Jump to ContentJump to Main Navigation
The Lock and Key of MedicineMonoclonal Antibodies and the Transformation of Healthcare$

Lara V Marks

Print publication date: 2015

Print ISBN-13: 9780300167733

Published to Yale Scholarship Online: January 2016

DOI: 10.12987/yale/9780300167733.001.0001

Show Summary Details
Page of

PRINTED FROM YALE SCHOLARSHIP ONLINE (www.yale.universitypressscholarship.com). (c) Copyright Yale University Press, 2018. All Rights Reserved. Under the terms of the licence agreement, an individual user may print out a PDF of a single chapter of a monograph in YSO for personal use (for details see www.yale.universitypressscholarship.com/page/privacy-policy). Subscriber: null; date: 16 January 2019

Hunting for the Elusive “Magic Bullet”

Hunting for the Elusive “Magic Bullet”

Chapter:
(p.1) Chapter One Hunting for the Elusive “Magic Bullet”
Source:
The Lock and Key of Medicine
Author(s):

Lara V. Marks

Publisher:
Yale University Press
DOI:10.12987/yale/9780300167733.003.0001

Abstract and Keywords

This chapter traces the history of the discovery of antibodies, from the late nineteenth-century theories of immunity to the development of monoclonal antibodies (Mabs) in 1975—a breakthrough that was made possible by the coming together of knowledge and techniques developed in many different geographic locations, laboratories, and disciplines, as well as at the bedside. The process was far from linear not only because of these logistical obstacles, but also because the evolving science was subject to the personal and theoretical rivalries among scientists. In many ways, the history of Mabs is as much about how substances, originally invisible to the naked eye, were imagined and then transformed into material entities.

Keywords:   Mabs, monoclonal antibodies, immunity, history

IN THE CLOSING DECADES of the nineteenth century, medical researchers, searching for a cure for infectious diseases, uncovered natural substances in the blood that seemed to act as very precise weapons against disease. Labeled “antibodies,” these substances aroused the hope that one day scientists would be able to harness their power and use them as “magic bullets” to fight these diseases without damaging healthy parts of the body.

The process of transforming antibodies into magic bullets was, however, beset with difficulties. For much of the twentieth century progress was slow, hampered not only by scientists’ inability to understand the immune system and how it produced antibodies, but also by their inability to isolate and purify individual antibodies from the billions produced by the body’s defense system. All of this changed in 1975 with the development of monoclonal antibodies (Mabs), a breakthrough discovery made possible by the coming together of knowledge and techniques developed in many different geographic locations, laboratories, and disciplines, as well as at the bedside. The process was far from linear not only because of these logistical obstacles, but also because the evolving science was subject to the personal and theoretical rivalries among scientists. In many ways, the history of Mabs is as much about how substances, originally (p.2) invisible to the naked eye, were imagined and then transformed into material entities.

The discovery of antibodies was built on years of medical and scientific inquiry into the nature of immunity and the development of techniques to fight infectious disease. Keen observers of epidemic diseases, such as pestilence and plague, had long noticed that individuals who suffered and survived one outbreak of disease appeared unscathed when it recurred.1 Such knowledge underpinned the development of vaccination against smallpox, and by the late nineteenth century doctors were using artificially weakened forms of a disease organism to confer immunity to that disease.2

Much of the early development of vaccines took place with little understanding of the immune mechanism that underlay their success. Until the late nineteenth century, most explanations rested on the belief that immunity stemmed not from a defense mechanism within the host, but from the infective agent itself. In 1883, however, Élie Metchnikoff, a Ukrainian biologist working at the Pasteur Institute, Paris, posited a new theory of immunity. He suggested that immunity occurred when white blood cells, known as phagocytes, sought out and ingested foreign invaders. This startled his contemporaries, particularly pathologists, who had hitherto believed that phagocytes contributed to the spread of disease by transporting foreign matter around the body. Nonetheless, Metchinkoff’s theory, later called “cellular immunity,” transformed our understanding of immunity, as scientists came to understand that immunity occurred due to processes taking place within the body, not outside of it.3

In the 1890s, this understanding of immunity took a new turn with the work of the German bacteriologist Hans Buchner, who discovered soluble components capable of destroying bacteria in blood serum. As a result, he argued that immunity was mediated by cell-free components, later defined as “complements,” floating in the blood, which he labeled “humoral immunity.” Buchner’s work set off an intense battle over whether cells or other blood-borne factors were more important in conferring immunity. As the immunologist and historian Arthur Silverstein has commented, the dividing line at times reflected the Franco-Prussian War, with French scientists defending Metchnikoff and German scientists promoting Buchner.4

(p.3) While these tensions continued into the early twentieth century, the newly emerging discipline of bacteriology began to provide another perspective on the disease process. For centuries, disease was thought to be caused by poisons from slimes and miasmas. In the late nineteenth century, however, bacteriologists began to discover poisons produced by bacteria, labeled toxins, that remained in the body even after the bacteria had died. Toxins were now seen as the cause of disease, and therapy moved from the destruction of bacteria to a concern with toxins. This approach took on a new importance following the work conducted by Henry Sewall, an American physiologist, and Albert Calmette, a French bacteriologist in the 1880s, who discovered that pigeons became resistant to relatively large amounts of rattlesnake poison if given gradually increasing amounts of the toxin beforehand.5

New ideas arose from the work of Emil von Behring, a German physician and physiologist. Steeped in military medicine and concerned with the treatment of wounds, Behring began to experiment with disinfectants, using iodoform on infections in animals. He discovered that while iodoform was incapable of killing pathogenic germs, animals exposed to it developed resistance to germs. Soon afterward, he noticed that anthrax bacteria could be destroyed in test tubes by adding serum taken from rats, animals known to be resistant to anthrax. This could not be repeated with blood serum from animals without anthrax resistance. Moreover, rat serum was ineffective against bacteria other than anthrax. Behring also found that blood taken from an animal previously exposed to a pathogen was more effective than blood taken from an unexposed animal.6

These observations led Behring to wonder whether something unknown in the organism was responsible for destroying bacteria while leaving other body tissues and organs unaffected. With this in mind, he and Kitasato Shibasaburō, a Japanese physician and bacteriologist, conducted further animal experiments at the Koch Institute in the summer of 1890, and discovered that blood serum taken from animals that had survived diphtheria or tetanus provided some protection when injected into animals with no previous exposure to such diseases. The serum also cured animals with diphtheria and tetanus. This evidence led them to hypothesize the presence of “forces” in the blood of the exposed animals, but not in those uninfected.7 Additional research indicated that toxins injected (p.4) into nonimmune animals remained in the animals’ blood and other bodily fluids long after their death. These findings confirmed the newly emerging theory of “humoral immunity” and opened up a radically new path for therapy.8

Soon after Behring and Shibasaburō’s findings were publicized, Paul Ehrlich, a German physician with expertise in structural chemistry, detected in blood a substance he called antibodies that seemed to confer immunity against plant toxins.9 He discovered that these antibodies were very specific—antibodies against ricin offered no protection against abrin. He observed that ricin and abrin toxins could not be distinguished by their toxicity but by the antibodies they generated, and noticed that antibodies responded in this way not just to bacterial toxins but to other toxins as well. Furthermore, he noted that large quantities of antibodies could be produced by only small amounts of toxin, and found that immunity developed six days after exposure to the toxin, but then remained for a long time.10

