Making monoclonal antibodies
The hunt for a single antibody
From the late 1960s onwards Milstein would devote much of his research to understanding somatic mutation in antibodies. The idea that somatic mutation could help explain antibody diversity was not new. Indeed, Milstein and Brenner's suggestion that this could be what determined antibody diversity was in many ways an extension of a hypothesis first formulated by the American molecular biologist Joshua Lederberg in 1959.
In his quest to understand somatic mutation and antibody diversity, Milstein was greatly helped by the availability of cells taken from the blood of patients with multiple myeloma. Such myeloma cells, which produce antibodies resembling normal antibodies, had been noted in 1951 by Henry Kunkel, an American immunologist based at the Rockefeller Institute in New York. Investigating the blood of myeloma patients, Kunkel was surprised to observe that malignant plasma cells of multiple myeloma appeared to produce just one antibody. This contrasted with normal plasma cells which produce a vast array of antibodies.
Kunkel's discovery was a significant breakthrough for antibody research. Ever since the early 20th century scientists had been struggling to isolate and purify single antibodies from the billions made by the body every day. One of the advantages of the antibodies produced by myeloma cells is that they are all identical. Moreover, they are fairly easy to obtain in large quantities from the blood or urine of patients with multiple myeloma. Based on this, Kunkel and his co-workers began using myeloma cells as a tool for investigating normal antibodies.
The availability of myeloma cells for antibody research greatly expanded after 1962 when the American molecular biologist Michael Potter made the serendipitous discovery that an injection of mineral oil into the peritoneal cavity of BALB/c mice, a particular strain of laboratory mice, induced the growth of myeloma cells. This meant that the cells could be grown easily and indefinitely. Following this, Potter and his colleagues at the National Cancer Institute in Bethesda established a collection of myeloma cells lines for distribution to researchers around the world.
By the 1970s scientists' access to myeloma cells was made much easier by the work of Kengo Horibata and A.W. Harris under the supervision of Melvin Cohn at the Salk Institute in San Diego. They found a way of adapting Potter's mouse myeloma cells to grow in tissue culture, a technique that allows for the growth of tissues or cells outside an organism. Essentially, tissue culture provides for the growth of cells under controlled conditions outside their natural environment. The growth of such cells is helped by the use of a suitable medium, which provides the nutritional elements they require.
The first tissue culturing techniques date back to 1885, and had become a common method in laboratories by the 1940s and 1950s. Each cell type necessitated different conditions, such as temperature, and various types of culture medium for optimum growth. This was perfected by a process of trial and error. The tissue culture developed for the growth of Potter's myeloma cells is described in K. Horibata and A.W. Harris, 'Mouse myelomas and lymphomas in culture', Experimental Cell Research, 60 (1970), 61-77.
Overall the work undertaken by Horibata and Harris freed scientists from the laborious process of growing myeloma cells in mice. With the cultivation of myeloma cells in vitro, scientists now had access to a continuous supply of such cells.
Milstein had obtained Potter's mouse myeloma cell line (MOPC21) that Horibata and Harris had adapted to long-term tissue culture during the early 1970s from his LMB colleague Alan Munro, who had originally obtained them from the Salk Institute where he had spent a sabbatical year. Milstein grew these cells together with George Brownlee for the purposes of harvesting mRNA and as a source for studying the structure of antibodies.
Based on their growth of the myeloma cells, Brownlee and Milstein were able to establish the existence of a leading sequence in the RNA of the light chain. They reported their results in C. Milstein, G. Brownlee, G.G. Harrison and T.M. Mathews, 'A possible precursor of immunoglobulin light chains', Nature New Biology, 239 (1972), 117-120.
Fusing myeloma cells to pinpoint somatic mutation
After his letter with Brenner in Nature, which had hypothesised that antibody diversity was the result of somatic mutation, Milstein began looking for a way to carry out experiments to validate the theory. This he decided to do using the MOPC21 myeloma cell line. He was joined in the venture by David Secher who joined the LMB to do a doctorate in the summer of 1970.
