Scientific Research

César Milstein: Hybridoma Cells to Create Monoclonal Antibodies

by David Warmflash, MD

On December 8, 1984, immunologist César Milstein arrived at the Karolinska Institutet in Stockholm, Sweden, to accept the Nobel Prize in Physiology and Medicine. Outside the world of immunology, his name is not well-known, but Milstein’s work advanced medicine and science in very profound ways. From routine applications like home pregnancy tests to more exotic jobs like searching for life on Mars, Milstein’s discovery of monoclonal antibodies and their uses has enabled humans to do things that otherwise might not be possible. Beyond tests, monoclonal antibodies are used ever more frequently in medicine – Zmapp, a treatment for Ebola, is just one example.

Milstein in the lab
Figure 1: César Milstein in the Medical Research Council laboratory. image © MRC Laboratory of Molecular Biology

Monoclonal antibodies come from specially developed cells called “hybridomas.” A hybridoma is a combination, or fusion, of two different types of cells: one with the genes needed to make the desired antibody, the other a cancer cell that makes the hybridoma “immortal.” This means that the hybridoma can be reproduced over and over to produce cells identical to itself and provide a specific antibody. The idea of a combining a cancer cell with another cell for a good purpose may sound counterintuitive, but in the 1970s Milstein and his colleagues realized the enormous potential of this technology to change medicine and biology.

Snake venom as inspiration

Born October 8, 1927 in Bahía Blanca, Argentina, César Milstein was the middle of three brothers in a family of Jewish immigrants from Eastern Europe. His father, Lazaro, hailed from a village in Ukraine; his mother Maxima was born on Argentinian soil, but she herself was a child of Ukrainian Jews. Like many East European Jews of that time period, Milstein’s parents identified as Jews strongly in the cultural sense, and not at all in the religious sense. They read Yiddish literature and associated socially with other Jews, which included working in non-religious Jewish organizations. They were part of socialist movements, concerned with workers’ rights, and did not attend synagogue or view the world from a religious perspective. They also spoke Yiddish with one another at home, but raised their sons to speak only Spanish.

As a child, Milstein loved reading books and became fascinated with science at age 8 because of a conversation with an older cousin. The cousin worked at the Instituto Malbran as a biochemist, and during their conversation she explained to the young César how she was developing serum from snake bite victims to be used as a snake bite treatment. This was the beginning of Milstein’s fascination with the immune system, and it had a major impact on his future work. Back then, scientists did not know the mechanism underlying the effect of a snake bite antiserum. But later, Milstein learned that the immune system produced antibodies, proteins that gave the antiserum its beneficial effect against the toxins that people received in a snake bite.

A year after the conversation with his cousin about the snake serum, Milstein read a Spanish translation of Microbe Hunters by Paul de Kruif. The book described Antony van Leeuwenhoek, Louis Pasteur, and other pioneers of biology as adventurers. This drew Milstein further into the world of life science. He knew at that point that he wanted a biological career.

Microbe Hunters (Spanish)
Figure 2: Los Cazadores de Microbios, the Spanish version of The Microbe Hunters by Dr. Paul de Kruif.

A student and activist in Buenos Aires

Milstein moved to Argentina’s capital, Buenos Aires, for his high school education. In 1945, he entered the University of Buenos Aires as a chemistry major. He was a brilliant science student, but politics and funding shortages forced him to think about other matters alongside his studies. His secular Jewish, left-wing upbringing lay at the foundation of his identity and his interest in science did not replace that, but added to it. As an undergraduate, he naturally gravitated into student movements against the Perón government that ruled Argentina. This government was right-wing economically and generally did not support scientific research at the university. On top of that, the right-wing government was complex when it came to relations with the country’s Jewish community. The founder of the rightwing party, Juan Perón, sympathized with the Axis powers of World War II, and Nazis fleeing Europe were allowed refuge in Argentina. On the other hand, Perón spoke in favor of Jewish rights and established diplomatic relations with the state of Israel early in 1949, and there were noticeable numbers of Jewish Peronists. But Milstein’s Jewish family was socialist. He was raised to believe that lack of wealth should not be a barrier to the benefits of civilization, especially education.

