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The advent and rise of monoclonal antibodies

A 1975 Nature paper reported how cell lines could be made that produce an antibody of known specificity. This discovery led to major biological insights and clinical successes in treating autoimmunity and cancer.
Klaus Rajewsky is at the Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, 13125 Berlin, Germany.
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In their 1975 Nature paper1, the immunologists Georges Köhler and César Milstein described the production of monoclonal antibodies of predetermined specificity, each made by a continuously growing cell line that had been generated by the fusion of an antibody-producing cell from an immunized mouse with an immortal cancer cell specialized for antibody secretion. Hearing from César about this work before it was published, on the way to an obscure meeting in San Remo in Italy, I knew immediately that our research field had reached a turning point.

Antibodies were discovered in 1890 by the physiologist Emil von Behring and the microbiologist Shibasaburo Kitasato as protective antitoxins in the blood of animals exposed to diphtheria or tetanus toxin2. Ever since, antibodies have been a major research subject, given their key role in adaptive immunity (specific immune responses against, for example, invading disease-causing agents) and their wide range of specificities, essentially covering the universe of chemical structures. This had stood out from early on as a major genetic puzzle. How can our limited genome encode a seemingly limitless repertoire of specificities? And in medical (and industrial) practice, antibodies have been used ever since their discovery as the basis for serum therapy (the treatment of infectious diseases using blood serum from immunized animals), as diagnostic tools to monitor infectious disease, and in innumerable other contexts.

But antibodies specific for any given molecule (called an antigen in the context of an antibody response) came, with a few notable exceptions, as mixtures of antibodies, produced by thousands of antibody-producing cells in an immunized animal or infected person. Each of these cells produced an antibody of its own kind, so that ‘antibody specificity’ usually referred to the properties of antibody populations rather than those of individual antibodies. The inability to produce molecularly defined, homogeneous antibodies of predetermined specificity was a major hurdle that needed to be overcome.

Nature PastCast: Antibodies’ ascendency to blockbuster drug status

This changed overnight with Köhler and Milstein’s paper. Köhler had joined Milstein’s group at the MRC Laboratory of Molecular Biology in Cambridge, UK, as a postdoc, to study the mechanism of somatic mutation that operates in antibody diversification. The plan was to use mouse myeloma cells for this purpose. These are tumour cells originating from antibody-secreting immune cells. The cancer immunologist Michael Potter at the National Cancer Institute in Bethesda, Maryland, had shown years before that myelomas could be induced in a particular mouse strain by the injection of mineral oil3. The Milstein team was propagating and fusing to each other cells obtained from cell lines derived from various such tumours. However, the myeloma antibodies were ill-defined in terms of specificity. Could one perhaps fuse antibody-producing cells from immunized mice to myeloma cells, to produce continuously dividing cells that make antibodies specific for the immunizing antigen? To detect such fused cells, an approach offered itself which Köhler had become acquainted with during his PhD at the Basel Institute for Immunology in Switzerland and that had been developed by the institute’s director, Niels Jerne4. This was a simple technique in which cells secreting antibodies in response to, and specific for, sheep red blood cells (SRBCs) can be identified by the formation of a clearance (called a plaque)in SRBC-containing agar plates.

With this, the stage was set for the Köhler–Milstein experiment (Fig. 1). Large numbers of plaque-forming hybrid cells secreting anti-SRBC antibodies appeared when spleen cells from SRBC-immunized mice were fused with myeloma cells. The fused cells had acquired expression of a single type of anti-SRBC antibody from a spleen cell and preserved the immortality and high rate of antibody secretion of the myeloma fusion partner. Myeloma and spleen cells were unable to multiply under the chosen experimental conditions, and the myeloma cells apparently preferred antigen-activated spleen cells over others for fusion, a prerequisite for the striking success of the experiment.

Figure 1 | The production of monoclonal antibodies. Köhler and Milstein’s 1975 Nature paper1 solved the problem of how to generate clones of continuously dividing cells that make antibodies of a known specificity. The ability to generate such monoclonal antibodies revolutionized antibody research and paved the way to clinical advances. The authors injected mice with sheep red blood cells and isolated spleen cells, including those that produce antibodies. Different antibody colours indicate antibodies specific for different molecules (antigens), and produced by different cells. The authors had the idea of fusing antibody-producing spleen cells of limited lifespan with myeloma cells — immortal cancerous immune cells secreting antibodies of unknown specificity. Spleen cells that had been activated upon antigen recognition fused preferentially with the myeloma cells, generating hybrid cells called hybridomas. Unlike unfused cells, the hybridoma cells could grow on the selective agar plates used, and formed colonies of identical cells. Hybridomas that secreted antibodies specific for sheep red blood cells were identified by their ability to destroy such cells when added to the agar, generating a clearance (plaque). These original hybridoma cells made two types of antibody, one that recognized sheep red blood cells and another of unknown specificity.

