. At the tim e Lazaro m et Maxim a she was a school teacher. Soon
`after their m eeting, Maxim a rose to becom e a head m istress. From 1926 to
`1933, Maxim a directed School No.3, the first co-educational school
`established in Bahía Blanca. Milstein, the m iddle of three brothers, was born
`at the fam ily hom e on this school's prem ises. He also attended the school in
`his early childhood. Both of Milstein's parents spoke Yiddish at hom e, but
`Milstein was raised speaking only Spanish.
`
`During his early childhood Milstein preferred playing with other children in
`the streets to reading books. W ith his m other's encouragem ent, however, he
`soon began to find pleasure in books, particularly adventure stories such as
`Rudyard Kipling's Jungle Book. Milstein developed a desire to pursue science
`at the early age of 8. This was prom pted by a discussion he had with one of
`his cousins who had just com pleted her degree in Chem istry and was then
`working as a biochem ist at the Instituto Malbran. Milstein was particularly
`fascinated by his cousin's description of her attem pts to extract snake
`venom to treat snake bite victim s. Milstein's interest in science deepened when on his ninth birthday he was given a Spanish translation of
`Paul de Kruif's Microbe H unters by his m other. This book awakened his desire to have the sam e type of adventurous life like that of the
`scientists Antoni van Leeuwenhoek and Louis Pasteur described in the book.
`
`This photograph was taken when Milstein was a young
`m an.
`
`Photograph credit: Celia Milstein.
`
`Milstein grew up in a fam ily which prized knowledge and education. Until his last year of school, Milstein attended schools close to hom e in
`Bahía Blanca, including the Colegio Nacional. In his final year, however, he m oved to a secondary school in Buenos Aires to prepare for the
`entrance exam of the University of Buenos Aires.
`
`Milstein's parents always supported his research, his m other helping to type up his first PhD thesis and his father offering him econom ic
`assistance so that he could dedicate him self to his doctoral research. Fiercely independent, Milstein declined his father's financial support.
`
`Education
`
`In 1945 Milstein started to study chem istry at the University of Buenos
`Aires. His undergraduate studies, however, were interrupted when,
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`The Story of César Milstein and Monoclonal Antibodies:Milstein's early life
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`Bahía Blanca
`Ba hía Bl a nc a ,Buenos A ir es P r ovinc e,
`A r g entina S a ve
`V iew l a r g er
`m
`a p
`
`Sign in
`
`during a faculty picnic, he suffered severe injuries to his pancreas when
`he hit a log while diving into a shallow pool and had to take off som e
`tim e off to recover. He finally received his BSc in chem istry in 1952.
`
`During his undergraduate years Milstein was active in cam paigns against
`the Peronist governm ent's policies aim ed at privatising education and
`their m ore general im positions on universities and student life and rose
`to be President of the Student Union. At the tim e the governm ent was
`clam ping down on any political activity, and the atm osphere was
`particularly tense. In 1951, for exam ple, a chem istry student, Ernesto
`Mario Bravo, was arrested and tortured for 20 days as a result of
`protesting against the governm ent. His arrest sparked a m ajor student
`strike. More than 150 strikers were arrested and university
`adm inistrators expelled the m ore prom inent leaders of the student
`m ovem ent. In the end, however, Bravo was released. The student
`m ovem ent considered this a m ajor achievem ent.
`
`Three years later student unrest erupted once again when the Peronist
`regim e im posed even greater control over the m edia, education system ,
`trade unions and the legislative and the judiciary. In October 1954
`students joined workers striking against the then deep econom ic and
`
`M a p da ta © 2015 G oog l e
`Google Map showing the location of Bahia Blanca in Argentina.
`The town is a m ajor trans-shipping and com m ercial centre,
`known for its large export trade of grains, wool, oil and fruit.
`Click to view a larger m ap.
`
`social crisis and increasing unem ploym ent.
`
`Shortly after Milstein returned to the University of Buenos Aires from his several m onths of convalescence, Milstein m et Celia Prilleltensky,
`a fellow chem istry undergraduate. Their first encounter was at the laboratory bench, where they found them selves working alongside each
`other. Celia not only shared Milstein's scientific interests, but was sim ilarly an ardent student cam paigner for free education. A year after
`their graduation in 1952, Milstein and Celia m arried.