In 1897 Ehrlich published a theory that would transform understandings of the mechanism behind the body’s immune system and production of antibodies. Drawing on knowledge gained from his earlier investigation of dyes, he hypothesized that all cells possessed a wide variety of special receptors, which he termed “side chains,” and that these functioned like gatekeepers or locks for each cell. Each side chain, he argued, permitted entry of substances only with structures matching their own. Such side chains served two purposes. The first and primary one was to let nutrients into the cell; the second was to act as a defense mechanism when a cell was attacked by foreign substances, or “antigens.”11 Cells would also generate additional side chains when they encountered an antigen and these would break off to become antibodies that bound and neutralized free-floating antigens. He argued that each antibody possessed receptors designed to match specific antigens in the way that a key fits a particular lock. Ehrlich borrowed this metaphor from Hermann Emil Fischer, a German chemist and former colleague, to explain the specific binding of enzymes to substrates (Figure 1.1).12

Ehrlich’s theory was not without critics.13 One of the strongest was Jules Bordet, a Belgian immunologist and microbiologist based at the Pasteur Institute, Paris, who detected the presence of another substance when heating fresh serum containing antibacterial antibodies during the 1890s. (p.5)

Hunting for the Elusive “Magic Bullet”Hunting for the Elusive “Magic Bullet”

Figure 1.1. Paul Ehrlich, ca. 1908, with his depiction of his side-chain theory of immunity. Ehrlich believed that immune cells had a vast array of receptors (1), each specific to a particular substance (2). When a toxin interacted with the relevant receptor (3), the cell would be activated and would react by producing more receptors, which would then be released into the bloodstream as antibodies to neutralize the toxin (4).

(Photograph from Bildarchiv Bayerische Staatsbibliothek, Porträt-und Ansichtensammlung, Bild-Nr. Port-003494; illustration from P. Ehrlich, “Croonian Lecture: On Immunity with Special Reference to Cell Life,” Proceedings of the Royal Society of London 66 [1899]: 424–48)

Initially called “alexin” and later renamed “complement,” this substance appeared to act as an accessory to antibodies in destroying antigens. Bordet’s finding sparked an intense debate over the nature of the interaction between antibodies and their complements.14

The accuracy of the details of Ehrlich’s theory was debated well into the twentieth century. Nonetheless, Ehrlich had laid the basis for a new understanding of immunity. His hypothesis not only explained the origin of protective antibodies, but also powerfully depicted, in words and later in diagrams, how antibodies functioned. Their ability to target precisely certain chemical groups on specific molecules led Ehrlich to prophesy that one day antibodies would be developed that, like “magic bullets,” could seek out and destroy specific disease-causing microorganisms without harming the rest of the body. The term “magic bullets” came from Weber’s romantic opera Der Freischütz (The marksman), in which (p.6) a man sells his soul to the devil in exchange for magic bullets with which he hoped to win a marksmanship contest to gain a lady’s hand.15 Although Ehrlich soon turned to organic arsenic compounds for therapeutics, his dream about antibodies was to inspire other scientists. In 1901, Behring was awarded a Nobel Prize, followed by Ehrlich and Metchnikoff in 1908. Many mysteries about antibodies, however, lingered.16

A key question was when and how antibodies formed in the body and acquired their ability to bind to particular antigens. For much of the early twentieth century there were two competing schools of thought on this question, neither of which appreciated the importance of the other’s work. Each view was shaped by a scientist’s particular discipline training and outlook. On one side were the biologists who were interested in unraveling the interaction of the antigen with the cell and the implications this had for understanding the biological phenomenon behind the antibody response. On the other were the chemists, whose preoccupation with structural and quantitative relationships made them keen to determine the size of the antibody repertoire and the mechanism which established the specificity of antibodies.17

During the early twentieth century Ehrlich’s side-chain hypothesis held center stage. According to this view, antibodies existed in the body independently of any exposure to antigens. Each antibody, Ehrlich argued, had a unique three-dimensional configuration with certain functional domains and affinity. This hypothesis, later known as the “selection theory,” suggested that antigens selected antibodies with compatible receptors. Ehrlich’s idea was soon challenged, however, by the “instruction theory,” which portrayed antibodies as completely new entities, formed as the result of some form of an antigen template. Antigens were thought to interact and impress their specificity on normal substances, which in turn became antibodies. Champions of this idea were Karl Landsteiner, an Austrian biologist and physician based initially in Holland and then New York, and Oskar Bail, a German hygienist, bacteriologist, and immunologist at the German University in Prague. Doubts about Ehrlich’s notion of a preexisting repertoire of antibodies strengthened after 1917 as a result of Landsteiner’s experiments, which demonstrated that the body could produce antibodies against new synthetic antigens. Scientists wondered how, if Ehrlich’s model were true, the body could prepare in (p.7) advance effective antibodies to these novel antigens, as well as the wide diversity of antigens encountered during a lifetime.18

A vehement critic of Ehrlich’s theory was Ludwick Fleck, a Polish microbiologist and immunologist. During the 1930s he rejected the then growing consensus that antibodies were chemical substances, arguing that a body’s defense did not rest with any individual type of molecule like an antibody, but was inherent in the global property of serum. He was particularly dismissive of the “lock and key” concept, believing it oversimplified the host-pathogen interaction. From his perspective, the interaction between host and parasite was not to be considered as “attack” and “defense,” but rather as a process “akin to development, ageing or cyclic fluctuations in life cycles of parasites and bacteria.”19

Debates about antibodies and their purpose entered a new phase during the 1930s, spurred on by new quantitative analytical methods and new biochemical techniques, notably the introduction of ultra-centrifugation and electrophoresis. These helped shift the concept of antibodies from an “ill-defined set of serum activities” to definable protein molecules. It now became possible to describe the chemical structure of antibodies and antigens, and to establish how they bound to each other in molecular terms.20

At the forefront of the new research was Linus Pauling, an American chemist at the California Institute of Technology (Caltech). In 1940 he postulated that the binding of an antibody to an antigen was determined by the molecules’ shape rather than their chemical composition. Echoing Ehrlich’s earlier notion that antibodies and antigens worked together like a lock and a key, Pauling suggested that the structure and specificity of an antibody was molded by its physical interaction with a particular antigen rather than by its chemical composition.21

By the 1950s many scientists had become dissatisfied with the theories so far proposed because none adequately accounted for the simultaneous diversity and specificity of antibodies. In 1955 Niels Jerne, a Danish physician and immunologist then involved in serum measurement and standardization at the Danish National Serum Institute and for the World Health Organization, published a new theory. Reflecting his strong mathematical grounding, Jerne questioned the supposition underlying many contemporary theories: that there existed an infinite number of antibodies (p.8) and antigens. He argued instead that initially mammals possess a small repertoire of antibodies in their blood, and that copies of antibodies are produced as a result of the successful binding of an antibody to an antigen. Jerne developed this idea, which he called the “natural selection theory,” as a result of his exploration of the differential binding between antibodies and antigens, which had revealed that a single antibody could bind to many antigens. Borrowing the lock and key metaphor, he explained that keys did not have to fit 100 percent perfectly to open a lock.22

Initially, Jerne’s hypothesis attracted little support because scientists could not see how a protein such as an antibody could replicate itself. Within two years, however, Jerne’s insights provided the foundation for what was later known as “clonal selection theory.” This theory was formulated independently by David Talmage at the University of Colorado and Frank Macfarlane Burnet at the Walter and Eliza Hall Institute of Medical Research in Melbourne. Like Jerne, Talmage argued that a small number of antibodies could distinguish between a larger number of antigen determinants, and stressed the importance of differentiating between an antiserum containing many different specificities and the individual antibodies it contained. Overall, clonal selection theory showed that the cell provided the mechanism for multiplying antibodies. Awarded a Nobel Prize in 1960 for his part in the formulation of clonal selection, Burnet surmised that the body possessed certain cells dedicated to making antibodies, and that these cells were where antibody diversity was generated, stored, and expressed. In this context, the antibody repertoire was produced by cells naturally, without any dependence on external antigens, and this repertoire was encoded by a small number of genes that were in place during the fetal stage of development and could expand through somatic mutation.23