University of Buenos Aires Chem Lab
Figure 3: A chemistry lab at the University of Buenos Aires in 1947. image © Digital Library / Program FCEN History, Faculty of Natural Sciences, University of Buenos Aires

The right-wing Peronist policies aggravated most students because they put restrictions on universities and student life and sought to privatize education. Privatizing would mean that only students from wealthy families would be able to attend college. Milstein was popular on campus because he sided with left-wing student movements that favored free education, and in 1951 he ended up as president of the student union. It was a big risk for Milstein because student leaders were being arrested and the student union president was expected to help them. The most famous arrested student was Ernesto Mario Bravo. Like Milstein, Bravo was a chemistry student. On May 17, 1951, Bravo was abducted by the police (not actually arrested) and tortured by the government for the next 20 days. Students and university administrators protested, demanding Bravo’s return, and the protests culminated in a two-day university strike. As student union president, Milstein was under the government spotlight but the protests and strike led to Bravo’s release.

Along with intense campus politics, Milstein’s undergraduate years were difficult because of an accident that he suffered from diving into a pond and hitting a log. To recover, he had to take some time off. After returning to school, he fell in love with Celia Prilleltensky, a fellow chemistry student. The two students graduated in 1951 and were married one year later. At that time, Milstein enrolled as a graduate student at the same university and found a faculty advisor, Professor Andrés Stoppani, to guide him through a PhD program in biochemistry. When he began the PhD program in 1951, Milstein was shocked and disappointed to learn how underfunded Stoppani and his research lab were. As with the issues that Milstein had faced as an undergraduate, the underfunding of research was the result of the Peronist right-wing policies. Stoppani suspected that Milstein’s political perspective, and his history campaigning against the Peronist education policy, would get the young man into trouble. Stoppani advised Milstein to take time off with his wife until the political environment changed.

By 1954, the political situation had calmed enough for Milstein to start working with Stoppani. At the time, Stoppani still had no funding to support a graduate student. He was forced to use a portion of his very low salary to acquire materials for experiments and he could not afford needed equipment. Milstein worked on enzyme research in the Stoppani lab for his dissertation. However, to support his studies, he had to work part-time in a private clinical biochemistry laboratory. Only in 1955 did funding from the government improve enough for the biochemistry department to buy some basic equipment, such as a refrigerated centrifuge, but other essential machines were still out of grasp. To use one particularly important machine – a spectrophotometer – Milstein had to walk several blocks between buildings. This used up a lot of precious time and one day also caused him to break some expensive glassware, which almost got him expelled from the department.

Milstein’s devotion to politics on campus as an undergraduate and the lack of funding that plagued his day-to-day life as a graduate student drew time away from his studies. Nevertheless, he performed brilliantly as a student. In 1957, he earned his doctorate based on research concerning a type of chemical bond in enzymes called a disulfide bridge. Along with earning his PhD, the disulfide bridge research won him an Argentinian Chemical Association award for best thesis. From that point on, he continued working with Stoppani and the two published groundbreaking scientific papers resulting from Milstein’s doctoral work.

Comprehension Checkpoint

In Argentina, science education was well-funded during the Peron regime.

Professional advances, but more political difficulties

In 1958, Milstein moved to Cambridge, England to continue his research at the Sir William Dunn School of Biochemistry, supported by a fellowship from the British Council. Within a couple of years, this led Milstein to be awarded a second doctorate, this one from Cambridge University, based on his research of an enzyme called phosphoglucomutase – research that revealed a very unexpected mechanism through which the enzyme is activated. During this period, Milstein also met and formed professional bonds with the famed biochemist Fred Sanger, winner of the 1958 Nobel Prize. Soon, Milstein and Sanger started working together on the phosphoglucomutase enzyme. In 1960, the pair published a paper revealing the sequence of amino acids (building blocks of proteins) that made up an important region of the enzyme.

Milstein and Sanger
Figure 4: César Milstein and Fred Sanger image © MRC Laboratory of Molecular Biology

In 1961, Milstein returned to Argentina where he became head of the new Department of Molecular Biology at the Instituto Malbran, the same research center where his older cousin had worked on snake antiserum. His wife Celia also was appointed to the new department. It was a time of reform, with many other scientists returning to Argentina alongside the Milsteins, because the Perón government had fallen. In addition to continuing to research phosphoglucomutase, Milstein started studying another enzyme, alkaline phosphatase. Very soon, however, the new government was overthrown by a coup d'etat. This put in place another right-wing government, one hostile to Milstein because he was an academic and his Jewish name, which in the mind of the authorities made him a suspected communist. And so, along with thousands of other scientists and academics, Milstein left Argentina once again and returned to Cambridge.