`
`At the sam e tim e as getting m arried, Milstein began to look for a suitable doctoral supervisor. Initially he sought to work with Professor Luis
`Leloir, a distinguished Argentinian enzym ologist. To this end he visited Leloir's workplace, an old house in Buenos Aires. On arrival he m et
`what seem ed to him an unassum ing m an carrying a basket. This turned out to be Leloir. Having no space to take Milstein on, Leloir instead
`referred him to the Argentinian biochem ist Professor Andrés Stoppani.
`
`Milstein recalled that Stoppani was 'one of the few and perhaps
`the only full-tim e Professor of the Faculty of Medicine in the
`University of Buenos Aires, perhaps the m ost im portant universities
`in Latin Am erica, a full tim e professor who probably had a salary
`of about the sam e order of m agnitude as a janitor, trying to do
`serious and honest research in a laboratory with no funds at all'.
`
`Stoppani advised Milstein to take som e tim e off before he started
`his doctorate in view of the tense political clim ate which was
`hostile to students such as Milstein who had actively cam paigned
`against the Peronist governm ent's policies in education. His advice
`encouraged Milstein to take a year long honeym oon with Celia
`exploring Europe.
`
`By 1954 the political environm ent had begun to im prove and
`Milstein began researching enzym es for a doctorate in
`biochem istry although he had no funding as there was none for
`students in this period. He was forced to support his studies by
`working half-days in the Laboratorios Liebeschutz, a clinical
`biochem istry laboratory. In later years Milstein argued that this
`part-tim e job had taught him the value of organising his tim e.
`
`During his doctoral research Milstein had access to only the m ost
`basic equipm ent. Som e idea of how poor the facilities were at the
`tim e can be seen from his recollections that Stoppani had 'to pay,
`from his own m eagre salary, for the pound of yeast … needed from
`tim e to tim e in order to prepare … [the enzym e] aldehyde
`dehydrogenase.'He recalled, 'W e survived on what was inherited
`from the golden days … from the Medical School and with reagents
`justified by teaching requirem ents'. The m ost precious piece of equipm ent in the departm ent was a W arburg apparatus, which Stoppani did
`not allow anyone but him self to use.
`
`This photograph shows Milstein early on in his courtship with Celia
`Prilleltensky.
`
`Photograph credit: Celia Milstein.
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`Click here to see the
`additional photographs from
`Milstein's early life.
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`By 1955, the political situation in Argentina had im proved still further and with this the conditions in
`Stoppani's departm ent becam e slightly easier. This m eant, for exam ple, that the departm ent was able to
`purchase a refrigerated centrifuge. The centrifuge proved useful to Milstein in his enzym e preparations.
`Nonetheless, problem s rem ained. The nearest spectrophotom eter, an instrum ent that Milstein needed for
`m easuring enzym e activity, was located three blocks away from his departm ent and Milstein had to trek every day between departm ents
`carrying reagents and enzym e preparations. Early on in his doctoral studies he cam e close to losing his position in the departm ent when, in
`the process of m aking his enzym e preparation, he succeeded in consecutively breaking three of the departm ent's five very expensive 5-litre
`flasks.
`
`Despite these hurdles, Milstein com pleted his doctoral research and was awarded a prize in 1957 by the Associación Q uim ica Argentina for
`the best thesis in chem istry that year. His doctorate was an investigation of the enzym e dehydrogenase. He had focussed his research on
`one of the enzym e chem ical bonds, known as a disulphide bridge. Between 1957 and 1959 Milstein would publish several papers with
`Stoppani arising from his doctoral research.
`
`The relationship with Cambridge begins
`
`In 1958, funded by a British Council scholarship, Milstein joined Malcolm Dixon and Edwin W ebb at the Sir W illiam Dunn School of
`Biochem istry in Cam bridge. In part the decision was influenced by the fact that Stoppani had worked with Dixon before the Second W orld
`W ar.
`
`Initially, Milstein had difficulty understanding what Dixon and W ebb were
`saying because he lacked fluency in English, but with their advice he set out
`to study the kinetics and heavy m etal activation of the enzym e
`phosphoglucom utase. This was inspired by Dixon's suggestion that Milstein
`follow up an odd observation m ade som e years earlier in the departm ent
`that phosphoglucom utase required two m etals for full activity, m agnesium
`and a trivalent m etal like chrom ium .