What Milstein and Secher wanted to determine was the rate of somatic mutation that occurred in the antibodies produced by the myeloma cell line and to identify and characterise any variants that emerged. This they hoped would help them unravel the process of antibody diversity as it occurred in nature.
Shortly after Milstein and Secher began their project to investigate somatic mutation in MOPC21 myeloma cells, they were joined by Dick Cotton, a postdoctoral scientist from Australia who was interested in immunogenetics. Rapidly picking up the skills for cell culture, with the help of Abraham Karpas who worked in a nearby laboratory, Cotton soon succeeded in cloning MOPC21 cells in soft agar. This laid the basis for the isolation of MOPC21 mutants.
With Cotton's technique in place, the team then spent many months looking for somatic mutants. However, this proved frustrating. The difficulty was that such mutants rarely occur. Just five structural variants were isolated, for example, after three months of continuous culture and analysis of antibodies produced by 7000 clones.
While providing a disappointing yield of mutants, the myeloma cells nevertheless provided scientists with a window through which to see somatic mutation in cells. Importantly, mutation had occurred without any addition to the myeloma cell line. It had taken place on its own accord. The difficulty, the team found out later, was that the mutants observed in the myeloma cell line bore no resemblance to the major types of mutants found in nature.
Alongside this experiment a second project was launched. Undertaken by Cotton, this involved the fusion of two different myeloma cell lines. It was carried out using inactivated Sendai virus obtained from Abraham Karpas. This reagent was used for the promotion of cellular fusion.
One of Cotton's objectives was to understand why antibody-producing cells appeared to use only one set of parental genes to produce a functional antibody. Called allelic exclusion, this phenomenon was particularly puzzling as in most cases cells inherit a copy of both sets of genes from their parental cells. What scientists assumed was that in the process of antibody reproduction one gene was silenced while the other was transferred across. By fusing two myeloma cells lines, Cotton and Milstein wanted to see which genes would be transferred and which would be silenced. They were also interested in what effect such fusion would have on the structure of antibodies in terms of their variable and constant regions.
In the fusion experiment, the first myeloma cell line was a variant of the mouse MOPC21 line that was sensitive to the chemical bromodeoxyuridine. The second was a rat myeloma cell line obtained from Hervé Bazin in Belgium.
To Cotton and Milstein's surprise, the fusion of the mouse and rat myeloma cells resulted in a hybrid clone, known as a hybridoma, that produced antibodies carrying the genes of both parental cells. Indeed, they could find no evidence for allelic exclusion. Moreover, there appeared to be no scrambling of the variable and constant regions in the structure of the antibodies produced. Based on this, they theorised that the linkage between variable and constant regions occurred early in the process of cell differentiation, before antibody production began.
Following the fusion of the rat and mouse myeloma cells, Cotton, with the help of Shirley Howe, one of Milstein's laboratory technicians, continued to perfect the fusion method. One of the fusions resulted in hybridomas derived from mouse-mouse myeloma cell fusions. These yielded the same results as the earlier mouse-rat fusions.
Cotton and Howe's fusion efforts were greatly enhanced by the selection of a different myeloma clone, known P3-X63Ag8. The clone was a variant of a sub-clone of the P3 myeloma cell line originally developed by David Secher. Secher had made this from the cell line originating from Horibata and Harris's adaptation of Potter's MOPC21 myeloma cell line. A key advantage of P3-X63Ag8 was that it was resistant to azaguanine (Ag), a reagent that helps promote fusion. Other myeloma cell lines were liable to be destroyed by azaguanine.
In 1973 Milstein presented the results from the myeloma cellular fusion experiments conducted with Cotton to the Basel Institute of Immunology. It was here that Milstein met Georges Köhler. At the time Köhler was on a temporary secondment to the Institute while completing a doctorate at the University of Freiburg. Excited by Milstein's presentation and taking an instant liking to him, Köhler asked to join Milstein's research team in Cambridge as a post-graduate. This he did in April 1974, and he soon joined in the research efforts to understand somatic mutation and the mechanism underlying antibody diversity.
