The Story of César Milstein and Monoclonal Antibodies: Introduction
`
`Page 1 of 3
`
`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.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein
`
`9/8/2015
`
`1 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Introduction
`
`Page 2 of 3
`
`Portrait of César Milstein.
`Photo credit: Godfrey Argent Studio
`
`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.
`
`Milstein's early life and work >>
`
`http://www.whatisbiotechnology.org/exhibitions/milstein
`
`9/8/2015
`
`2 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Introduction
`
`Page 3 of 3
`
`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.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein
`
`9/8/2015
`
`3 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early life
`
`Page 1 of 5
`
`Milstein's early life
`
`The journey from Argentina
`
`The son of Jewish immigrants, César Milstein grew up in Bahía Blanca, a port town located by the Atlantic ocean some 500 miles south of
`Buenos Aires. Jews had begun to settle in Bahía Blanca from around 1900, many of them coming from central and eastern Europe.
`
`Family background
`
`Milstein's father, Lazaro, was born in a village in the Ukraine and migrated
`to Argentina in 1913 at the age of 14 with his aged aunt and uncle. For many
`years he lived in Jewish settlements near Bahía Blanca trying his hand at
`different trades, including farm labour, carpentry and railway work. During
`this time he taught himself 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, some with
`anarcho-syndicalist connections.
`
`Lazaro met Maxima, his wife-to be, in Bahía Blanca. Maxima was born in
`Argentina. She was the daughter of poor Ukrainian immigrants who made
`great sacrifices to ensure she had a secondary school education and went to
`college. At the time Lazaro met Maxima she was a school teacher. Soon
`after their meeting, Maxima rose to become a head mistress. From 1926 to
`1933, Maxima directed School No.3, the first co-educational school
`established in Bahía Blanca. Milstein, the middle of three brothers, was born
`at the family home on this school's premises. He also attended the school in
`his early childhood. Both of Milstein's parents spoke Yiddish at home, but
`Milstein was raised speaking only Spanish.
`
`During his early childhood Milstein preferred playing with other children in
`the streets to reading books. With his mother's encouragement, 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 prompted by a discussion he had with one of
`his cousins who had just completed her degree in Chemistry and was then
`working as a biochemist at the Instituto Malbran. Milstein was particularly
`fascinated by his cousin's description of her attempts to extract snake
`venom to treat snake bite victims. Milstein's interest in science deepened when on his ninth birthday he was given a Spanish translation of
`Paul de Kruif's Microbe Hunters by his mother. This book awakened his desire to have the same 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
`man.
`
`Photograph credit: Celia Milstein.
`
`Milstein grew up in a family which prized knowledge and education. Until his last year of school, Milstein attended schools close to home in
`Bahía Blanca, including the Colegio Nacional. In his final year, however, he moved 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 mother helping to type up his first PhD thesis and his father offering him economic
`assistance so that he could dedicate himself to his doctoral research. Fiercely independent, Milstein declined his father's financial support.
`
`Education
`
`In 1945 Milstein started to study chemistry at the University of Buenos
`Aires. His undergraduate studies, however, were interrupted when,
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/early
`
`9/8/2015
`
`4 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early life
`
`Page 2 of 5
`
`Bahía Blanca
`Bahía Blanca, Buenos Aires Province,
`Argentina
`View larger map
`
`Save
`
`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 some
`time off to recover. He finally received his BSc in chemistry in 1952.
`
`During his undergraduate years Milstein was active in campaigns against
`the Peronist government's policies aimed at privatising education and
`their more general impositions on universities and student life and rose
`to be President of the Student Union. At the time the government was
`clamping down on any political activity, and the atmosphere was
`particularly tense. In 1951, for example, a chemistry student, Ernesto
`Mario Bravo, was arrested and tortured for 20 days as a result of
`protesting against the government. His arrest sparked a major student
`strike. More than 150 strikers were arrested and university
`administrators expelled the more prominent leaders of the student
`movement. In the end, however, Bravo was released. The student
`movement considered this a major achievement.
`
`Three years later student unrest erupted once again when the Peronist
`regime imposed even greater control over the media, education system,
`trade unions and the legislative and the judiciary. In October 1954
`students joined workers striking against the then deep economic and
`
`Map data ©2015 Google
`Google Map showing the location of Bahia Blanca in Argentina.
`The town is a major trans-shipping and commercial centre,
`known for its large export trade of grains, wool, oil and fruit.
`Click to view a larger map.
`
`social crisis and increasing unemployment.