`
`Milstein was left to pursue his research on his own within Cam bridge. The
`work was not without its pitfalls. Milstein lost his first large-scale enzym e
`preparation in an electric cold bath. According to Milstein this was caused
`by the distraction of attending a cham pagne party to celebrate the awarding
`of a Nobel Prize to Fred Sanger in 1958. Sanger had been awarded the Prize
`two weeks after Milstein's arrival in Cam bridge. A central figure in the
`Cam bridge Biochem istry Departm ent, Sanger's achievem ent had been to
`show that proteins have a defined chem ical com position.
`
`Despite his early disaster with his enzym e preparation, within a year
`Milstein's experim ents on phosphoglucom utase had provided sufficient data
`for him to write up his research. This led to the award of a second
`doctorate, this tim e from Cam bridge University. Based on this research he
`published three papers. Contrary to contem porary opinion, Milstein
`discovered that the activation of phosphoglucom utase was caused by the
`displacement of heavy m etals by m agnesium . Prior to Milstein's finding,
`scientists believed the enzym e was activated by the heavy m etals
`them selves.
`
`During his British Council fellowship in Cam bridge, Milstein form ed a strong
`bond with Sanger. At the tim e Sanger was a pivotal figure in the Departm ent
`of Biochem istry, as he possessed the only functional pH m eter. W hile an
`unassum ing figure, Sanger was a dom inant influence within the departm ent.
`Milstein, for exam ple, rem em bered a warning sign to the entrance of the
`departm ent's high-voltage electrophoresis room reading 'Danger – High
`Power'which was altered by som eone in the departm ent to read 'Sanger –
`High Power'.
`
`Malcolm Dixon, the biochem ist who supervised Milstein at
`the Sir W illiam Dunn School of Biochem istry, Cam bridge
`University.
`
`Milstein was quickly drawn to Sanger not only because of the equipm ent he
`possessed but also by the fact that they shared the sam e research interests.
`They soon collaborated to define the active site of phosphoglucom utase and
`published a joint paper on this research. It appeared as C. Milstein, F. Sanger, 'An am ino acid sequence in the active centre of
`phosphoglucom utase', Biochemistry Journal, 79 (1960), 456-69.
`
`Photo credit: Sir W illiam Dunn School of Biochem istry,
`Cam bridge.
`
`W hen it cam e tim e for Milstein to return to Argentina, Sanger offered to secure Medical Research Council m oney for him to extend his tim e
`in Cam bridge. Milstein, however, decided to return to Argentina to take up a position he had been offered prior to com ing to Cam bridge.
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`This photograph was taken around 1980. It shows Milstein together with Fred Sanger.
`
`Photo credit MRC, Laboratory of Molecular Biology.
`
`A new chapter in molecular biology in Argentina
`
`In 1961 Milstein departed from Cam bridge for the Instituto Malbran, where he headed up a newly-created Departm ent of Molecular Biology
`in the National Institute of Microbiology. Celia also had a post in the departm ent. The return of the Milsteins to Argentina coincided with a
`period of reform in the country following the fall of Peron, when m any academ ic scientists who had been sidelined (or expelled) during
`Peron's rule returned; they included Bernardo Houssay, a physiologist who won the Nobel Prize in 1947, and Leloir who would go on to win
`the Nobel Prize in Chem istry in 1970.
`
`As head of the Departm ent of Molecular Biology, Milstein had a wide range of responsibilities, from em ploying his carpentry skills to install a
`laboratory to acting as m entor to 25 young scientists and bringing bacterial genetics into the research orbit of the departm ent. In addition
`to his day-to-day to m anagem ent of the departm ent, he continued the research he had begun in Cam bridge around phosphoglucom utase,
`developing techniques for studying the sequence of the enzym e and m arking its active centres. He also started investigating another
`enzym e: alkaline phosphatase of bacteria. Much of this work was focused on understanding the enzym e's m echanism of action. His notable
`achievem ent at this tim e was elucidating the sequence around the active site of alkaline phosphate of bacteria with Noé Zwaig. This they
`accom plished ahead of scientists in the USA. They published their results in N. Zwaig, C. Milstein, 'On the nature of the phosphoenzym e
`interm ediate in the phosphoglycerom utase reaction', Biochimica et Biophysica Acta, 73 (1963), 676-9.