Finding an antibody with defined specificity
Prior to Köhler's arrival, much of Milstein's research into somatic mutation and the diversity of antibodies had focused on a search for mutant genes within the variable region of antibodies, as this was the portion of the antibody understood to be responsible for binding to antigens. Yet this had proven a laborious process, akin to looking for a needle in a haystack.
What was needed was an antibody with a clearly defined specificity. This would provide the most effective means for detecting slight differences caused by such mutations. Such an antibody, however, was not readily available.
Until the mid-1970s most of the studies Milstein and his team had undertaken on somatic mutation in antibodies were still being conducted with myeloma cells, dictated by their abundance and the fact that this was the closest scientists had come to a source of natural antibodies. However, such cells had certain limitations. The difficulty was that no one knew which specific antigens the myeloma cells bound to. Part of the problem was the fact that such cells are triggered by malignancy, a process that affects cells at random.
Not knowing which antigen was targeted by myeloma cells was a major problem for Milstein and others interested in understanding the molecular basis of antibody specificity. Some scientists had attempted to get around the problem by trying to induce tumours to produce antibodies to an injected antigen. These efforts, however, had come to nothing.
This shows a confocal image of the spleen of a mouse showing B cells stained in blue. Photo credit: Peter Lane and Fiona McConnell, Wellcome Images B0003949.
By the time that Milstein and Köhler began their collaboration, a number of scientists had begun to devise ways to make antibodies with known specificity to a particular antigen.
Discovery of a natural hybrid cell producing antibodies
One of the earliest was Joseph Sinkovics, a Hungarian immunologist based at the M.D. Anderson Hospital and Tumor Institute in Texas funded by the National Cancer Institute (NCI). Between 1960 and 1962, while conducting research into simple viral mouse leukaemia, Sinkovics happened to come across a mouse lymphoma cell with virus like particles on its surface, a highly unusual characteristic, which prompted an immune response in mice able to destroy the cell. Keen to learn more Sinkovics and his team began looking for ways to grow the cells in tissue culture. Initially this proved an uphill struggle, but by 1966-67 they had managed to grow a new cell line in suspension spinner cell cultures. This they achieved by cultivating the original cells in culture with tissue taken from the spleen of mice that had rejected such cells. Much to the surprise of the team the new cells appeared to contain sets of chromosomes unlike the previous lymphoma cells which had just 2 sets of chromosomes. The new cells also grew more vigorously when injected into the adult mouse than the previous cells, forming huge tumours which presented as abnormal fluid or ascites in their abdomens. This work was published in J.G. Sinkovics, B. Drewinko, E Thornell, 'Immunoresistant tetraploid lymphoma cells', Lancet (17 Jan 1970), 139-40.
Joseph Sinkovics, 1960s, credit: Sinkovics. Born in Budapest in 1924, Sinkovics obtained his medical degree from the University of Petrus Pazmany. Following this he set up a laboratory to study viruses in the Institute of Microbiology in Budapest. In 1956 Sinkovics left Hungary following the uprising against the Soviet imposed communist government and took up a Rockefeller Fellowship in the US. In 1979 he was appointed a consultant oncologist in the University of Texas M.D. Anderson Hospital and Tumor Insitute, where he was to remain for the rest of his career. For more about Sinkovics click here.
On further investigation the team discovered that the new cells not only produced virus particles but also antibodies against the mouse leukaemia virus. Further experiments with mice indicated the antibodies were highly specific against the virus. Indeed, they were better at neutralising the mouse leukaemia virus than antiserum taken from rabbits or mice immunised with the virus. Sinkovics hypothesised that the antibodies were the result of a natural fusion of a splenic plasma cell with the mouse lymphoma cell. Such a fusion he argued was highly exceptional and would only occur under special conditions. He believed it was due to a 'specific immune reaction between the budding virus still embedded in the cell membrane and the antibodies retained before their release on the surface of the immune spleen cells'. This process generated a hybrid cell (later known as a hybridoma) capable of producing antibodies which incorporated the immortal qualities of the malignant cell. (For more details see J. Sinkovics, 'Discovery of the hybridoma principle in 1968-69. Immortalization of the specific antibody producing cell by fusion of a lymphoma cell', Journal of Medicine, 16 (1985), 16: 509-524).