The principle underlying clonal selection theory was soon supported experimentally. In 1958 two scientists, the molecular geneticist Joshua Lederberg and the biologist Gustav Nossal, published results of an experiment, originally launched to disprove the theory, that instead confirmed that one cell was responsible for the production of just one type of antibody.24 The following year Lederberg elaborated the genetic framework described by Burnet, and showed the existence of a specific antibody gene that could mutate rapidly to a full repertoire, a process that took place not only during fetal development but throughout life.25

(p.9) The clonal theory echoed the earlier findings of Astrid Fagreaus, a Swedish immunologist who had discovered in 1948 that antibodies were generated by B cells, a type of white blood cell in bone marrow.26 Further research by Jacques Miller and Graham Mitchell in the early 1960s confirmed the view that bone marrow generates antibody cells (bone marrow lymphocyte B cells), with help from cells in the thymus (thymus lymphocyte T cells).27

Clonal selection explained not only antibody formation, but also observations made since the early twentieth century that antibody responses to a particular antigen were exponentially higher and faster after the initial encounter. Additionally, it appeared to provide a clue to immunodeficiency diseases as well as the mechanisms underlying auto-immunity and selftolerance that had been puzzling scientists since the early twentieth century.28 Overall the theory helped bring together the humoral and cellular theories used to explain the function of the immune system as a whole.

As theories about antibody formation twisted and turned, the application of antibodies to clinical problems was taking on a life of its own. By the mid-twentieth century antibodies had become a major tool in medical treatment in the form of serum therapy. The foundation for this work went back to Behring, who had started to apply his antibody discoveries to find a cure for diphtheria in the 1890s. Known colloquially as “the strangler of children,” diphtheria was a pressing concern, claiming the lives of around fifty thousand German children annually. By the summer of 1891, Behring had demonstrated the therapeutic possibilities of blood serum taken from animals immunized against diphtheria, but was unable to develop a supply of strong enough serum in high enough quantities to treat humans. To resolve this problem Behring entered a partnership in 1893 with Ehrlich and the pharmaceutical manufacturer Farbwerke Hoechst. Within a year they had developed a standardized, potent serum from horses that proved clinically safe for use in humans. Their success was based on Ehrlich’s observation that a toxin needed to be injected over a long period and in steadily increasing doses to secure a high potency of antibodies in animal serum, and that this potency varied over time. The trick was to capture an animal’s serum when the antibodies had reached their maximum strength.29

The new serum therapy helped to decrease diphtheria mortality from 50 to 25 percent after its introduction to Paris, offering great hope in (p.10) eliminating a much feared disease. Next Behring turned his attention to developing similar therapies for tetanus and streptococcal infections. By 1896 Hoechst was marketing tetanus serum for immunizing humans and animals. Yet demand for the therapy remained small, reflecting the low incidence of tetanus. Efforts to develop serum therapies against streptococcal infections and tuberculosis also had little success.30

Optimism about serum therapy was reignited with the successful emergence of a treatment for meningococcal meningitis, an epidemic disease that was taking lives across the world in the early twentieth century. The treatment, which reduced mortality by half, resulted from the separate efforts of the German physician Georg Jochmann and the American physician and pathologist Simon Flexner. By the late 1920s antipneumococcal sera had also become a central component of pneumonia control in six U.S. states. This was facilitated by a precipitation technique developed by Lloyd Felton, a scientist at Harvard Medical School, which enabled greater purification of antibodies in serum.31

By the 1930s serum therapy had become the choice treatment for many infectious diseases. In addition to those already mentioned, it included treatments for erysipelas, scarlet fever, whooping cough, anthrax, botulism, gas gangrene, brucellosis, dysentery, tularemia, measles, poliomyelitis, mumps, influenza meningitis, and chickenpox. Serum therapy was not without its side effects, however. Labeled “serum sickness,” symptoms included fever, rashes, joint pains, and sometimes anaphylactic complications. Behring observed such problems as early as 1893. Almost all patients receiving serum therapy manifested some form of adverse reaction, even if mild. These were attributed to the fact that serum preparations were drawn from animals, so contained proteins foreign to humans.32

Over the years, scientists tried various solutions. Early on, Behring discovered that water, salts, proteins, and ferments could break down diphtheria serum, and developed a fractionation technique that separated the antibodies found in the serum. This method enriched the antibodies in the serum, thus lowering the dosage needed and decreasing the frequency of side effects. Reactions in patients were also significantly reduced with the introduction of Felton’s precipitation technique in 1924 and the application of ultra-centrifugation and electrophoresis from 1939, all of which provided more purified antibodies.33

(p.11) While preparations from animal serum were being improved, human serum also began to be investigated. By the second decade of the twentieth century, two French physicians, Charles Nicolle and Ernest Conseil, had shown that it was possible to induce immunity against infectious diseases like typhus and measles by using serum taken from patients convalescing from these diseases. In 1920 it was found that patients who recovered from diseases like measles retained protective antibodies into adult life. Use of human serum for treatment nonetheless remained constrained because large quantities of serum were required. In 1933, however, Charles McKhann and his colleagues at Harvard devised a new technique of obtaining purified antibodies from human placentas. This not only ensured greater purity, but also a more highly concentrated source of antibodies than before. The development of new fractionation methods deployed by Edwin Cohn in 1944 for military blood supplies led to further progress. It enabled the production of concentrated gammaglobulin, a class of proteins discovered in 1939 to contain most of the antibodies in human blood. Cohn’s approach made it possible to give much smaller doses of human serum, which reduced the risk of serum sickness. His method was adopted quickly for the prevention and treatment of measles.34

Alongside efforts to improve the purification of animal and human serum, artificial antibodies were explored. As early as 1894 Behring hypothesized that it might be possible one day to produce antibodies “without the aid of an animal body.” Many scientists subsequently attempted to make antibodies in vitro. By 1929 at least ten successful attempts to create artificial antibodies had been reported, and a hundred clinics and research laboratories were thought to be active in this area. An application for a patent for an artificial diphtheria antibody was also filed in Germany during this period.35

The drive to make artificial antibodies gathered momentum in the 1940s with the work of Pauling, who established a research program at Caltech in 1941 for this purpose, funded by the Rockefeller Foundation. Within a year he had announced the successful production of an artificial antibody and applied for a patent, establishing its commercial exploitation with Lederle Laboratories. Excitement for the project soon dissipated, however, when other scientists, notably Landsteiner, found it impossible to reproduce Pauling’s results.36

(p.12) The inability to make artificial antibodies was particularly disappointing given the difficulties of standardizing animal and human serum and the expense of serum production. Because animals and humans cannot be stimulated to produce specific antibodies, their serum contains thousands of different antibodies, each differing in affinity and specificity. Serum varies not only batch to batch, but between the particular animals or humans, because of the wide differences in exposures to toxins over a lifetime. These variables mean that each new batch requires extensive characterization and testing. Furthermore, serum preparations required intravenous injection, demanding considerable expertise from the physician. Not surprisingly, then, interest in serum therapy dwindled with the arrival of sulphonamides in the 1930s and then penicillin in the 1940s. These medications proved not only easier and cheaper to produce but also more straightforward to administer and less toxic. They were also far more effective against infectious diseases.37