`
`In 1962 Milstein's life, like that of m any other Argentinians, was thrown into turm oil as a result of a m ilitary coup. Most disturbingly for
`Milstein, Ignacio Pirosky, the director of his institute, was dism issed, as were m any colleagues in Milstein's departm ent who had defended
`Pirosky. W ith the coup, persecution began to m ount against both political dissenters and Jews in Argentina. This had m ajor im plications for
`Milstein. Bearing a Jewish nam e, authorities im m ediately associated him with com m unist activists. W hen four of his own staff were
`expelled from his departm ent, he found him self no longer able to concentrate on his own scientific research and he decided to resign from
`his position and return to Cam bridge. He was just one am ongst the m any tens of thousands of intellectuals and scientists who left Argentina
`during the m ilitary regim e.
`
`More historical background can be found on Milstein and the rise and fall of m olecular biology in Argentina in the late 1950s and late 1960s
`in P. Kreim er and M. Lugones, 'Pioneers and Victim s: The Birth and Death of Argentina's First Molecular Biology Laboratory', Minerva, 41/1
`(2003), 47-69.
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`Milstein's career begins at the Laboratory of Molecular Biology
`
`Milstein departure from Argentina was facilitated by Fred Sanger. On hearing of Milstein's difficulties in Argentina, Sanger invited him to
`join him at the Medical Research Council (MRC) Laboratory of Molecular Biology, where he now headed the Protein Chem istry division.
`Milstein arrived at the Laboratory in 1963 on a three year contract paid for by the MRC. This was one year after the Laboratory had m oved
`into its own prem ises.
`
`For m ore on Milstein's recollections of his early days see C. Milstein, 'Messing about with isotopes and enzym es and antibodies', Lynen
`Lecture, Miam i W inter Sym posium Proceedings, in W . W helan, ed., From G ene to Protein: Translation into Biotechnology (New York and
`London, 1982).
`
`<< Introduction to the exhibition
`
`Milstein's early antibody research >>
`
`Sponsored by the Medical Research Council as part of its Centenary Programme.
`
`Supported by the Department of Social Science,
`Health & Medicine, King's College, London
`
`About W hat is Biotechnology | Advisory Board | Contact us | Term s and Conditions
`
`Website design by Silico Research the creative minds behind BioPartnering.
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`The Story of César Milstein and Monoclonal Antibodies: Milstein's early antibody research
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`Early antibody research
`
`The quest to understand antibodies diversity
`
`Soon after arriving at the Laboratory of Molecular Biology (LMB) Milstein began an investigation into the Bence-Jones protein. His prim e
`objective was to understand the m olecular structure behind the form ation and diversity of antibodies.
`
`This photograph
`shows a crystal of a
`Bence-Jones
`protein.
`
`Photo credit: Alex
`McPherson,
`University of
`California, Irvine;
`National Institutes
`of General Medical
`Sciences, ID 2399.
`
`The Bence-Jones protein is a substance found in the urine and blood of patients suffering from m ultiple m yelom a, a cancer that results in
`the softening of bones. The protein was first described by the English physician Henry Bence-Jones in 1847 and its physical characteristics
`were determ ined during the 1950s. By the early 1960s, scientists had discovered that the Bence-Jones protein possessed the sam e structure
`as a light-chain, a sub-unit of an antibody m olecule. Thereafter scientists began to use the protein as a tool for investigating the structure
`and function of norm al antibodies. One of the advantages in exploring the Bence-Jones protein was that it was easily available. Scientists
`could gain access to the protein collected from the urine and blood of patients in hospitals.
`
`Milstein's decision to investigate Bence-Jones proteins for the purposes of investigating antibodies was prom pted in part by his supervision
`of a doctoral student working on antibodies when in Argentina and in part by discussions he had with Sanger on arriving at the LMB.
`
`At the start of his research into Bence-Jones proteins, Milstein was one of m any scientists then trying to understand the body's im m une
`system by unravelling the structure and function of the billions of antibodies m ade by the body every day to fight off infections.
`
`An antibody works by specifically targeting a foreign
`intruder, known as an antigen. Such antigens can range
`from bacteria, viruses and fungi, to pollen, dust, or food
`proteins that cause allergic reactions. Not all antigens are
`foreign bodies, however; they can also be cancer cells
`m ade in the body itself.