The fused cells could be cloned and propagated indefinitely as what were later termed hybridomas, producing unlimited amounts of monoclonal antibodies. The first-generation hybridomas secreted two types of antibody: the desired one, plus an antibody of unknown specificity originating from the myeloma fusion partner. But this two-antibody problem was soon solved through the isolation of myeloma lines that had lost antibody expression5,6.

Antibodies against any desired antigen could now be generated, investigated and used as homogeneous molecular entities. In 1984, Köhler and Milstein won the Lasker Award together with Potter, and that same year Köhler, Milstein and Jerne were awarded the Nobel Prize in Physiology or Medicine.

The impact of the Köhler–Milstein paper on biomedical and, specifically, immunological research was dramatic, propelled by scientific developments that occurred around the time the paper appeared. Thus, it became clear shortly afterwards that the variable and constant regions of antibodies are encoded by separate gene segments. Antibody diversity arises when somatic recombination joins gene segments together, and when a subsequent process called somatic hypermutation operates, during the course of the antibody response, on the recombined gene segments encoding antibody variable regions. Together, these mechanisms generate a vast repertoire of antibody specificities, as well as distinct classes of antibody, which mediate their various roles (effector functions) through their differing constant regions.

These insights were accompanied by the explosive development of new molecular and genetic tools that allowed the isolation and manipulation of antibody genes in multiple ways. Together with the hybridoma technology, they fuelled a rapidly growing and still expanding field of investigation, in which basic research on antibody diversification and effector function goes hand-in-hand with the production and engineering of monoclonal antibodies for diagnostic and therapeutic purposes.

In the early days, the production of monoclonal antibodies was entirely based on hybridoma technology and used for two main purposes: to study the somatic evolution of the antibody repertoire and the molecular basis of antibody specificity; and to generate reagents that bind to specific proteins or other molecules expressed by cells of the body or by pathogens. In both cases, completely new insights and technical advances resulted. Thus, affinity maturation of antibodies (the increase of antibody affinity during the course of an antibody response) began to be understood at the molecular level. And the technique of fluorescence-activated cell sorting was revolutionized by monoclonal antibodies, allowing the separation of different cell types at an unprecedented level of specificity and resolution. Recent highlights in this area include approaches allowing gene-expression profiling of single cells that have been characterized by the expression of large arrays of surface-marker proteins through cocktails of DNA-tagged, ‘barcoded’ monoclonal antibodies7.

In medicine, monoclonal antibodies have an ever-increasing role and have generated a multibillion-dollar market, which is expected to grow substantially in the future. In addition to their impact on medical diagnosis, the therapeutic application of antibodies has led to spectacular successes in the treatment of autoimmune diseases and cancer. The 2018 Nobel Prize in Physiology or Medicine was awarded for the “discovery of cancer therapy by [antibody-mediated] inhibition of negative immune regulation”. As often happens in biology, both the mechanisms and the efficient induction of the inhibitory processes underlying this type of immunotherapy are still unclear, with ongoing research providing challenges and new perspectives that are driving the development of monoclonal antibodies against additional targets.

Monoclonal antibodies are also being developed to control infectious diseases — following the concept of protective antibodies that goes back to von Behring and Kitasato. Prevalent diseases such as malaria, influenza and AIDS call for the development of what are termed broadly neutralizing monoclonal antibodies, which, applied individually or in cocktails, might provide broad protection8.

Intensive work in this direction has yielded promising results, including engineering antibody specificity through the substitution of variable domains by ligand-binding domains from non-antibody receptors9. Yet the immune system itself uses similar tricks10 and, by and large, antibody design is still unable to outdo it in terms of generating and selecting antibody specificities11. Nevertheless, the manifold modern molecular, cellular and genetic approaches to selecting and engineering antibodies have had, and continue to have, a tremendous impact on the field, whether by producing partly or fully human antibodies of different classes, making bi-specific or toxin-conjugated antibodies for specific therapeutic purposes, or incorporating antibody variable regions into chimaeric antigen receptors on T cells for use in an anticancer treatment called CAR-T cell therapy.

Monoclonal antibodies are nowadays often generated by isolating or transforming antibody-producing cells taken directly from immunized animals or patients, and transplanting the antibody-encoding genes of these cells into suitable producer cell lines, rather than using hybridoma technology1214. But they started their spectacular career in 1975, secreted by hybridoma cells in Köhler and Milstein’s SRBC-containing agar plates.

