Nobel Prize in Medicine/Physiology 1984 for Theories Concerning the Specificity in Development and Control of the Immune System and the Discovery of the Principle for Production of Monoclonal Antibodies
César Milstein was born on October 8, 1927, into a family of Ukrainian descent. His father, Lázaro Milstein, was a Jewish Ukrainian immigrant who came to Argentina at the age of 14, in 1913, with an aunt and uncle, living in a Jewish settlement near Bahía Blanca. Although Lázaro learned Spanish, he retained a strong connection to his cultural roots, being active in social and cultural activities and helping preserve Yiddish literature. He worked as a farm laborer, a carpenter, and as a traveling salesman. César’s mother, Máxima Vapniarsky, was born in Argentina to Ukrainian immigrants. She was a schoolteacher who, in 1926 at the age of 33, became the director of the first co-educational school in Bahía Blanca.
Milstein and his two brothers were raised speaking Spanish. Both parents emphasized the importance of education. Other factors that influenced the direction Milstein would take in life were his cousin and a book. His cousin was a biochemist who told Milstein about the work she did at the Instituto Malbrán, doing research into snake venom to develop a treatment for snake bites. The book was a Spanish translation of The Microbehunters (Los Cazadores de Microbios) by Pau de Kruif. The book depicted the lives of such scientists as Pasteur and Leeuwenhoek, which led Milstein to think that scientists led exciting lives.
Milstein spent most of his high school years at the Colegio Nacional in Bahía Blanca. In the mid-1940s, he went to Buenos Aires to prepare for the entrance exam to enroll in the Universidad de Buenos Aires. He started his undergraduate studies in chemistry. There he met a fellow chemistry student, Celia Prilleltensky, who shared not only an interest in chemistry with Milstein but also a spirit of activism in support of free education and against the Peronist government’s policies to privatize education. His activism included a period as the President of the Student Union.
Between 1950 and 1956, both Milstein and Celia worked part-time as clinical analysts at the Laboratorio Liebeschultz. This covered the time during which they graduated from the Universidad de Buenos Aires (1952) and got married (1953). In 1953, Milstein was planning to start his doctoral work and, on the recommendation of Luis Federico Leloir, asked Andrés Stoppani to supervise his post-graduate work. Stoppani suggested that Milstein take a year off before starting his graduate studies, as the political climate under the Peronist regime was not favorable to academia. Milstein and Celia therefore took the opportunity to have a year-long honeymoon, traveling around Europe and working on a kibbutz in Israel. When they returned in 1954, they resumed their part-time work at the Laboratorio in the clinical biochemistry lab. Working there while pursuing his graduate studies gave Milstein a firm understanding of the value of organizing one’s time.
Although the political situation had improved in Milstein’s absence, there still was little support for the sciences at the Universidad de Buenos Aires. The academic laboratories had only the most basic equipment and the faculty were not fully staffed. Stoppani was the only full-time professor of chemistry, yet was paid at the level of a janitor and had to pay for basic laboratory supplies out of his own pocket. By 1957, Milstein had completed his doctoral research, which focused on the disulfide bridge, a chemical bond in the enzyme dehydrogenase. Milstein was awarded a prize for the best thesis in chemistry for the year by the Asociación Química Argentina. He also published several papers with Stoppani in the next few years.
In 1958, Milstein received funding from the British Council to work with Malcolm Dixon at the School of Biochemistry in Cambridge, England, even though Milstein did not speak English at this time. In Cambridge, Milstein studied the kinetics and heavy metal activation of the enzyme phosphoglucomutase, a subject that also interested Frederick Sanger (who won the Nobel Prize in 1958 for showing that proteins have a defined chemical composition). Milstein and Sanger developed a close working relationship. After one year of work, Milstein had enough data for a second dissertation, showing that activation of phosphoglucomutase is caused by displacement of heavy metals by magnesium. With this, Milstein was awarded a second doctorate, this time from Cambridge University.