More success was to be had in the use of antisera for diagnostics. Scientists had begun to investigate the use of antibody-containing sera for diagnostic tests since the late nineteenth century based on the observation that antibodies could disintegrate (lyse), separate (precipitate), or clump together (agglutinate) bacteria within a solution. With antigens and antibodies found to have predictable biochemical reactions, physicians soon deployed antisera as diagnostic probes to define, isolate, and measure a wide variety of immunological molecules. The first was the Widal test, which was introduced in 1896 for the diagnosis of typhoid. This was followed by the Landsteiner test for blood grouping (1900–1901), and the Wasserman test for detecting syphilis (1906). Many others followed, one of the most important being the Coombs anti-globulin test developed in 1945, which was used to detect antibodies causing the premature destruction of red blood cells. It proved important for both blood transfusions and detecting Rh incompatibility between mother and fetus. By the 1960s, the use of antisera for diagnostics was widespread in clinics and hospitals, aided in part by the emergence of electrophoresis and labeling techniques, which employed first enzymes and then radioisotopes.38

Despite the success of serological diagnostics, in the years after the Second World War many clinicians perceived immunology and its application as an auxiliary medical discipline of little relevance. During the 1950s, however, a deepening knowledge of the chemical structure of antibodies (p.13) and the mechanism behind their diversity provided a new avenue for improving the clinical utility of antibodies for diagnosis and therapy. A key to the antibody enigma came from an unexpected source: myeloma cell lines (also known as plasmacytomas). As early as 1951, while studying the blood of patients with myeloma (a type of cancer that develops from plasma cells in the bone marrow), Henry Kunkel, an American immunologist based at the Rockefeller Institute, New York, unexpectedly discovered that myeloma proteins resembled normal antibodies and that malignant plasma cells of multiple myeloma produced just one abnormal antibody. This contrasted with normal plasma cells, which produce a large array of antibodies. His observation, and the fact that each myeloma cell was identical and fairly easy to obtain in large quantities from blood or urine taken from multiple myeloma patients, led him to investigate myelomas as a model for normal antibodies. Soon Kunkel and his colleagues had unraveled the chain structure of myeloma proteins and divided them into different classes and subclasses.39

The use of myeloma cells for investigating normal antibodies spread beyond Kunkel’s laboratory, following a major advance in the production of such cells by the molecular biologist Michael Potter and colleagues at the National Cancer Institute in Bethesda from the late 1950s. Potter had found, serendipitously, 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. The method provided for the indefinite growth of myeloma cells in mice on an unprecedented scale. By 1962 Potter and his team had established a collection of myeloma cell lines for distribution to researchers around the world. Access to these cells was enhanced by various researchers, most notably Kengo Horibata and A. W. Harris under the supervision of Melvin Cohn at the Salk Institute in San Diego, who adapted Potter’s mouse myelomas to grow in tissue culture. With a ready supply of myeloma cells, scientists could much more easily investigate the normal immune response.40

Research into the immune system was further enhanced by the appearance of another tool in 1963: a plaque assay that helped scientists to see and count the antibody-producing cells with their naked eye. Devised by Niels Jerne at the University of Pittsburgh, with his postdoctoral researcher Albert Nordin, this technique provided a means to determine how many antibody cells were involved in the antibody response.41 (p.14) Theo Staehelin, a fellow colleague of Jerne and Nordin at Pittsburgh, who witnessed the test in its formative stage, recalled Niels walking “one day in November 1962 into our lab. He held a beaker with a turbid, dilute suspension of sheep erythrocytes in his hand and asked … [rhetorically] whether one might see the difference upon lysis of the cells. On the bench, I had a bottle of the detergent sodium desoxycholate (10%) which we used to solubilize rat liver microsomal membranes in order to liberate membrane-bound polysomes. With a pipette, I added a few drops to the red cell suspension under slight stirring. Within seconds, the turbid solution turned transparent like a light red wine. Niels looked quite pleased…. Just a few weeks later, Niels and Al Nordin announced and showed around in the department the result of a single experiment whose simplicity, beauty, and significance excited everyone. It was the “Plaque Formation in Agar by Single Antibody-Producing Cells.”42 The technique for viewing the antibodies spread rapidly to laboratories all over the world, and during the next two decades was cited in publications more than four thousand times.43

During the 1960s antibody research entered a new era as a result of the work of two scientists: Gerald Edelman, an American biologist and former student of Kunkel based at the Rockefeller Institute for Medical Research in New York City, and Rodney Porter, an English biochemist working initially at the National Institute for Medical Research in Mill Hill, London, and then at St. Mary’s Hospital in London. Each scientist was interested in deciphering the structure of antibodies to answer the long-standing question of how a group of antibody proteins that seemed almost identical could simultaneously target any one of a multitude of antigens. By early 1962 they had independently shown that the structure of antibodies consisted of heavy and light protein chains, which joined together to form three sections yielding a molecule shaped like the letter “Y.”44

The work of Edelman and Porter, which led to their being awarded a Nobel Prize in 1972, inspired further investigations into antibody structure across the world. By 1969 the amino-acid sequence of human antibodies had been unraveled, a detailed picture of how the antibody worked had been built up, and its composition of both constant and highly varying regions had been revealed. Central to these developments was the collaborative analysis of amino-acid sequences carried out by a young German (p.15)

Hunting for the Elusive “Magic Bullet”

Figure 1.2. Basic structure of an antibody

postdoctoral fellow Norbert Hilschman, together with Kunkel and the American chemist Lyman Craig at the Rockefeller Institute. By the end of the 1960s, they had determined that while the upper regions of the “Y,” each made up of a light chain paired with a long chain, varied between antibodies, the stem region of the “Y” shape, which is composed of the two long heavy chains, was constant (Figure 1.2). The top arms of the “Y” provided unique binding sites designed for each specific antigen, whereas the stem bound together other components required for attacking such targets. Finally scientists had an explanation for what had puzzled them so long. Variations in the amino-acid sequence of individual antibodies in the upper regions of their “Y” shape was responsible for their multiple binding shapes, which in turn enabled their attachment to different antigens.45

The foundation in 1969 of what was to become the world’s largest institute of immunological research in the 1970s, the Basel Institute of Immunology (BII), led to the further investigation of antibodies. The BII was one of a number of basic research institutes established and funded by the multinational pharmaceutical company F. Hoffmann–La Roche (p.16) in this period to keep the company abreast of developments in biology, cell biology, and biochemistry. Championed by Alfred Pletscher, the company’s director of medical research, the BII officially opened its doors in 1971.