Nature 575, 47-49 (2019)

doi: 10.1038/d41586-019-02840-w

References

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    Köhler, G. & Milstein, C. Nature 256, 495–497 (1975).

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    Behring, E. & Kitasato, S. Dtsch Med. Wochenschr. 49, 1113–1114 (1890).

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    Potter, M. & Boyce, C. R. Nature 193, 1086–1087 (1962).

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    Jerne, N. K. & Nordin, A. A. Science 140, 405 (1963).

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    Kearney, J. F., Radbruch, A., Liesegang, B. & Rajewsky, K. J. Immunol. 123, 1548–1550 (1979).

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    Galfrè, G, & Milstein, C. Methods Enzymol. 73, 3–46 (1981).

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    Todorovic, V. Nature Methods 14, 1028–1029 (2017).

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    Walker, L. M. et al. Science 326, 285–289 (2009).

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    Gardner, M. R. et al. Nature 519, 87–91 (2015).

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    Tan, J. et al. Nature 529, 105–109 (2016).

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    Verkoczy, L., Alt, F. W. & Tian, M. Immunol. Rev. 275, 89–107 (2017).

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    Scheid, J. F. et al. Nature 458, 636–640 (2009).

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    Steinitz, M., Klein, G., Koskimies, S. & Mäkelä, O. Nature 269, 420–422 (1977).

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    Traggiai, E. et al. Nature Med. 10, 871–875 (2004).

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Transcript

Nature PastCast: Antibodies’ ascendency to blockbuster drug status

Kerri Smith

This is the Nature PastCast, each month raiding Nature’s archive and looking at key moments in science. In this show, the beginnings of some blockbusters in the 1970s.

Music

Voice of Nature: John Howe

Nature, August 7, 1975.

Music

Lara Marks

From the late nineteenth century, scientists began to wonder whether antibodies could be the next magic bullet in medicine.

Greg Winter

I had no idea that it was going to have the impact that it has now. At that point, I don’t think anyone had realised the importance.

Voice of Nature: John Howe

Volume 256, Continuous cultures of fused cells secreting antibody of predefined specificity.

Lara Marks

Today, monoclonal antibodies are a very important part of the biotechnology industry, yet they’re very little known. It’s very interesting, if you mention to people the drug Herceptin, which is used for breast cancer, most people will have heard of that drug, but they will not understand that it’s actually based on a monoclonal antibody.

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Lara Marks

I’m Lara Marks. I’m a historian of medicine based at King’s College London. I’m working on a book on the history of monoclonal antibodies and their transformation of healthcare since the 1970s. The reason monoclonal antibodies are so useful is because they’re very particular about which cells they target and then attach to, and that means that they can be used very specifically.

Voice of Nature: John Howe

The manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. G. Köhler and C. Milstein, MRC Laboratory of Molecular Biology, Hills Road, Cambridge.

Lara Marks

César Milstein was the son of Jewish-Ukrainian immigrants in Argentina. He trained in chemistry and fled Argentina in the early 1960s after the political turmoil bought about by the military coup. He took up a position at the Laboratory for Molecular Biology in Cambridge in 1963. What I find fascinating is that everyone knows about Crick and Watson but don’t know about Milstein, and yet they were based in the same laboratory, although working a decade apart.

Music

Lara Marks

Once Milstein arrived at the Laboratory of Molecular Biology, he began investigating the mechanism behind antibody diversity. Like many other scientists at the time, he was puzzled about why it was that such an apparently almost identical group of proteins, the antibodies, could specifically target simultaneously any one of a multiple of foreign invaders like bacteria, viruses or pollen. Based on the theory that antibody diversity stemmed from a mutation in the DNA of antibodies, Milstein conducted experiments to validate that idea, and it was out of those experiments that monoclonal antibodies was born.

Greg Winter

So, my name is Greg Winter. I’m a scientist working at the MRC Laboratory of Molecular Biology, but now most of my time is spent of Master of Trinity College, Cambridge. My role in the story has been as a group leader working with Milstein as the Head of Division and picking up part of the antibody story that he started. Well, I started off at the MRC Laboratory of Molecular Biology in 1973 as a PhD student, and the first time I remember really meeting Milstein was when I was walking down the corridor and I saw what looked to me like my supervisor Hartley involved in some major argument with this chap who later turned out to be César Milstein, a small chap. They’d both got pipes and they would have these long periods where one of them would make a point and the other one, in struggling for an answer, would decide his pipe needed attention and puff it, bang it, ream it out, relight it, suck on it, and then continue the argument.