Credit: Sinkovics. This diagram outlines the hypothesis Sinkovics developed to account for the formation of the natural hybridoma in murine lymphomas. He presented the diagram to various international conferences between 1968 and 1969, including the International Tumor Conference in Perugia, Italy and at two annual conferences at the University of Texas M.D. Anderson Hospital, one on basic science and another for clinical medicine. The image was also published on the front of the Leukemia-Lymphoma Year Book (1970).
Between 1968 and 1969 Sinkovics presented his findings to several international and American conferences during which he showed a sketch of his hypothesis. Yet, to his disappointment, the significance of what he had found seemed to by-pass everyone who heard him. As he recalled, no one rose to ask a question or comment on what he had found. In spite of this, Sinkovics and his colleagues continued their work on the cells. (J.G. Sinkovics, 'On the threshold of the door of “no admittance”, in A. Szentivanyi, H. Friedman, eds., Immunologic Revolution: Facts and Witnesses (Florida, 1994).
Photograph showing the spontaneous fusion of lymphoma cells with leukaemia virus immune plasma cells which continued to secrete specific mouse leukaemia virus-neutralising antibodies. The double nucleated tetraploid cells are the new hybrid cells. Credit: J.G. Sinkovics, Cytolytic Immune Lymphocytes (Budapest, 2008), figure 7D, p.100.
By 1970 Sinkovics and his colleagues had shown that the fused cells could grow in suspension culture for many years and continued to produce specific mouse leukaemia virus-neutralising antibodies. Furthermore they had devised a method to purify the antibodies. That year, however, they were forced to abandon the project because the NCI refused funding to extend the project despite finding it to be scientifically sound. Moreover, the team could not envisage any therapeutic applications for the antibodies. (J.G. Sinkovics, 'On the threshold of the door of “no admittance”, in A. Szentivanyi, H. Friedman, eds., Immunologic Revolution: Facts and Witnesses (Florida, 1994).
Antibodies from cloned B cells
Soon after Sinkovics and his team had developed their antibodies, another group at the National Institute of Medical Research led by Brigitte Ita Askonas (1923-2013), an Austrian-Canadian biochemist, hit upon a way of generating antibodies by cloning B lymphocytes, a type of white blood cell, in genetically identical irradiated mice. This they did as part of a wider project to understand the process underlying the generation of B cells and antibody diversity. Called E9, these clones produced antibodies that bound to a hapten antigen, a small molecule that can only stimulate an immune response when attached to a larger molecule like a protein. Labelled E9, the clones had a major drawback – they could only be maintained for a maximum of six months. (B. A. Askonas, A. R Williamson, B. E. G. Wright, 'Selection of a single antibody-forming cell clone and its propagation in syngeneic mice', Proceedings US National Academy of Sciences, 67/3 (1970), 1398-1403).
Brigette Askonas, credit: Cambridge University. Born in Vienna in 1923 to Czech parents, Askonas undertook a biochemistry degree at McGill University and then landed up as a post-graduate researcher at Cambridge University and the Basel Institute of Immunology. In 1952 she joined the National Institute of Medical Research where she remained until 1989. Her research was focused on understanding the process underlying the generation of B cells and antibody diversity. For more about Brigitte Askonas click here.
Splenic fragment technique for antibody production
As had happened with Sinkovics, the generation of antibodies to a specific antigen by the Askonas team would remain largely overlooked by the scientific community. More attention was paid to a technique published in 1969 by Norman Klinman, an American immunologist based at the University of Pennsylvania with an attachment to the Wistar Institute. His method required a number of steps. First a mouse would be treated with radiation to destroy its antibody-producing capability. It would then be injected with new antibody-producing cells, some of which lodged in its spleen. Once this was done the mouse's spleen was cut into cubes and individually grown as hybrid cells in tissue culture with an added antigen. If an antibody-producing cell was present within a given fragment, it would produce antibodies specific to that antigen. Called 'monofocal antibodies', these antibodies could then be isolated from the culture medium and used for experiments.
