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A brief history of the antibody – part III, monoclonal antibodies

March 8, 2011 at 4:39 pm | Posted in Antibody, Learning Centre | Leave a comment

By Deborah Grainger

This installment of the “A brief history of the antibody” series follows on from part II – which discusses César Milstein’s original research motivation and other events leading up to the development of monoclonal antibodies. I recommend that you read the two in close and chronological succession to get the most from them.

The first monoclonal antibodies

In the background of the somatic mutation work, another project in César Milstein’s lab, and the main research effort of one of his reasearch staff Richard G. H. Cotton, had successfully fused two myeloma cells in culture and the resulting hybrid cell co-dominantly expressed the antibody chains of both parent cells. This eventually proved to be more useful to the Milstein lab than anticipated.

By now, the somatic mutation work made it obvious the lab could not go on looking for mutants in the P3 cell line: they had looked for suitable antigenic binding activity of its antibody but were unsuccessful.  The previous fusion experiments of R.G.H. Cotton gave Georges Köhler an idea. His attempts to work with a myeloma cell line, which produced a protein displaying promising antigen binding properties, had been unsuccessful: “In my hand[s] they did not grow, so I had to think about another project”. Taking note of the myeloma fusion experiments, Köhler, along with Milstein’s assistant Shirley Howe, attempted to fuse a myeloma cell with an antibody producing B cell; they were successful, soon generating a mouse myeloma HPRT^- line. The resulting hybrid was designated P3-X63 Ag8 and was to become the most important production of Milstein’s laboratory [1]. Two more myeloma hybrids, or hybridomas as they became known (see box 1), followed the first; All three of the first monoclonal antibody clones were raised against different antigens of sheep red blood cells (SRBC), a convenient choice due to the simplicity and sensitivity of the SRBC lytic assays available.

Media coverage

It began to dawn on Milstein that the discovery of “á la carte” antibodies was probably far more useful than just answering questions concerning antibody diversity. He and Köhler wrote a manuscript reporting the early hybrid experiments and approached Nature [2]. The referees were cautious but positive; however, the editors at Nature did not consider the subject matter to be of sufficient general interest to publish the manuscript as an article: they instructed the authors to trim the original text somewhat to fit the length of a letter [1]. It appeared that the scientific community was not taking much notice and the breakthrough did not have the immediate impact later attributed to it. However, there were exceptions, and one of those was a scientific correspondent for the BBC, John Newell. He telephoned Milstein on the day the monoclonal paper was published and his ensuing report was broadcast by the BBC World Service the following week. In this report Newell commented on the potential applications of monoclonal antibodies, including their use in the diagnosis of disease [3] (see box 2). However, the National Research and Development Corporation (NRDC), which was at the time the only support option for MRC scientists wanting to protect their inventions and develop them commercially, did not agree with Newell. They did not see the possibilities the BBC broadcaster had seen, and in October 1976 advised the MRC as follows: “It is certainly difficult for us to identify any immediate practical application which could be pursued as a commercial venture…” It is easy to see how wrong the NRDC were in hindsight.

  

Demonstrating the potential of monoclonal antibodies

Unfortunately, after initial success the lab ran into technical difficulties (as always seems to be the case after progress) and could not reproduce their fusion experiments for at least 6 months [1]. Köhler too, who had recently returned to Basel, was unable to derive new hybridomas. If ever there was a lesson to check your stock solutions on a regular basis, this would feature in there somewhere: the lab had been hindered by the contamination of one of their solutions with a toxic substance. After identifying this problem, they developed a reliable protocol for producing monoclonal antibodies. Milstein continued in his efforts to derive antibody diversity and fortunately, a separate but related project in his lab also came into fruition; this helped the matter greatly. A research scientist named Pamela Hamlyn (now Rabbitts) was working on the adaptation of DNA sequencing for the sequencing of antibody light chain mRNA. Combining this work with the hybridoma technology allowed the Milstein lab to sequence the light chains of a large number of monoclonal antibodies, each raised against the same antigen at different times after immunization. This allowed the lab to correlate the mutation rate of antibodies over the course of antigen exposure with the increase of antigen affinity. This ultimately helped our understanding of the role of hypermutation in the affinity maturation of antibodies. Meanwhile, in the background of this success, Milstein and his colleagues still wished to demonstrate the greater practical importance of monoclonal antibodies in both basic research and clinical diagnosis. With several collaborators, the Milstein lab was able to show hybridomas could produce antibodies for Human Leukocyte Antigens, that hybridoma produced antibodies were ideal for the study of cell surface and tumor antigens and to provide reagents for cell fractionation. Furthermore, monoclonal abntibodies were useful for radioimmunoassays and neuropharmacology investigations as well as blood group reagents. If the regulatory body of the NRDC were slow to catch on to the applications of monoclonal antibodies, the academic community certainly was not; Milstein’s lab became inundated with requests for suitable myeloma cell lines and the fusion partners and applications of the hybridoma technique grew rapidly.

Antibodies for therapy

Of course, we now know that monoclonal antibodies have been indispensible to both basic research and medicine. They form the basis of a wide spectrum of assays and detection methods and have been used in therapy since 1986 (The first therapeutic antibody was Muronomab CD3 against T cell CD3 receptor for reducing the risk of transplant rejection). Their technology has continued to grow and develop and these advancements have paved the way for the modern production methods of therapeutic antibodies. The development of antibody phage-display libraries by John McCafferty in 1990 revolutionized how antibodies are selected for drug discovery. This technology allows fragments of antibodies to be displayed on phage; those that show the highest antigen binding affinity can be used to develop highly-specific antibodies. This approach, for the derivation of specific antibodies to a pre-defined antigen, circumvents the host animal and because it is based on human genes has been invaluable to the use of antibodies in therapy.

 “A desire to understand nature”

It appears the NRDC were wrong in their initial assessment of the potential of monoclonal antibodies, but I concede it is somewhat difficult to predict the ultimate worth of any development in basic science outright. Looking at it from another angle, the general consensus when assessing most innovations in hindsight is that one (almost always) automatically assumes their present purpose is what was originally intended by their development – when this is often far from the truth. At the end of his Nobel lecture in 1984, Milstein had this to say on the matter: “The hybridoma technology was a by-product of basic research. Its success in practical applications is to a large extent the result of unexpected and unpredictable properties of the method. It thus represents another clear-cut example of the enormous practical impact of an investment in research which might not have been considered commercially worthwhile, or of immediate medical relevance. It resulted from esoteric speculations, for curiosity’s sake, only motivated by a desire to understand nature.” It would seem that the story behind the development of monoclonal antibodies is not just for the history books of science, but a lesson for its future as well.

[1] César Milstein, “The hybridoma revolution: an offshoot of basic research”,  BioEssays (1999) 21.11, 966-73.

[2] Köhler, G and Milstein C, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature (1975) 256, 495-97

[3] Winter, GP., Antibody engineering, Phil Trans R Soc Lon, (1989) B 324, 537-47

Other related resources:

Georges Köhler, Nobel lecture “Derivation and diversification of monoclonal antibodies”  8 December 1984 (www.nobelprize.org).