`
`Shortly after Milstein returned to the University of Buenos Aires from his several months of convalescence, Milstein met Celia Prilleltensky,
`a fellow chemistry undergraduate. Their first encounter was at the laboratory bench, where they found themselves working alongside each
`other. Celia not only shared Milstein's scientific interests, but was similarly an ardent student campaigner for free education. A year after
`their graduation in 1952, Milstein and Celia married.
`
`At the same time as getting married, Milstein began to look for a suitable doctoral supervisor. Initially he sought to work with Professor Luis
`Leloir, a distinguished Argentinian enzymologist. To this end he visited Leloir's workplace, an old house in Buenos Aires. On arrival he met
`what seemed to him an unassuming man carrying a basket. This turned out to be Leloir. Having no space to take Milstein on, Leloir instead
`referred him to the Argentinian biochemist Professor Andrés Stoppani.
`
`Milstein recalled that Stoppani was 'one of the few and perhaps
`the only full-time Professor of the Faculty of Medicine in the
`University of Buenos Aires, perhaps the most important universities
`in Latin America, a full time professor who probably had a salary
`of about the same order of magnitude as a janitor, trying to do
`serious and honest research in a laboratory with no funds at all'.
`
`Stoppani advised Milstein to take some time off before he started
`his doctorate in view of the tense political climate which was
`hostile to students such as Milstein who had actively campaigned
`against the Peronist government's policies in education. His advice
`encouraged Milstein to take a year long honeymoon with Celia
`exploring Europe.
`
`By 1954 the political environment had begun to improve and
`Milstein began researching enzymes for a doctorate in
`biochemistry 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
`biochemistry laboratory. In later years Milstein argued that this
`part-time job had taught him the value of organising his time.
`
`During his doctoral research Milstein had access to only the most
`basic equipment. Some idea of how poor the facilities were at the
`time can be seen from his recollections that Stoppani had 'to pay,
`from his own meagre salary, for the pound of yeast … needed from
`time to time in order to prepare … [the enzyme] aldehyde
`dehydrogenase.' He recalled, 'We survived on what was inherited
`from the golden days … from the Medical School and with reagents
`justified by teaching requirements'. The most precious piece of equipment in the department was a Warburg apparatus, which Stoppani did
`not allow anyone but himself to use.
`
`This photograph shows Milstein early on in his courtship with Celia
`Prilleltensky.
`
`Photograph credit: Celia Milstein.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/early
`
`9/8/2015
`
`5 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early life
`
`Page 3 of 5
`
`Click here to see the
`additional photographs from
`Milstein's early life.
`
`By 1955, the political situation in Argentina had improved still further and with this the conditions in
`Stoppani's department became slightly easier. This meant, for example, that the department was able to
`purchase a refrigerated centrifuge. The centrifuge proved useful to Milstein in his enzyme preparations.
`Nonetheless, problems remained. The nearest spectrophotometer, an instrument that Milstein needed for
`measuring enzyme activity, was located three blocks away from his department and Milstein had to trek every day between departments
`carrying reagents and enzyme preparations. Early on in his doctoral studies he came close to losing his position in the department when, in
`the process of making his enzyme preparation, he succeeded in consecutively breaking three of the department's five very expensive 5-litre
`flasks.
`
`Despite these hurdles, Milstein completed his doctoral research and was awarded a prize in 1957 by the Associación Quimica Argentina for
`the best thesis in chemistry that year. His doctorate was an investigation of the enzyme dehydrogenase. He had focussed his research on
`one of the enzyme chemical 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 Webb at the Sir William Dunn School of
`Biochemistry in Cambridge. In part the decision was influenced by the fact that Stoppani had worked with Dixon before the Second World
`War.
`
`Initially, Milstein had difficulty understanding what Dixon and Webb were
`saying because he lacked fluency in English, but with their advice he set out
`to study the kinetics and heavy metal activation of the enzyme
`phosphoglucomutase. This was inspired by Dixon's suggestion that Milstein
`follow up an odd observation made some years earlier in the department
`that phosphoglucomutase required two metals for full activity, magnesium
`and a trivalent metal like chromium.
`
`Milstein was left to pursue his research on his own within Cambridge. The
`work was not without its pitfalls. Milstein lost his first large-scale enzyme
`preparation in an electric cold bath. According to Milstein this was caused
`by the distraction of attending a champagne 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 Cambridge. A central figure in the
`Cambridge Biochemistry Department, Sanger's achievement had been to
`show that proteins have a defined chemical composition.