Klinman published his technique in R. Klinman, 'Antibody with Homogeneous Antigen Binding Produced by Splenic Foci in Organ Culture', Immunochemistry, 6/5 (1969), 757-9. The splenic fragment system marked a major milestone. Importantly, it showed that one cell produced only one antibody. The technique was soon adopted by a number of scientists. One of the first to do so was Walter Gerhard, a Swiss-trained physician, who had a post-doctoral research position in Klinman's laboratory in the early 1970s. By early 1975 Gerhard, by now based at the Wistar Institute, had successfully generated single antibodies with known specificity against influenza viruses.
The difficulty with Klinman's method, however, was that it produced only a minute quantity of antibodies. Moreover, the cells that produced the antibodies survived for a maximum of only three months.
The creation of monoclonal antibodies
On pondering how to take their investigations into the somatic mutation of antibodies further, Milstein and Köhler started searching for a means to create an antibody with the desired specificity. Their starting point was Cotton's myeloma cell fusions and the experiments by Jerrold Schwaber and Edward Cohen at the University of Chicago. In 1973, these men had succeeded in producing a hybrid cell line able to secrete both myeloma and lymphocyte-derived antibodies by fusing human lymphocytes with mouse myeloma cells. These earlier techniques, however, had a major limitation. Crucially the antibody producing cells did not survive long and the antigens that the antibodies targeted remained unknown.
Milstein and Köhler wondered whether one way to resolve some of the difficulties encountered by previous researchers was to fuse a normal B cell from the spleen of a mouse immunised with a certain antigen, which itself produced an antibody with known specificity, with a mouse myeloma cell. By doing this they hoped to transfer to the antibody the trait of immortality from the myeloma cell. If it worked, they would have access to a hybrid cell, or hybridoma, not only capable of secreting antibodies with known specificity but also of surviving indefinitely.
Initially three different myeloma cell lines (labelled P3, 289 and P1) were chosen as the fusion partners for the spleen cells in the experiment. After a number of trials and errors, however, Köhler determined P3-X63-Ag8, the cell line used by Cotton, to be the most promising myeloma fusion partner.
Köhler chose to grow the fused cells in a hypoxanthine-aminopterin-thymidine (HAT) medium. The medium had first been used for the fusion of tumour cells back in the 1960s. Like Cotton before him, Köhler also added inactivated Sendai Virus to the culture as this was known to disrupt the membranes of cells and thereby promote the fusion process.
The antigen Milstein and Köhler decided to target was sheep red blood cells (SBRC) because the mouse's immune system was known to react vigorously against them. Moreover, antibodies against such cells could be easily detected by a plaque essay test, a procedure developed in 1963 that had become common in laboratories by the mid-1970s.
Milstein and Köhler's fusion project was well under way by December 1974. It was undertaken with the skilled technical assistance of Shirley Howe. Towards the end of the month Köhler could see a number of cells growing in the medium, but he did not know if any had the desired specificity for the SRBC antigen. Before he could test the mixture with the plaque assay, however, Köhler spotted contamination of the mixture.
With the cells no longer viable, Köhler was forced to start the fusion process again. By the end of January 1975 he finally had some cells for testing and he decided to perform the first plaque assay test for the experiment on January 24 1975. This involved testing cells he had cultivated four days earlier through the fusion of X63 with the cells taken from a mouse immunised with SRBCs.
Köhler started the plaque assay test at 5pm and returned home in the expectation that the process would take some hours before the results would materialise. A few hours later he returned to the laboratory bringing his wife as company to see what he anticipated to be boring results. Much to his amazement, Köhler spotted green halos on the two plates on which the two tests had been performed. He was so happy with what he saw that he shouted and kissed his wife! The test not only showed the hybrid cells were capable of secreting antibodies to SRBCs, but they produced large amounts. The frequency of antibodies was far greater than anything he or Milstein had ever predicted.