By this time, scientists already knew a great deal about the immune system and how it worked, but questions remained. The BII’s overall objective was to conduct basic research into immunology, with a particular emphasis on the “molecular, cellular, genetic and regulatory problems of antibody formation and antibody structure and function.”46 Directed by Jerne, a major founding member, the institute rapidly became an international hub of collaborative research. Its turnover of scientists was kept deliberately high in order to keep research and ideas flowing. Theo Staehelin, one of BII’s first permanent members, described the institute as “the mecca of immunology.” Its influence extended well beyond its physical boundaries. Its Annual Reports soon provided the largest circulation of immunology news in the world, and many of its visitors, who spent their formative scientific years there, later became internationally renowned immunologists.47

In 1974, Jerne electrified immunological research with the publication of his “idiotypic network theory.” Jerne aimed to provide a blueprint for understanding the regulatory mechanism governing the immune system, looking specifically at how stimulatory and suppressive factors were balanced. His central tenet was that there was “a vast number of immune responses [in the body that were] … going on all the time, even in the absence of a foreign antigen.” This meant that antibodies primarily responded to each other, and treated external antigens as subordinate, merely disturbing the equilibrium normally existing between “idiotypes,” a term first coined by the French immunologist Jacques Oudin in 1966 to describe the unique antigenic regions of individual antibodies that elicit an antibody response. Importantly, Jerne argued that antibodies reacted not only to foreign antigens, but also to self-constituent antibodies, which could explain the paucity of autoimmune responses.48

Not everyone was convinced by Jerne’s theory. Many doubted whether it could indeed be tested empirically. In time, regulatory elements other than the idiotypes highlighted by Jerne proved to have a more prominent role in immune regulation, diminishing his theory’s relevance. Nonetheless, it immediately galvanized research, setting off the equivalent of what (p.17) the historian Anne-Marie Moulin has called a “Copernican revolution” in the field of immunology. Critically, it stimulated new experimental research dedicated to unraveling the nature and consequences of network regulation and to exploring antibody diversity.49

While the immunological community was impressed by Jerne’s new theory, the emergence of a more practical development excited them even more. This was the development of a technique to create hybrid cells capable of secreting monoclonal antibodies, each identical (clones) and derived from a single (mono) kind of lymphocyte B cell.

Scientists had experimented for years with antibodies taken from antibody-containing blood sera, using them as highly selective and sensitive reagents for the structural analysis of a wide variety of antigens. The information gained from these experiments was limited, however, by the inherent heterogeneity of antisera, which could result in crossreactions during the testing process. Devising single antibodies with known specificity to particular antigens was therefore a highly significant goal. The idea that someday it might be possible to adapt the myeloma line to secrete limitless amounts of antibody was first put forward in 1967 by Melvin Cohn. The task was not easy. One of the difficulties was finding a way of isolating an individual antibody from the billions produced by the immune system. The closest scientists had come to realizing this ambition was through the use of myeloma cells. But while myeloma cells provided an abundant source of single antibodies, it was not known which antigens they targeted. Part of the problem is that myeloma cells are triggered by malignancy, a process that hits cells at random. Attempts to induce tumors to produce antibodies to an injected antigen had also so far failed. Myeloma cells on their own were therefore unsuitable for experimental studies exploring the molecular basis of antibody specificity.50

Methods to create individual antibodies with known specificity were to be given a boost by new techniques for the fusion of cells developed in the 1960s. Cellular fusion had been of interest ever since the nineteenth century to both cellular biologists and geneticists. Research in this area took off in a new direction in 1960 when Georges Barski and colleagues at the Institut Gustave Roussy in Villejuif, France, spotted cellular fusion between two different tumor cell lines that had been taken from two different inbred strains of mice and grown as a cell mixture in tissue (p.18) cultures. This research quickly attracted international attention because it raised the hope that a new technique for cellular fusion could now be devised to replace time-consuming breeding methods. Crucially, the technique opened the way for more genetic analyses in mammals, particularly the investigation of mutated genes responsible for heritable human diseases. Inducing cellular fusion initially proved difficult. From 1962, however, the process became easier through the introduction of the Sendai virus to promote fusion, the use of myeloma cells, and the adoption of the selective hypoxanthine-aminopterin-thymidine (HAT) medium to separate the fused cells. Soon cellular fusion was being undertaken by scientists on a large scale, with many of them successfully fusing two different mammalian species: human and mouse.51

One of the earliest scientists to exploit the new methods to produce single antibodies with known specificity was Joseph Sinkovics, a Hungarian clinical pathologist and laboratory clinical virologist based at the M.D. Anderson Hospital in Houston. Starting his research in the mid-1960s, he successfully developed a cell line of antibodies with known specificity that could be grown indefinitely by fusing antibody-producing plasma cells with lymphoma cells. Such antibodies could be grown in continuous cultures in spinner bottles or as ascites tumors in mice.

Sinkovics and his team were unable to take his research further, however, due to a lack of funding. At the same time, in the early 1970s, at the National Institute of Medical Research in Mill Hill, London, Brigitte Ita Askonas, an Austrian-Canadian biochemist who had just spent a year at the BII, and the immunologists Alan Williamson and Brian Wright found a way to clone B cells (single antibodies with known specificity) in vivo, using spleen cells from mice immunized with haptenated carrier antigens.52 Their work was part of a wider project to understand the process underlying the generation of B cells and antibody diversity. While their work revealed many aspects of B cells, the antibodies had a major drawback—they survived for only a short time.53

The successful development of antibodies in Texas and London was largely ignored. What attracted greater interest was a splenic fragments culture technique devised by Norman Klinman, an American immunologist based at the University of Pennsylvania with an attachment to the Wistar Institute, an independent research institution internationally renowned for its development of vaccines against polio, rabies, and rubella. (p.19) Klinman’s technique, which was published in 1969, provided a powerful tool for the isolation of single B lymphocytes secreting a single type of antibody. The method involved irradiating mice spleens, thereby destroying their antibody-producing capability, then injecting them with new antibody-producing cells, some of which lodged in the spleen. Cubes of the spleen were then grown individually in tissue culture with an added antigen. If a given fragment had an antibody-producing cell, it would produce antibodies, called “monofocal” antibodies, that were very specific to that particular antigen.54

One of the first to adopt Klinman’s technique was Walter Gerhard, a Swiss-trained physician who moved from the BII to take up postdoctoral research under Klinman in the early 1970s. He was trying to develop antisera as a tool to understand the antigenic structure of hemagglutinin, a glycoprotein found on the surface of influenza viruses that was considered an important mechanism in the recurrence of influenza in man. Frustrated by the heterogeneity of the antisera he was using, Gerhard was looking for a means to improve them. Klinman’s technique offered him a major way forward.55

By 1975 Gerhard had successfully cultivated monofocal antibodies with known specificity against influenza viruses. Some of his best hybrid cells produced between two hundred and three hundred nanograms of monofocal antibodies. This was a sufficient quantity to test different strains of the influenza virus and to determine immune responses to influenza. Disappointingly, however, Gerhard’s hybrid cells began to decrease their secretion of antibodies after thirty to forty days and usually petered out altogether after ninety days.56

At the same time, Ron Levy, an oncologist at Stanford University and former colleague of Klinman, deployed the method to produce tumor antigens in order to find a way of improving cancer diagnostics and treatment. With Klinman’s technique, Levy recounted, “We got really highquality antibodies but only for a short period of time. The cells would die, and then we would have to make them again.” Overall, too, the technique yielded very few antibodies, so it was unsuitable for use as a long-term diagnostic and therapeutic tool.57

More progress was made at the Laboratory of Molecular Biology (LMB) in Cambridge, England, by the Argentinian-born British immunologist César Milstein and the German postdoctoral biologist Georges Köhler who (p.20) joined him from the BII in 1974. Both were interested in finding mutant genes in the variable region of antibodies that bind to antigens, and they shared the belief that this approach was important to understanding the process of somatic mutation (the genetic alteration of cells after conception), which they believed underlay the diversity of antibodies. Milstein and researchers elsewhere had already carried out some groundwork for this research. But they faced a laborious hunt for the mutant genes because they lacked an antibody with defined specificity. Given that an antibody with clearly defined specificity to a target would provide the most effective means of detecting the slight differences caused by such mutations, Milstein and Köhler quickly turned their attention to devising one.58