`
`Despite his early disaster with his enzyme preparation, within a year
`Milstein's experiments on phosphoglucomutase had provided sufficient data
`for him to write up his research. This led to the award of a second
`doctorate, this time from Cambridge University. Based on this research he
`published three papers. Contrary to contemporary opinion, Milstein
`discovered that the activation of phosphoglucomutase was caused by the
`displacement of heavy metals by magnesium. Prior to Milstein's finding,
`scientists believed the enzyme was activated by the heavy metals
`themselves.
`
`During his British Council fellowship in Cambridge, Milstein formed a strong
`bond with Sanger. At the time Sanger was a pivotal figure in the Department
`of Biochemistry, as he possessed the only functional pH meter. While an
`unassuming figure, Sanger was a dominant influence within the department.
`Milstein, for example, remembered a warning sign to the entrance of the
`department's high-voltage electrophoresis room reading 'Danger – High
`Power' which was altered by someone in the department to read 'Sanger –
`High Power'.
`
`Malcolm Dixon, the biochemist who supervised Milstein at
`the Sir William Dunn School of Biochemistry, Cambridge
`University.
`
`Milstein was quickly drawn to Sanger not only because of the equipment he
`possessed but also by the fact that they shared the same research interests.
`They soon collaborated to define the active site of phosphoglucomutase and
`published a joint paper on this research. It appeared as C. Milstein, F. Sanger, 'An amino acid sequence in the active centre of
`phosphoglucomutase', Biochemistry Journal, 79 (1960), 456-69.
`
`Photo credit: Sir William Dunn School of Biochemistry,
`Cambridge.
`
`When it came time for Milstein to return to Argentina, Sanger offered to secure Medical Research Council money for him to extend his time
`in Cambridge. Milstein, however, decided to return to Argentina to take up a position he had been offered prior to coming to Cambridge.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/early
`
`9/8/2015
`
`6 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early life
`
`Page 4 of 5
`
`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 Cambridge for the Instituto Malbran, where he headed up a newly-created Department of Molecular Biology
`in the National Institute of Microbiology. Celia also had a post in the department. The return of the Milsteins to Argentina coincided with a
`period of reform in the country following the fall of Peron, when many academic 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 Chemistry in 1970.
`
`As head of the Department of Molecular Biology, Milstein had a wide range of responsibilities, from employing his carpentry skills to install a
`laboratory to acting as mentor to 25 young scientists and bringing bacterial genetics into the research orbit of the department. In addition
`to his day-to-day to management of the department, he continued the research he had begun in Cambridge around phosphoglucomutase,
`developing techniques for studying the sequence of the enzyme and marking its active centres. He also started investigating another
`enzyme: alkaline phosphatase of bacteria. Much of this work was focused on understanding the enzyme's mechanism of action. His notable
`achievement at this time was elucidating the sequence around the active site of alkaline phosphate of bacteria with Noé Zwaig. This they
`accomplished ahead of scientists in the USA. They published their results in N. Zwaig, C. Milstein, 'On the nature of the phosphoenzyme
`intermediate in the phosphoglyceromutase reaction', Biochimica et Biophysica Acta, 73 (1963), 676-9.
`
`In 1962 Milstein's life, like that of many other Argentinians, was thrown into turmoil as a result of a military coup. Most disturbingly for
`Milstein, Ignacio Pirosky, the director of his institute, was dismissed, as were many colleagues in Milstein's department who had defended
`Pirosky. With the coup, persecution began to mount against both political dissenters and Jews in Argentina. This had major implications for
`Milstein. Bearing a Jewish name, authorities immediately associated him with communist activists. When four of his own staff were
`expelled from his department, he found himself no longer able to concentrate on his own scientific research and he decided to resign from
`his position and return to Cambridge. He was just one amongst the many tens of thousands of intellectuals and scientists who left Argentina
`during the military regime.
`
`More historical background can be found on Milstein and the rise and fall of molecular biology in Argentina in the late 1950s and late 1960s
`in P. Kreimer and M. Lugones, 'Pioneers and Victims: The Birth and Death of Argentina's First Molecular Biology Laboratory', Minerva, 41/1
`(2003), 47-69.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/early
`
`9/8/2015
`
`7 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early life
`
`Page 5 of 5
`
`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 Chemistry 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 moved
`into its own premises.