Lara Marks

Looking through Milstein’s papers, you get a very strong sense of a man who wanted to do the right thing for the world, who was very keen to improve the world but also was fascinated by basic science, and he really married the ability of pursuing basic science and then finding its application for practical uses. Georges Köhler arrived at the Laboratory of Molecular Biology in April 1974, and thereafter partnered with Milstein.

Music

Lara Marks

The creation of monoclonal antibodies involves several steps. In the first instance, an animal has to be immunised against a particular foreign substance or what is known as an antigen. The animal’s antibodies are then harvested from its spleen for fusion with a myeloma cancer cell to create what is known as a hybrid cell or hybridoma, and it is that hybridoma that secretes the monoclonal antibodies.

Voice of Nature: John Howe

We describe here the derivation of a number of tissue culture cell lines which secrete anti- sheep-red-blood-cell antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor.

Lara Marks

The test not only showed the hybrid cells were capable of secreting antibodies, but they produced large amounts.

Voice of Nature: John Howe

The above results show that cell fusion techniques are a powerful tool to produce specific antibody directed against a predetermined antigen.

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Lara Marks

In May 1975, they submitted a paper to Nature. They pointed out that their technique could have valuable medical and industrial uses.

Voice of Nature: John Howe

Such cells can be grown in vitro in massive cultures to provide specific antibody. Such cultures could be valuable for medical and industrial use.

Greg Winter

Of course, nowadays it seems entirely prophetic, but at that stage I think everyone was clear that quite a lot more work needed to be done.

Lara Marks

When they submitted it, Nature’s editors missed the ramifications and asked for the article to be shortened. Shortly, after submitting their article, Milstein and Köhler suddenly had a crisis because they could not replicate their technique. The crisis was so bad that Milstein considered withdrawing their article from Nature.

Greg Winter

Sometimes it worked and sometimes it didn’t. I think Milstein told me when they first started it, they got it to work. He said at some point that they got it to work and then they wrote the paper up and they got it into Nature, and then to their horror, they couldn’t get it to work again for a period of time, and it was an awful lot of fiddling around that they had to do.

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Lara Marks

The possibilities that monoclonal antibodies provided for medical and industrial uses was not only missed by Nature’s editors. The National Research Corporation, which was responsible for patenting research, funded by British councils, also did not see its commercial value.

Greg Winter

I hadn’t actually quite worked out what happened, but they never responded to César. Someone forgot to do it – these were very different days. César actually didn’t mind too much but he had actually done his bit. And what they said is, ‘Look, we don’t think it’s worth filing because the antibodies you’ve described were antibodies against sheep-red-blood cells, which aren’t really useful for anything. When you’ve got a useful antibody, tell us and we’ll file a patent on it.’

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Lara Marks

Well, the NRDC did not see any reason to pursue a patent. Scientists at an American institution known as the Wistar Institute, led by Hilary Koprowski, were quick to move on patenting monoclonal antibodies. Their patenting of the technique was to cause huge furore because in fact, their work was based on myeloma cell lines that had been sent to Koprowski by Milstein.

Voice of Nature: John Howe

Using suitable detection procedures, it should be possible to isolate tissue culture cell lines making different classes of antibody.

Lara Marks

The patents that Koprowski and his colleagues took out were for monoclonal antibodies against viral antigens and cancer. The NRDC’s decision not to patent Milstein and Köhler’s technique was to lead to a huge political storm in the late 1970s and early 1980s.

Extract from Margaret Thatcher speech

Mr President, my Lords, Ladies and Gentlemen…

Lara Marks

At the forefront of the debate was Margaret Thatcher, who was scandalised that such a technique that had been developed in Britain had been patented in America and was being commercialised there. The furore over the issue was partly fuelled by the fact that manufacturing was in decline in Britain.

Extract from Margaret Thatcher speech

In the 1940s when I took a science degree, the new emerging industries were plastics, mad-made fibres and television. Later it will be satellites, computers and telecommunications, and now it is biotechnology and information technology; and today our universities and science parks are identifying the needs of tomorrow. So there are new industries and new jobs in the pipeline. Because it is the spirit of enterprise that provides jobs. It is being prepared to venture and build a business.

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Lara Marks

Milstein quickly realised that for monoclonal antibodies to have an impact, he would need to collaborate with others to demonstrate their utility. Milstein began to get numerous requests for different cell lines to make monoclonal antibodies from his laboratory, and he was becoming so inundated with requests, he did not know quite what to do. By chance, in February 1977, Milstein answered the knock on the door of a salesman called David Murray, who was founder of Seralab.