Comprehension Checkpoint

Milstein focused his research primarily on

Antibody research

Milstein was able to move back to Cambridge quickly because of his friend and colleague Fred Sanger, who directed the protein chemistry division of the Medical Research Council Laboratory of Molecular Biology (LMB). Milstein’s focus at LMB was on the formation of antibody molecules, working on the very topic that had inspired him as a boy.

Antibody_2
Figure 5: Schematic diagram of an antibody and antigens with light and heavy chains noted.

Scientists had known about antibodies since the late 19th century. They knew that antibodies protected against disease, but they had no idea how antibodies worked. By the time Milstein started his research at LMB in the 1960s, scientists knew that antibodies were proteins. They knew that antibodies were shaped almost like the letter Y and that this shape had something to do with how antibodies worked. The top part of the Y shape was known to vary enormously between antibodies. This variability allows the immune system to produce antibodies able to grab onto a wide range of molecules foreign to the body. The upper part of the Y shape grabs a particular foreign molecule the way that a lock connects perfectly with the shape of the key that matches it. Scientists in the 1960s knew that antibodies worked this way, but how so many different shapes could be produced was a mystery.

To solve the mystery, Milstein designed experiments to test the possibility that the diversity could be the result of mutations occurring in the DNA sequences. This hypothesis was developed based on an earlier, simpler idea proposed in 1959 by another pioneering molecular biologist, Joshua Lederberg. Milstein was not talking about the kind of mutations that are passed on to future generations. Instead, he meant somatic mutations, changes in gene sequences in the DNA of body cells, which are all cells other than reproductive cells (called gametes). These mutations not passed on to the next generation, but are important because they often lead to cancer. In particular, Milstein set out to investigate somatic mutations in the immune system cells that make antibodies.

Comprehension Checkpoint

In antibodies, the top of their Y-shape

Tackling research problems

During the 1960s, numerous scientists were studying the DNA of immune system cells and they all, including Milstein, encountered the same two problems. First, experiments required a large number of cells and cells that made antibodies were hard to isolate. Second, the research required figuring out how the DNA in a cell that made one type of antibody differed from the DNA of a cell that made a different antibody. Every person has billions of antibody-producing immune cells in his or her blood. Each cell that is dedicated to making a specific antibody is called a clone. To compare one clone with another, Milstein knew he would need multiple copies of each clone.

To overcome both problems, Milstein investigated Bence-Jones proteins, which are produced by a type of cancer called multiple myeloma. Antibodies are composed of smaller units that form the upper part of the Y-shape called heavy chains and light chains. Since the Bence-Jones proteins appeared to have the same structure as a section of the light chains of antibodies, this solved the problem of isolating antibodies. The Bence-Jones proteins could also be obtained in large quantities from the urine and blood of multiple myeloma patients, solving the problem of acquiring large numbers of cells. Furthermore, Bence-Jones protein molecules from the same patient were known to be identical to one another. Essentially, multiple myeloma makes a whole bunch of the same immune cell clone and a piece of that antibody, the light chain, accumulates in the person’s blood and urine.

Bence-Jones Protein
Figure 6: A crystal of Bence-Jones protein created with X-ray crystallography, which can reveal detailed, three-dimensional protein structures. image © NIH / Alex McPherson, University of California, Irvine

By utilizing Bence-Jones proteins from patient samples, Milstein believed he might figure out faster than other scientists how antibodies worked and how they were formed. The hope was that it would lead him to devise experiments to test his somatic mutation hypothesis, that the diversity of antibody shapes is the result of mutations in the DNA sequences. Like the protein enzymes that Milstein had studied, antibodies and Bence-Jones proteins are made of chemical building blocks called amino acids. The sequence of amino acids of each protein depends on DNA sequences, or genes, available in each cell. Milstein’s long-term tactic was to compare the sequences of DNA and amino acids with the structure of the Bence-Jones protein. If he could do this, he could prove that the diversity of antibodies does indeed come from somatic mutations.