`
`For more on Milstein's recollections of his early days see C. Milstein, 'Messing about with isotopes and enzymes and antibodies', Lynen
`Lecture, Miami Winter Symposium Proceedings, in W. Whelan, ed., From Gene 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 What is Biotechnology | Advisory Board | Contact us | Terms and Conditions
`
`Website design by Silico Research the creative minds behind BioPartnering.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/early
`
`9/8/2015
`
`8 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early antibody research
`
`Page 1 of 5
`
`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 prime
`objective was to understand the molecular structure behind the formation 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 multiple myeloma, 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 determined during the 1950s. By the early 1960s, scientists had discovered that the Bence-Jones protein possessed the same structure
`as a light-chain, a sub-unit of an antibody molecule. Thereafter scientists began to use the protein as a tool for investigating the structure
`and function of normal 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 prompted 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 many scientists then trying to understand the body's immune
`system by unravelling the structure and function of the billions of antibodies made 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
`made in the body itself.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/antibodies
`
`9/8/2015
`
`9 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early antibody research
`
`Page 2 of 5
`
`Once an antibody recognises a particular antigen it will
`attach itself to a specific marker on the cell surface of the
`antigen so that the latter can be targeted for destruction.
`In many ways the binding of an antibody to an antigen can
`be likened to the insertion of a key in a lock.
`
`By the time 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
`formation composed 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, forming
`the stem of the Y, and one that was variable on the tip of
`the arms of the Y.
`
`Nonetheless, while the basic molecular structure of
`antibodies was beginning to be solved, what remained a
`puzzle was how such an apparently almost identical group
`of proteins could specifically target simultaneously any
`one of a multiple 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.
`
`What lay behind the heterogeneity of antibody molecules would be a recurrent subject for most 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 simple experiments,
`essentially comparing primary DNA sequences of two different antibodies. This gave Milstein the means to elucidate the diversity of
`antibodies at the level of their amino acid sequences.
`
`Dissecting the antibody structure
`
`Having decided upon his topic of research, Milstein began investigating different chemical techniques to dissect the structure of antibodies.
`He was assisted by John Jarvis, a biochemistry technician who had joined the LMB when he did. Jarvis would work as Milstein's research
`assistant until Milstein retired.
`
`One of the first experiments Milstein conducted was to determine the amino acid sequence of the Bence-Jones protein in order to unravel
`the function of the disulphide linkages within the overall molecular structure of the antibody. He published his first results in C. Milstein,
`'Disulphide bridges and dimers 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 many 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 amino-acid sequences, the positions of
`carbohydrate attachments and mutations. 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 determining the structure of antibodies was the chromatography column. In column
`chromatography a sample is put through a glass tube filled with a liquid known to separate the different components within the sample.
`Because of molecule size and polarity, different components in the sample travel through the column at different speeds.
`
`In Milstein's experiments, the chromatography column was used to
`separate out the subunits of light and heavy chains and other
`components of the antibody protein. Once put through the column,
`the separated components of the antibody were then collected and
`purified. After this, an enzyme was added to the collected sample in
`preparation for sequencing. This enabled digestion of the sample to
`separate its constituent elements. After being treated with an
`enzyme, the sample was run again through a chromatography
`column. Together these steps enabled Milstein to begin to determine
`the sequence of individual genes and larger genetic regions within
`the antibody.
`
`Overall the sequencing of an antibody's amino acids was a laborious
`and time-consuming process. The procedure would become much
`easier and faster with the development of new sequencing methods
`in the early 1970s.
`
`http://www.whatisbiotechnology.org/exhibitions/milstein/antibodies
`
`9/8/2015
`
`10 of 63
`
`BI Exhibit 1024
`
`

`

`The Story of César Milstein and Monoclonal Antibodies: Milstein's early antibody research
`
`Page 3 of 5
`
`In his elucidation of antibody diversity at the level of amino acid
`sequences, Milstein was aided by Richard Pink, his first English
`graduate student, and Blas Frangione, an American 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 1970s Milstein had begun to shift his research towards
`investigating the role played by messenger ribonucleic acid (mRNA)
`and the encoding DNA in determining the diversity of antibodies. The
`mRNA is a type of chemical 'blueprint' for a protein product.
`
`Somatic mutation and antibody diversity
`
`One of the people Milstein worked with to understand what lay
`behind antibody diversity was Sydney Brenner. Milstein had been
`introduced to Brenner in the mid-1960s by Francis Crick, co-
`discoverer of the double helix of DNA and a close colleague of
`Brenner. Together Milstein and Brenner developed a theory to
`explain the phenomenon. This was pu