Instead of staying on in Cambridge after receiving his degree, Milstein returned to Argentina to be in charge of the Department of Molecular Biology at the Instituto Nacional de Microbiologia (INM — National Institute of Microbiology), which was headed by Ignacio Pirosky. Celia also had a position there. The timing was right, as in 1961 there was a period of reform following the fall of Peron, when many Argentine scientists who had been exiled or sidelined were able to return to work. At INM, Milstein supervised and mentored 25 young scientists and introduced the subject of bacterial genetics into the laboratory there.
This more open period for scientists, however, was short lived, as a new military coup in 1962 again brought restrictions to academia. There was a heightened level of political pressure against Jews and political dissent. Milstein therefore returned to Cambridge in 1963 to work with his friend Sanger at the Medical Research Council (MRC), Laboratory of Molecular Biology (LMB), where Sanger was head of the Protein Chemistry division. Milstein switched his focus from enzymes to antibodies, as he started to look at the molecular structure related to the formation and diversity of antibodies.
Milstein and his assistant John Jarvis looked specifically at the way that light-chain and heavy-chain units of antibodies are held together by disulphide bridges. They looked at the differences in amino acid sequences to examine the diversity of antibodies at the level of DNA. At first they had to use a time-consuming method based on the chromatography column. Milstein’s wife Celia was another assistant who helped in this work and co-authored a paper with Milstein in 1970. In the early 1970s, a new method of sequencing was developed that made the procedure more efficient. Milstein’s team also began to look at the role of mRNA and the encoding of DNA in the diversity of antibodies.
As early as the 1950s, it was known that normal plasma cells of multiple myeloma produce a large variety of antibodies. Malignant plasma cells, however, produce just one antibody. In the 1970s, scientists at the Salk Institute in California developed a method to adapt myeloma cells from mice to grow in tissue culture. Milstein used mouse myeloma cells originally grown at the Salk Institute for his basic research into the structure of antibodies. He wanted to see if antibody diversity results from a mutation in the DNA of an antibody, a process referred to as somatic mutation. Milstein’s experiments involved fusing together different myeloma cell lines, creating hybrid clones known as hybridomas. In April 1974, he presented the results of his myeloma cellular fusion experiments at the Basel Institute of Immunology, in Switzerland, where he met Georges Kohler. Interested in Milstein’s work, Kohler joined the research team in Cambridge.
In the mid-1970s, Milstein and Kohler had fused mouse myeloma cells with normal B cells from the spleen of a mouse immunized with a known antigen so that the mouse produced a specific antibody. They cultivated the fused cells in a culture medium and, after several months, found that the hybrid cells could secrete antibodies to the specific antigen in large amounts in a cell line that continually reproduced. These “immortal” cell lines could produce a limitless supply of identical antibodies with a known specificity. The process became known as hybridoma technology and the resulting identical antibodies as monoclonal antibodies (mAbs). Milstein and Kohler published their results in the August 1975 issue of Nature.
Milstein described the work he conducted at this time in the Lynen Lecture, entitled “Messing about with isotopes and enzymes and antibodies,” published by W. Whelan in the Proceedings of the Miami Winter Symposium From Gene to Protein: Translation into Biotechnology (1982).
The work Milstein described in his articles was not a straightforward process, as complications could arise from contamination of the culture medium the cells were grown in, and it could be difficult to fuse the cells to create the different hybridomas. There seemed to be as much art or skill as science involved in the process, as Milstein and his team learned through trial and error. Despite the difficulties of the work, Milstein shared cell lines with other scientists who wanted to conduct their own research. This was done in the spirit of openness common in the scientific community but with the condition that the scientists acknowledge the source of the cell lines, that they ask permission before sharing them with a third party, and that they would not patent any products that came from the cell lines.
Under such conditions, Milstein shared some cell lines with Hilary Koprowski, who was director of the Wistar Institute in Philadelphia. Because of the different procedures for and timing of patent applications, Koprowski applied for a broad patent in the United States in June 1977 before the MRC was able to apply for a patent in the United Kingdom through the National Research Development Corporation (NRDC).