Voice of Nature: John Howe

Seralab, monoclonal antibodies derived from hybrid myelomas.

Lara Marks

A company he had founded in the early 1970s to sell antisera as a laboratory agent to scientists in the research community.

Voice of Nature: John Howe

Remember that monoclonal means the same antibody against the same determinant every time.

Lara Marks

David Murray was an interesting entrepreneur. He had started life as a general manager for his father’s cabaret club in Soho. Murray was already aware of Milstein’s work, having read his paper in Nature, and was particularly interested in monoclonal antibodies because he saw is as a means of standardising the antisera products he was selling through his company. Murray quickly suggested that he form a collaboration with Milstein to distribute Milstein’s cells. In fact, it was the collaboration between David Murray from Seralab and Milstein that laid the basis for the very first commercialisation of monoclonal antibodies.

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Lara Marks

One of the first monoclonal antibodies developed as a drug was Orthoclone, which was approved in 1986 for the prevention for kidney transplant rejection. After the approval of Orthoclone, it would take a number of years, however, for other monoclonal antibody drugs to hit the market. One of the problems was that all the monoclonal antibodies at this point where mouse or murine monoclonal antibodies, and so could course immunoreactions in patients.

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Greg Winter

It took me more than a year with some help, but we managed to create this humanised antibody. At the same time, I was looking out for therapeutic possibilities because I thought, well, this looks to me like these things, as far as I can tell, are as close to human as we’re going to be able to get.

Voice of Nature: John Howe

Nature, 24th March, 1988. Reshaping human antibodies for therapy.

Greg Winter

César had been working with a young medic a year or two earlier who had been interested in making an antibody against T and B cells, and this chap was Herman Waldmann and he made an antibody called Campath antibody, and he was interested in using this as a blockbuster. So, we both went to talk to each other and we collaborated then on humanising the antibody that he had the right antibody and that was humanised Campath antibody, otherwise known as Alemtuzumab. I look back and I think I just don’t know how we did it.

Voice of Nature: John Howe

The Lancet. 17th December, 1988. A genetically reshaped human monoclonal antibody, Capath-H1, was used to treat two patients with Non-Hodgkin lymphoma.

Greg Winter

I got introduced to the patients, so I was allowed to go and see her and I went and had a chat and she was a lovely old lady. She said, ‘You must be very pleased about this.’ ‘Well, I am,’ I said, ‘but I’m also extremely worried.’ She asked me why I worried and I said, ‘Well, I hope it’s going to continue.’ I had no idea, none of us had any idea, whether this was going to be a short-term remission or longer, hadn’t a clue. She said, ‘Oh, it doesn’t matter dear. If it buys me a few months, that will be fine. My husband is dying and I want to be with him when he dies.’ I sort of choked.

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Lara Marks

With efforts to reengineer monoclonal antibodies, scientists now had to hand, by the early 1990s, a much safer and effective monoclonal antibody for therapeutics. Between 1997 and 2011, the Food and Drugs Administration approved between one and four new monoclonal antibodies per year. Amongst those antibodies were Rituxan or rituximab which was developed for non-Hodgkin’s lymphoma and today, it is the bestselling biological cancer drug in the world. In 1998, Herceptin was approved for breast cancer, which today again, is another blockbuster drug. Most people will have heard of that drug, but they will not understand that it’s actually based on a monoclonal antibody. Moreover, most people will not understand that monoclonals are not just being used for drugs which are becoming blockbusters but are also a vital component of our diagnostics today. For example, monoclonal antibodies exist in home-testing kits for pregnancy, ovulation, menopause, and equally at a global level, governments are using monoclonal antibodies in diagnostics for assessing whether we’re having pandemic flu. They’re also used, for example, in the diagnosis of AIDs and in other infection diseases.

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Greg Winter

It transformed what I thought about science. I thought to myself, well, I need to make sure that the work that I do in the future isn’t necessarily directed to immediate practical or medical gain, but I should be mindful of it.

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Lara Marks

As a historian, you’re meant to be cynical and you’re meant to say it’s all a revolution overnight, but it was revolutionary. It did transform things. Not immediately, but it did happen. When you look at monoclonals, it’s not hype, it really has transformed things but in a very quiet way.

Music

Voice of Nature: John Howe

Nature, volume 256. August 7, 1975. Cost: 45 pence.

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Kerri Smith

This Nature PastCast was produced by me, Kerri Smith, with contributions from historian Lara Marks and scientist Greg Winter. Next month in episode six of this twelve-part series, scientists in the 1960s begin to feel the Earth move.