In 1962, Michael Potter, a molecular biologist at the US National Cancer Institute (NCI), discovered accidentally that a certain strain of laboratory mice (BALB/c mice) grows myeloma cells if injected with mineral oil. This made multiple myeloma cells and Bence-Jones proteins much easier to obtain compared with getting them from the blood and urine of human volunteers. Potter and the scientists working with him at NCI started growing large supplies of myeloma cells and made them available to scientists around the world. During the 1970s a team at San Diego’s Salk Institute developed a way to grow Potter’s cells in tissue culture; this meant that large numbers of the cells could be grown without even using mice. One line of these cells was called MOPC21 and it gave scientists like Milstein the freedom to focus their time and attention on doing creative experiments rather than having to devote much of their time into caring for the cells and keeping them alive in mice. Milstein collaborated with pathologist George Brownlee to extract a molecule called RNA from the MOPC21 cells. RNA is produced from DNA and acts as an intermediate in the construction of a protein, including an antibody, from amino acid building blocks. So, by examining the cells’ RNA, the genetic sequence for antibodies made by the cell could be obtained.

Comprehension Checkpoint

Every person has ______ antibody-producing immune cells in his or her blood.

Searching for mutations

In the early 1970s Milstein also began working with two younger scientists, David Secher and Dick Cotton. The research that took shape consisted of two major components. The first was to clone the MOPC21 cells to reproduce numerous exact genetic copies of the cell line. These needed to grow on a substance called “soft agar” because this would allow individual clones to be sampled easily.

The team needed to determine the rate at which mutations occurred once clones were growing consistently. Clones that mutated from a parent clone – called variants – could then be pulled from the agar, and the effects on the antibodies made by the cells could be compared. But after three months of culturing and analyzing antibodies from 7,000 clones, only five variants in antibody structure were observed. The method was too slow, although the occurrence of any mutations was a good finding.

A new type of cell: Hybridomas

The second component of the research sought to fuse two cells, each from a different myeloma cell line. This project depended largely on the laboratory skills of Dick Cotton, who was a postdoctoral scientist visiting Milstein’s LMB lab from Australia. All body cells have a mix of genes; half from the individual’s mother and half from the father. By the 1970s, however, scientists knew that each antibody-making immune cell used just one of the two sets of parental genes to make its antibody. Either the maternal genes were used or the paternal genes, while the other set of genes was turned off.

Cotton wanted to understand why this was the case, and together with Milstein he decided that merging two cells into one might reveal something about which set of genes turns on or off in different situations. In the resulting fusion or hybrid cell, they wanted to see which genes would be turned on and which would be turned off. They also wanted to see what effect the turning on or off of different genes in the fused cell would have on the structure of the antibodies made by the hybrid cell.

For one of the parent cells to make the hybrid cell, they used a mutant grown from the mouse MOPC21 cell line, while the other parent cell was from a line developed by a Belgian researcher. But rather than showing genes from one parent turned on and genes from the other parent turned off, the cell made by fusing the mouse and rat cell did something surprising. It manufactured antibodies using genes from both parent cells. Unlike in conventional immune cells, the new hybrid cell, which they called a hybridoma, did not shuffle the genes for different regions of the antibody to make new kinds of antibodies. Instead, they decided that the gene shuffling must occur early in the development of an immune cell before the cell starts making antibodies.

Using the same technique, but tweaking procedures to perfect it, the team made other types of mouse-rat hybridomas and also mouse-mouse hybridomas. The results were the same as in the first hybridoma: antibody genes from both parents were used to make antibodies. There was a problem, however. The new hybridomas revealed a lot about the genetics of antibody production, but there was no way to control which antibody was produced. In normal physiology, the immune system makes antibodies that specifically recognize and bind with a particular foreign entity called an antigen. Since the hybridomas were made from myeloma cells, the antibodies produced were the same antibodies that the myeloma cells were born to make as cancer cells. Milstein needed a way to trigger the hybridomas to make new antibodies, meaning antibodies against a particular antigen, just like the immune system does. Also, the hybridomas were very short lived, which made continuous experiments difficult over time.