Milstein’s own work had started out as pure science but soon was seen to have practical applications. In the spring of 1977, his work on rat histocompatibility and antigens showed that mAbs could be used to test compatibility between an organ donor and recipient as well as for tissue typing. With approval from the MRC, Milstein formed an agreement for Sera-lab to distribute mAbs in the United Kingdom and give part of the profit back to the MRC. He made a similar arrangement with the Salk Institute for distribution in the United States.
Milstein collaborated with other scientists for other ways in which mAbs could be used in medicine. He had a long-lasting friendship and collaboration with a fellow Argentine, Claudio Cuello, in a variety of mAb applications, such as detecting Substance P, involved in pain and neurotransmission, and other neural proteins like serotonin. Their work improved the understanding of diseases like Alzheimer and Parkinson and the development of neuropharmacological drugs and immuno-based diagnostic tests. Milstein collaborated with Leonard Herzenberg on methods to use mAbs for automated separation of cells, with David Secher for using mAbs to purify natural products like interferon, and with Douglas Voak and Steven Sacks for using mAbs for blood group typing.
For all of his work, Milstein was well-known internationally as a top researcher in the field. He received such recognition as membership in the Royal Society (1975), an honorary doctorate (1977) from the Universidad Nacional del Sur in his hometown of Bahía Blanca, the Wolf Prize in Medicine (1980), and the Royal Medal (1982). In 1983 he became the Head of the Protein and Nucleic Acid Chemistry Division in Cambridge. His most note-worthy award, however, was the Nobel Prize in Physiology and Medicine, along with Georges Kohler, in 1984, for their theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies. Milstein entitled his Nobel Prize lecture “From the Structure of Antibodies to the Diversification of the Immune Response.”
In his later years and even at the time he retired in 1995, Milstein was interested in the origin of somatic mutation in antibodies. After retirement he remained an active member of the LMB and collaborated with Michael Neuberger to determine the enzymatics involved in somatic mutation. He contributed to a paper on the mutational process and its mechanism, which was submitted for publication to PNAS shortly before his death in 2002.
In the 1970s, Milstein had been diagnosed with a chronic heart condition that had afflicted his mother, as well. He modified his diet and incorporate exercise to the extent that he was well known as an avid walker. Despite such lifestyle changes, he died of a heart attack at the age of 74. He was remembered by his colleagues as not only an excellent research scientist, but also as someone who throughout his life devoted himself to helping science and scientists, especially in the less developed countries. In March 2000, Milstein said that “Science will only fulfill its promises when the benefits are equally shared by the really poor of the world.” With that as inspiration, the MRC created a scholarship in Milstein’s name to help support Argentine scientists studying in Cambridge.
Resources:
“César Milstein – Biographical,” “Cesar Milstein – Facts,” and “Cesar Milstein Nobel Lecture” at http://www.nobelprize.org
“Professor César Milstein” at http://www.whatisbiotechnology.org
“The Story of César Milstein and Monoclonal Antibodies”. Collated and written by Dr. Lara Marks, content from an exhibit on César Milstein, at http://www.whatisbiotechnology.org (makes reference to a film about Milstein “Un Fuegito,” produced by Ana Fraile, Pulpo films, available on vimeo.com)
“César Milstein” at en.wikipedia.org
“César Milstein: Argentine Immunologist” at http://www.britannica.com
“César Milstein, PhD.” at http://www.aai.org (American Association of Immunologists)
“César Milstein, 74, Who Won Joint Nobel Prize in Medicine” New York Times, 26 March 2002
“César Milstein (1927-2002)” at http://www.jewishvirtuallibrary.org
“César Milstein: Pasión por la Ciencia” at http://www.serargentino.com
“César Milstein” at http://www.bionity.com
“César Milstein: Oct 8, 1927 – March 24, 2002” by Rabbitts, T.H., Cell Press, 2002 at http://www.cell.com
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