To achieve their goal they decided to use tissue culturing and a hybrid cell line that Milstein had created with his postdoctoral researcher Dick Cotton by fusing a human lymphocyte with two myelomas (one from a mouse and the other from a rat). The significance of the cell line was that it could express antibodies to the parental cells. Milstein and Köhler also drew from the work of Jerrold Schwaber and Edward Cohen based at the University of Chicago, who in 1973 had produced a hybrid cell line able to secrete both myeloma and lymphoctye-derived antibodies through the fusion of human lymphocytes and mouse myeloma cells.59

In order to overcome some of the drawbacks of previous efforts, Milstein and Köhler tried to create a hybrid cell by fusing a myeloma cell with a normal spleen cell taken from immunized mice. This they hoped would generate an immortal cell line capable of secreting antibodies with known specificity. As Milstein explained, “We would be applying the wellestablished cell-fusion technique to a new purpose, namely to fix in a permanent cell line a function that is normally expressed only in a ‘terminal’ cell: the plasma cell derived from a B lymphocyte stimulated by an antigen.”60

The antigen they decided to target was sheep red blood cells (SRBC) because the mouse’s immune system was known to react vigorously against them. Antibodies against SRBCs could also be easily detected by Jerne and Nordin’s plaque essay test, a procedure that Köhler had learned from Herman Waldmann at Cambridge University. With the skilled technical assistance of Shirley Howe, by December 1974 various experiments were being conducted in earnest. Initially three different myeloma cell lines (P3, 289, and P1) were selected as fusion partners for the spleen cells. (p.21)

Hunting for the Elusive “Magic Bullet”

Figure 1.3. Microscope photo of a hybridoma cell secreting monoclonal antibodies

(Geoff Hale)

Eventually, however, Köhler determined that the most successful myeloma fusion partner was X63, his variant of a subclone of the P3 myeloma cell line originally prepared by David Secher, then a postdoctoral student at LMB. It originated from Horibata and Harris’s adaptation of Potter’s myeloma cell lines in Cohn’s laboratory at the Salk Institute. The advantage of X63 was that it was resistant to azaguanine, a reagent used to promote fusion. Köhler grew the cells in a HAT medium and added inactivated Sendai virus to promote fusion.61

Next, Köhler adapted Jerne and Nordin’s plaque test to determine whether any of the hybrid cells he produced would bind to SRBCs, by linking SRBCs with a fluorescein dye that glows green when put under an ultraviolet light. Should any of the antibodies produced in the experiment lock on to the surface of the SRBCs, he would be able to detect a bright green halo.62

By the end of December Köhler could see a number of cells growing in the HAT medium, but was unsure whether any had generated hybrid cells secreting antibodies with specificity for the SRBCs. Furthermore, by the time he could analyze the cells, some fungi had contaminated the culture so he was forced to go back to square one. Finally, on January 24, 1975, he was ready to try out his plaque test on some cells he had created by fusing X63 with the spleen cells taken from a mouse immunized against SRBCs. Expecting the process to take several hours, Köhler started (p.22)

Hunting for the Elusive “Magic Bullet”

Figure 1.4. Petri dishes showing the first Mabs grown by César Milstein and Georges Köhler

(G. Köhler and C. Milstein, “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256, no. 5517 [1975]: 495–97, fig. 2)

the test at 5 p.m., then went home. He returned some hours later with his wife as company because he expected boring results. He recalled, “I looked down at the first two plates. I saw these halos. That was fantastic. I shouted, I kissed my wife. I was all happy. The other results were positive as well. It was the best result I could think of.” Not only had he created hybrid cells that secreted antibodies that bound to SRBCs, but the number of antibodies they had generated was also far greater than anticipated.63

Following this success, Köhler and Milstein repeated their experiment twice more to see if the technique was reproducible. When these experi (p.23)

Hunting for the Elusive “Magic Bullet”

Figure 1.5. César Milstein (left) and Georges Köhler in 1984, around the time of their Nobel Prize

(Celia Milstein/MRC Laboratory of Molecular Biology)

ments proved positive, they excitedly realized that they possessed a tool that scientists had been striving to make for many years: an immortal cell line capable of producing endless quantities of identical antibodies with known specificity. Their method would later be dubbed “hybridoma technology” and the antibodies it produced “monoclonal antibodies,” to signify that they were derived from a single hybrid cell (Figure 1.3). 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 which they declared their technique to be a promising development for both medicine and industry. Yet the significance of their achievement eluded the journal’s editors, who asked Köhler and Milstein to shorten their article and did not include it in the section reserved for findings of major importance. The article was published in August 1975.64

The hybridoma technology marked the culmination of years of research into fighting infectious disease and understanding the (p.24) immunological mechanism by which living organisms defended themselves from foreign invaders. In 1984, Milstein and Köhler were awarded the Nobel Prize jointly with Jerne “for theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies” (Figures 1.4, 1.5). Their discovery was achieved despite many blind alleys and fierce theoretical battles. Now, at last, it seemed possible that scientists had found the powerful “magic bullet” against infectious disease that Paul Ehrlich had envisaged many decades earlier. But exactly how monoclonal antibodies could be applied in this way remained unknown.

Notes:

(1.) A. Silverstein, A History of Immunology (San Diego, 1989), ch. 1.

(2.) C. P. Gross and K. A. Sepkowitz, “The Myth of the Medical Breakthrough: Smallpox, Vaccination, and Jenner Reconsidered,” International Journal of Infectious Diseases 3, no. 1 (1998): 54–60; S. Riedel, “Edward Jenner and the History of Smallpox and Vaccination,” Baylor University Medical Center Proceedings 18, no. 1 (2005), 21–25; P. Debré, Louis Pasteur (Baltimore, 2000), chs. 14–15.

(3.) Silverstein, History, 18, 29, and ch. 2; L. Chernyak and A. I. Tauber, “The Idea of Immunity: Metchnikoff’s Metaphysics and Science,” J Hist Biol 23, no. 2 (1990): 187–249; L. Chernyak and A. I. Tauber, “History of Immunology: (p.239) The Birth of Immunology; Metchnikoff, the Embryologist,” Cellular Immunology 117 (1988): 218–33.

(4.) Silverstein, History, 30–32; A. Silverstein, “Cellular versus Humoral Immunology: A Century-Long Dispute,” Nat Immunol 4–5 (2003): 425–28; A. C. Hüntelmann, “Two Cultures of Regulation? The Production and State Control of Diphtheria Serum at the End of the Nineteenth Century in France and Germany,” Hygiea Internationalis 6, no. 2 (2007): 99–120; U. Klöppel, “Enacting Cultural Boundaries in French and German Diphtheria Serum Research,” Science in Context 21, no. 2 (2008): 161–80.

(5.) J. E. Alouf, “A 116-year Story of Bacterial Protein Toxins (1888–2004): From ‘Diphtheric Poison’ to Molecular Toxicology,” in J. E. Alouf and M. R. Popff, eds., The Comprehensive Source Book of Bacterial Protein Toxins (Waltham, Mass., 2006): 3–4; U. Lagerkvist, Pioneers of Microbiology and the Nobel Prize (London, 2003), 91.

(6.) F. Winau and R. Winau, “Emil von Behring and Serum Therapy,” Microbes and Infection 4 (2002): 185–88; D. S. Linton, Emil von Behring: Infectious Disease, Immunology, Serum Therapy (Philadelphia, 2005), 28–37, 58–63; J. Simon, “Emil Behring’s Medical Culture: From Disinfection to Serotherapy,” Medical History 51 (2007): 201–18.