While presenting a paper in Basel, Switzerland, Milstein met another researcher: Georges Köhler. For about a decade, Köhler had been working on his own kind of fusion cells using normal B lymphocytes. Also called B-cells, these are immune system cells that normally make antibodies in response to an infection. When this happens, they’re called plasma cells; when plasma cells become cancerous, they become multiple myeloma, the kind of cells that Milstein was fusing into hybridomas. A new collaboration developed as a result of this meeting and after many experiments Milstein and his colleagues created a new kind of hybridoma: a fusion of a healthy human B-cell and a mouse myeloma cell. They did this through a trial and error process, trying different combinations of cells and observing what the different, resulting hybridomas could do. The human B cell/mouse myeloma hybridoma turned out to be a groundbreaking achievement. It could manufacture the antibody that normally could be made by its human B-cell parent, but, somehow, merging with the mouse myeloma cell made it immortal. This meant that a hybridoma could be used to generate a cell line that could exist for long periods of time and be used to create the antibody of the immune cell in large quantities.

Hybridoma
Figure 7: An illustration of the steps in creating the human B cell/mouse myeloma hybridoma. (1) Mouse immunized; (2) B-cells isolated from the spleen; (3) Myeloma cells cultivated; (4) Myeloma and B-cells fused; (5) Cell lines separated; (6) Cell lines screened into those that bind with specific antigens (a) and those that do not (b); (7) Multiplication of cells in vitro (a) or in vivo (b); (8) Antibodies harvested image © Martin Brändli

This achievement set up Milstein, Köhler, and a growing number of scientific colleagues for the next step: making a hybridoma that secretes antibodies against a chosen antigen. If they could do this, it would mean that, by using a common procedure, antibodies could be made to recognize and bind virtually anything chosen as an antigen. The mouse immune system was known to react strongly to sheep red blood cells (SRBCs), meaning that mice easily make antibodies against SRBCs. Additionally, antibodies against SRBCs could be detected easily in laboratory tests used routinely in the 1970s. For these reasons, the team chose SRBCs for the test antigen. The goal was to create a lymphocyte-myeloma hybridoma that manufactured antibodies against SRBCs.

In January 1975, the project succeeded in creating hybridoma cells that not only made antibodies against SRBCs but made them in huge quantities. In their 1975 scientific paper, published in the journal Nature, Milstein and Köhler emphasized the importance of their achievement by discussing potential applications of the hybridoma antibody technology to industry and medicine. Antibodies produced this way are called monoclonal antibodies, because they recognize one antigen (in fact, they recognize one part of a specific antigen, called an epitope). This is in contrast with polyclonal antibodies, which are made naturally by the immune system when it is stimulated by antigen exposure. In the case of a polyclonal immune response, the antibodies made vary in terms of the physical form and characteristics for binding antigens. In contrast, monoclonal antibodies are all exactly the same and recognize the exact same molecule and the exact same part of a molecule.

Comprehension Checkpoint

To make antibodies, regular immune cells use genes from

Monoclonal antibodies revolutionize healthcare

Because of this specificity of monoclonal antibodies and the large numbers of them that can be made with a hybridoma, they can be used as detectors in medicine and biology. Thus, during the 1980s and 1990s, they revolutionized health care, the pharmaceutical industry, and biology research. Virtually every test for something biological – a blood test for HIV (the virus that causes AIDS), hepatitis, influenza, etc. – depends on some kind of monoclonal antibodies, manufactured with the techniques developed by Milstein and his colleagues. Antibodies produced this way also are used for infectious agents in public health settings, detection for defense against biological weapons, and have even been studied for use in detection of life on Mars and other planets. They’re also being employed ever more frequently as therapies against disease, including Ebola.

Milstein and Kohler
Figure 8: César Milstein and Georges Köhler, along with Niels Kaj Jerne (not pictured), won the Nobel Prize in Physiology or Medicine in 1984. image © Celia Milstein/MRC Laboratory of Molecular Biology

Though perhaps not realizing just how far into the future Milstein’s work could take medicine, the scientific community understood the importance of the discovery fairly early. Thus, in 1984, together with Köhler and another colleague, Milstein was awarded the Nobel Prize for Physiology and Medicine.

Summary

From home pregnancy tests to treatment for Ebola, the discovery of monoclonal antibodies has greatly advanced science and medicine. This module traces the work of immunologist César Milstein, who successfully created “hybridoma” cells capable of producing specific antibodies in mass quantities. The module describes Milstein’s life as a student in Argentina during the regime of Juan Peron and his later research as he became a world-class scientist, winning the Nobel Prize in 1984.