Following this success, Köhler and Milstein repeated their experiment twice more to check the technique was reproducible. When these experiments proved positive, the two scientists realised that they possessed a tool scientists had been striving to make for many years. Critically, they had created an immortal cell line capable of producing an endless supply of identical antibodies with known specificity. The method would later be dubbed 'hybridoma technology' and the antibodies it produced 'monoclonal antibodies' to signify the fact that they were derived from a single hybrid cell.
In May 1975 the two scientists submitted a paper announcing their experiment to Nature, one of the most prestigious scientific journals in the world. In this paper they pointed to the fact that their technique could have major benefits for both medicine and industry. Yet the importance of their achievement was missed by the journal's editors, who asked for the article to be shortened and failed to include it in the section reserved for findings considered to be of leading significance. In the end the article was published in Nature in August 1975.
In later years there would be much speculation as to who originally conceived of the experiment to generate the first successful monoclonal antibodies. Köhler and Milstein responded in 1984 when they were jointly presented with the Albert Lasker Basic Medical Research Award, stating publicly: 'both the conception and execution of the work was the result of close collaboration between us with the skilled technical assistance of Shirley Howe'. Milstein would later emphasise that monoclonal antibodies had been made as a tool for answering a basic scientific question and not for any particular practical application.
A crisis in reproducibility
Just at the point that Köhler and Milstein's paper was accepted for publication by Nature, the two scientists faced a major crisis. Suddenly their technique stopped working and they were unable to achieve any fusions. This came as a total surprise as it had worked on seven previous occasions. Increasingly concerned as subsequent attempts failed, Milstein began to wonder whether in fact they should withdraw their paper from Nature. With the validity of their results threatened, the two scientists faced six stressful months trying to fathom what had gone wrong.
Their anxiety was not helped by the very basic conditions in which they were performing their experiments. In his manuscript, entitled 'In the early days', Milstein's recalls:
'In the early days, the “tissue culture laboratory” was a room in the basement where the power switches of the laboratory were located. Effectively we only had one hood, and several feet of bench. The laboratory where we were working was so overcrowded, and I was so concerned about the bacteria which were being used all over the place that I preferred the situation, located four floors away from my own laboratory. It contained the electricity switch board so we were not allowed to have water services inside, or any other device which could mean flooding or splashing. It had no windows, the covering of the floor and painting of the walls was totally unsuitable. My only argument in its favour was a report I read somewhere saying that in a microbiological laboratory which had been suitably tested, the least contaminated area was found to be the lift shaft. How did my assistant Shirley Howe survive those conditions? I don't know. But I should point out that at one stage we became somewhat concerned about her health.' (Milstein's early manuscript for 'Messing about with Isotopes', presentation to Miami Winter Symposium, 1981, Churchill Archives Centre, Milstein Papers, file MSTN/D23).
Not only did Milstein and his team have to work in very basic and hazardous conditions, the technique for producing monoclonal antibodies required multiple steps and ingredients. While some of these procedures and ingredients could be carefully pretested and monitored, others could not.
The process for teasing out what had gone wrong was further hampered because by then Köhler had returned to the Basel Institute of Immunology. In the end, it would be Giovanni Galfré, a postdoctoral student who had recently joined Milstein, who identified the source of the problem – an incorrectly-prepared stock solution of HAT medium which was proving toxic to the cells.
Soon after identifying the problem with the culture medium, Galfré had to resolve another issue. This was the fact that the original preparations of Sendai virus were running out. He chose to resolve the shortage by using instead polyethylene glycol (PEG), a reagent that had been used successfully in other cell-cell fusions.
Following Galfré's changes, Milstein and Köhler's hybridoma technique began working again. In fact Galfré's introduction of PEG helped enhance the success of the method. Scientists now had a viable technique for generating immortal monoclonal antibodies on an unprecedented scale. Their arrival heralded a major advance over conventional polyclonal antibodies.
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