(7.) Kitsato and Behring did not use the term antitoxin. Behring viewed the agent as a form of disinfectant. See J. Lindenmann, “Origin of the Terms ‘Antibody’ and ‘Antigen’,” Scandinavian Journal of Immunology 19, no. 4 (1984): 281–85; A. Cambrosio, D. Jacobi, and P. Keating, “Ehrlich’s “Beautiful Pictures” and the Controversial Beginnings of Immunological Imagery,” Isis 84, no. 4 (Dec. 1993): 662–99, esp. 666–67; Simon, “Emil”; Linton, Behring, 108–109.

(8.) P. Gronski, F. R. Seiler, and H. G. Schwick, “Discovery of Antitoxins and the Development of Antibody Preparations for Clinical Uses from 1890–1990,” Molecular Immunology 28, no. 12 (1991): 1321–32; A. Mendelsohn, “Cultures of Bacteriology: Formation and Transformation of a Science in France and Germany, 1870–1914,” Ph.D. diss., Prince ton University, 1996, 299–309; Winau and Winau, “Behring”; J. Simon, “Emil Behring’s Medical Culture”; Lagerkvist, Pioneers, 93–95.

(9.) The word “antibody” took time to be established. See Lindenmann, “Origin.”

(10.) A. M. Silverstein, “Paul Ehrlich’s Passion: The Origins of His Receptor Immunology,” Cellular Immunology 194 (1999): 213–21Linton, Behring, 105–106

(11.) The term “antigen,” a label used to denote a foreign invader, originated from research into antagonist products of bacteria in France from 1893. See Lindenmann, “Origin.”

(12.) Ehrlich’s immunity theory drew on his earlier discovery that dyes have side chains relating to their coloring properties and that each side chain had specific functions and affinity. See Silverstein, “Paul”; S. H. E. Kaufmann, “Elie (p.240) Metchnikoff’s and Paul Ehrlich’s Impact on Infection Biology,” Microbes and Infection 10, nos. 14–15 (2008): 1417–19; C. R. Prüll, “Part of a Scientific Master Plan? Paul Ehrlich and the Origins of His Receptor Concept,” Medical History 47 (2003): 332–56. Prüll argues that Ehrlich took time to develop his side-chain theory because as a Jew he could not obtain secure employment. See also T. Travis, “Emil Fischer and the Key to Specificity,” Chemistry and Industry (18 Apr. 1994).

(13.) Silverstein, History, chs. 4–7, p. 14; A. M. Moulin, Le dernier langage de la médicine: Histoire de l’immunologie de Pasteur au SIDA (Paris, 1991), 67–97; Cambrosio, Jacobi, and Keating, “Ehrlich’s ‘Beautiful Pictures’ ”; E. Crist and A. I. Tauber, “Debating Humoral Immunity and Epistemology: The Rivalry of the Immunochemists Jules Bordet and Paul Ehrlich,” J Hist Biol 30 (1997): 321–56.

(14.) A. Petterson, ceremony speech, Nobel Prize in Physiology or Medicine 1919, available at http://nobelprize.org/nobel_prizes/medicine/laureates/1919/press.html (accessed 17 Sept. 2014); A. B. Laurell, “Jules Bordet—A Giant in Immunology,” Scandinavian Journal of Immunology 32, no. 5 (1990): 429–32; Crist and Tauber, “Debating”; C. Schmalstieg and A. S. Goldman, “Jules Bordet (1870–1961): A Bridge between Early and Modern Immunology,” Journal of Medical Biography 17, no. 4 (2009): 217–24.

(15.) J. Drews, “Paul Ehrlich: Magister Mundi,” Nat Rev Drug Discov 3 (2004): 1–5; H. P. Vollmers and S. Brändlein, “The “Early Birds”: Natural IgM Antibodies and Immune Surveillance,” Histology Histopathology 20 (2005): 927–37; J. Drews et al., “Drug Discovery: A Historical Perspective,” Science 287 (2000): 1960–64; D. D. Boyden, An Introduction to Music (London, 1959), 339.

(16.) U. Lagerkvist, Pioneers of Microbiology and the Nobel Prize (London, 2003).

(17.) N. Jerne, “Waiting for the End,” Cold Spring Harbor Symposium on Quantitative Biology, 32 (1967): 591–603; Silverstein, History, 60–61; I. Löwy, “The Epistemology of the Science of an Epistemologist of the Sciences: Ludwik Fleck’s Professional Outlook and Its Relationships to His Philosophical Works,” in R. S. Cohen and T. Schnelle, eds., Cognition and Fact: Materials on Ludwik Fleck (Dordrecht, 1986), 421–24.

(18.) Silverstein, History, 65–67; L. E. Kay, “Molecular Biology and Pauling’s Immunochemistry: A Neglected Dimension,” History and Philosophy of the Life Sciences 11 (1989): 211–19, esp. 216; D. R. Forsdyle, “The Origins of the Clonal Selection Theory of Immunity,” FASEB J 9 (1995): 164–66; P. D. Hodgkin, W. R. Heath, and A. G. Baxter, “The Clonal Selection Theory: 50 Years since the Revolution,” Nat Immunol 8, no. 10 (2007): 1019–23, esp. 1019.

(19.) Löwy, “Epistemology,” 433–44I. Löwy, “Immunology in the Clinics: Reductionism, Holism, or Both?” in K. Kroker, P. Mazumdar, and J. Keelan, eds., Crafting Immunity: Working Histories of Clinical Immunology (Aldershot, 2008), 165–77.

(20.) L. Van Epps, “Michael Heidelberger and the Demystification of Antibodies,” JEM 203, no. 1 (2006): 5; J. Carneiro, “Towards a Comprehensive View of the (p.241) Immune System,” Ph.D. diss., Unité d’Immunobiologie Institut Pasteur, 1996, 11; M. Heidelberg, “A ‘Pure’ Organic Chemist’s Downward Path: Chapter 2—The years at P. and S,” Annual Review of Biochemistry 48 (1979): 1–21; H. L. Van Epps, “How Heidelberger and Avery Sweetened Immunology,” JEM 202, no. 10 (2005): 1306; J. Cruse, “A Centenary Tribute: Michael Heidelberger and the Metamorphosis of Immunologic Science,” JI 140 (1988): 2861–63.

(21.) Kay, “Molecular Biology and Pauling’s Immunochemistry,” 216; L. E. Kay, The Molecular Vision of Life (Oxford, Eng., 1993), 174–85; D. M. Knowles, Neoplastic Hematopathology (Philadelphia, 2001), 44; A. Cambrosio, D. Jacobi, and P. Keating, “Arguing with Images: Pauling’s Theory of Antibody Formation,” Representations 89 (2005): 94–130; T. Söderqvist, Science as Autobiography: The Troubled Life of Niels Jerne (London, 2003), 176; Silverstein, History, 72–76.

(22.) M. Cohn, “Reflections on the Clonal-Selection Theory,” Nature Reviews: Immunology 7 (2007): a23–a30; Söderqvist, Science, 167–85; A. M. Silverstein, “Splitting the Difference: The Germline–Somatic Mutation Debate on Generating Antibody Diversity,” Nat Immunol 4, no. 9 (2003): 829–33; D. R. Forsdyke, “The Origins of the Clonal Selection Theory of Immunity,” FASEB J 9 (1995): 164–66; J. M. Cruse and R. E. Lewis, “David W. Talmage and the Advent of the Cell Selection Theory of Antibody Synthesis,” JI 153 (1994): 919–24.

(23.) Silverstein, History, 75–76, 79–80.

(24.) C. Viret and W. Gurr, “The Origin of the ‘One Cell-One Antibody’ Rule,” JI 182 (2009): 1229–30.

(25.) Silverstein, History, 80–88; Silverstein, “Splitting.”