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`The Story of César Milstein and Monoclonal Antibodies: Milstein's early antibody research
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`Once an antibody recognises a particular antigen it will
`attach itself to a specific m arker on the cell surface of the
`antigen so that the latter can be targeted for destruction.
`In m any ways the binding of an antibody to an antigen can
`be likened to the insertion of a key in a lock.
`
`By the tim e that Milstein began his research into
`antibodies, scientists were beginning to unravel the basic
`structure of an antibody. This they viewed as a Y-shaped
`form ation com posed of two protein sub-units, labelled as
`light and heavy chains (appearing as red and blue in the
`diagram ), held together by disulphide bonds (indicated by
`green lines). They believed the antibody structure was
`divided into two regions, one that was constant, form ing
`the stem of the Y, and one that was variable on the tip of
`the arm s of the Y.
`
`Nonetheless, while the basic m olecular structure of
`antibodies was beginning to be solved, what rem ained a
`puzzle was how such an apparently alm ost identical group
`of proteins could specifically target sim ultaneously any
`one of a m ultiple of antigens. This specificity as well as the diversity of antibodies was a question that had intrigued scientists ever since
`the late 19th century when antibodies were first observed.
`
`This shows the basic structure of an antibody.
`
`W hat lay behind the heterogeneity of antibody m olecules would be a recurrent subject for m ost of Milstein's laboratory investigations from
`the 1960s onwards. One of the attractions of this research for Milstein was that it could be achieved through very sim ple experim ents,
`essentially com paring prim ary DNA sequences of two different antibodies. This gave Milstein the m eans to elucidate the diversity of
`antibodies at the level of their am ino acid sequences.
`
`Dissecting the antibody structure
`
`Having decided upon his topic of research, Milstein began investigating different chem ical techniques to dissect the structure of antibodies.
`He was assisted by John Jarvis, a biochem istry technician who had joined the LMB when he did. Jarvis would work as Milstein's research
`assistant until Milstein retired.
`
`One of the first experim ents Milstein conducted was to determ ine the am ino acid sequence of the Bence-Jones protein in order to unravel
`the function of the disulphide linkages within the overall m olecular structure of the antibody. He published his first results in C. Milstein,
`'Disulphide bridges and dim ers of Bence-Jones Protein', Journal of Molecular Biology, 9 (1964), 836-8. The paper provided the first sequence
`data for the Bence-Jones protein and was the first of the m any papers that Milstein would write on antibodies over the next forty years.
`
`Having analysed the dilsulphide bridges of antibodies, Milstein began to explore the differences in am ino-acid sequences, the positions of
`carbohydrate attachm ents and m utations. All of this work was directed towards understanding the nature of the diversity of antibodies at
`the DNA level.
`
`One of the tools that proved vital to Milstein's work in determ ining the structure of antibodies was the chrom atography colum n. In colum n
`chrom atography a sam ple is put through a glass tube filled with a liquid known to separate the different com ponents within the sam ple.
`Because of m olecule size and polarity, different com ponents in the sam ple travel through the colum n at different speeds.
`
`In Milstein's experim ents, the chrom atography colum n was used to
`separate out the subunits of light and heavy chains and other
`com ponents of the antibody protein. Once put through the colum n,
`the separated com ponents of the antibody were then collected and
`purified. After this, an enzym e was added to the collected sam ple in
`preparation for sequencing. This enabled digestion of the sam ple to
`separate its constituent elem ents. After being treated with an
`enzym e, the sam ple was run again through a chrom atography
`colum n. Together these steps enabled Milstein to begin to determ ine
`the sequence of individual genes and larger genetic regions within
`the antibody.
`
`Overall the sequencing of an antibody's am ino acids was a laborious
`and tim e-consum ing process. The procedure would becom e m uch
`easier and faster with the developm ent of new sequencing m ethods
`in the early 1970s.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/antibodies
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`9/8/2015
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`PETITIONER'S EXHIBITS
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`Exhibit 1024 Page 10 of 63
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`The Story of César Milstein and Monoclonal Antibodies: Milstein's early antibody research
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`Page 3 of 5
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`In his elucidation of antibody diversity at the level of am ino acid
`sequences, Milstein was aided by Richard Pink, his first English
`graduate student, and Blas Frangione, an Am erican postdoctoral
`scientist. He was also helped by his wife Celia, with whom he
`published a joint paper in the Journal of Molecular Biology in 1970.
`
`By the early 19