(26.) Silverstein, History, 75–76.

(27.) C. A. Janeway, “The Discovery of T Cell Help for B Cell Antibody Formation: A Perspective from the Thirtieth Anniversary of This Discovery,” Immunology and Cell Biology 77 (1999), 177–79.

(28.) A. Silverstein, “The Clonal Selection Theory: What It Really Is and Why Modern Challenges Are Misplaced,” Nat Imunol 3 (2002): 793–96.

(29.) A. C. Hüntelmann, “Diphtheria Serum and Serotherapy: Development, Production and Regulation in Fin-de-Siècle Germany,” Dynamis 27 (2007): 107–31Linton, Behring, ch. 4

(30.) Z. An, ed., Therapeutic Monoclonal Antibodies (Hoboken, N.J., 2009), 21; Linton, Behring, 121–47; A. Glatman-Freedman and A. Casadevall, “Serum Therapy for Tuberculosis Revisited: Reappraisal of the Role of Antibody-Mediated Immunity against Mycobacterium Tuberculosis,” Clinical Microbiology Review 11, no. 3 (1998): 514–32.

(31.) Rockefeller University, “The First Effective Therapy for Meningococcal Meningitis,” n.d.; Semp Inc., “Texas Cerebrospinal Meningitis Epidemic of 1911–12: Saving Lives with New York City Horse Immune Serum”; Anon., “The Specific Antibodies of Anti-Pneumococcal Sera,” American Journal of Public (p.242) Health 14, no. 9 (1924): 767–68; Anon., “Medicine: Pneumonia Cure?,” Time (May 19, 1924); H. F. Dowling, “The Rise and Fall of Pneumonia-Control Programs,” J Infect Dis 127, no. 2 (1973): 201–206; S. H. Podolsky, Pneumonia before Antibiotics: Therapeutic Evolution and Evaluation in Twentieth-Century America (Baltimore, 2006), ch. 1; H. Marks, The Progress of Experiment: Science and Therapeutic Reform in the United States (Cambridge, Eng., 2000), 62–67.

(32.) A. Cassadevall and M. D. Scharff, “Return to the Past: The Case for Antibody-Based Therapies in Infectious Diseases,” Clin Infect Dis 21 (1995): 150–61, table 1; Cruse, “Centenary Tribute to Michael Heidelberger”; Gronski, Seiler, and Schwick, “Discovery;” Linton, Behring, 328–40, 423–24; An, Therapeutic, 21; M. B. Llewelyn, R. E. Hawkins, and S. J. Russell, “Discovery of Antibodies,” BMJ 305 (1992): 1269–72.

(33.) Gronski, Seiler, and Schwick, “Discovery of Antitoxins”L. Harris, “Public Health Administration: Progress in the Treatment of Pneumonia,” American Journal of Public Health (1924): 620Anon., “Medicine: Pneumonia Cure?”Carneiro, Towards, 11

(34.) C. Nicolle, “Investigations on Typhus: Nobel Lecture,” 1928; C. F. McKhann and F. T. Chu, “Antibodies in Placental Extracts,” J Infect Dis 52, no. 2 (1933): 268–77; C. F. McKhann and F. T. Chu, “Use of Placental Extract in Prevention and Modification of Measles,” American Journal of Diseases of Children 45 (1933): 475–79; E. J. Cohn et al., “The Characterization of the Protein Fractions of Human Plasma,” Journal of Clinical Investigation 23, no. 4 (1944): 417–432; C. W. Ordman, C. G. Jennings, and C. A. Janeway, “The Use of Concentrated Normal Human Serum Gamma Globulin (Human Immune Serum Globulin) in the Prevention and Attenuation of Measles,” Journal of Clinical Investigation 23, no. 4 (1944): 541–49; J. Stokes, E. P. Maris, and S. S. Gellis, “The Use of Concentrated Normal Human Serum Gamma Globulin (Human Immune Serum Globulin) in the Prophylaxis and Treatment of Measles,” Journal of Clinical Investigation 23, no. 4 (1944): 531–40; H. Ganguli and S. N. Mukherjee, “Placental Globulin in the Prevention of Measles,” BMJ (11 Dec. 1954): 1395–97; A. N. H. Creager, “Producing Molecular Therapeutics from Human Blood: Edwin Cohn’s War time Enterprise,” in S. de Chadarevian and H. Kamminga, eds., Molecularizing Biology and Medicine: New Practices and Alliances, 1910s–1970s (Amsterdam, 1998), 107–139.

(35.) W. H. Manwaring, “Biochemical Relativity,” Science 72, no. 1854 (1930): 23–27; W. H. Manwaring, “Renaissance of Pre-Ehrlich Immunology,” JI 19, no. 2 (1930): 155–63; Gronski, Seiler, and Schwick, “Discovery,” 1329; Silverstein, History, 67–68, 84.

(36.) Kay, “Molecular Biology,” 216Kay, Molecular Vision, 174–75D. M. Knowles, Neoplastic Hematopathology (Philadelphia, 2001), 44, 176.

(37.) Cassadevall and Scharff, “Return,” 151A. Cassadevall, D. L. Goldman, and M. Feldmesser, “Antibody-Based Therapies for Infectious Diseases: Renaissance for an Abandoned Arsenal,” Bulletin de l’Institut Pasteur 95 (1997): 247–57.

(p.243) (38.) S. Deshpande, Enzyme Immunoassays: From Concept to Product Development (1996), 8–9.

(39.) Löwy, “Epistemology,” 424–25; H. G. Kunkel, R. J. Slater, and R. A. Good, “Relation between Certain Myeloma Proteins and Normal Gamma Globulin,” Proceedings of the Society for Experimental Biology and Medicine 76 (1951): 190–193; R. J. Slater, S. M. Ward, and H. G. Kunkel, “Immunological Relationships among the Myeloma Proteins,” JEM 101 (1955): 85–108; J. B. Natvig and J. D. Capra, “Henry J. Kunkel,” available online at the National Academies Press website, http://www.nap.edu/openbook.php?record_id=12042&page=224 (accessed 17 Sept. 2014); Rockefeller University, “Henry G. Kunkel (1916–1983), 1975 Albert Lasker Basic Medical Research Award, available online at http://www.rockefeller.edu/about/awards/lasker/hkunkel (accessed 17 Sept. 2014); Rockefeller University, “The Discovery of the Classes and Structures of Immunoglobulin Molecules,” available online at http://centennial.rucares.org/index.php?page=Immunoglobulin (accessed 17 Sept. 2014); K. Eichmann, Köhler’s Invention (Basel, 2005), 39–40.

(40.) R. G. Lynch, “Plasmacytomas and Basic Immunology,” in R. Lynch, ed., Milestones in Investigative Pathology (Bethesda, Md., 2009), 17–18. This work was an extension of work that Potter had begun in 1956. See Albert Lasker Basic Medical Research Award to Köhler, Milstein, and Potter, 1984, available online at http://www.laskerfoundation.org/awards/1984_b_description.htm (accessed 17 Sept. 2014); K. Horibata and A. W. Harris, “Mouse Myelomas and Lymphomas in Culture,” Experimental Cell Research 60, no. 1 (Apr. 1970): 61–77; Eichmann, Köhler’s, 26, 42; A. Cambrosio and P. Keating, Exquisite Specificity: The Monoclonal Antibody Revolution (Oxford, Eng., 1995), 26–27, A. Cambrosio and P. Keating, “Monoclonal Antibodies: From Local to Extended Networks,” in A. Thackray, Private Science: Biotechnology and the Rise of the Molecular Sciences (Philadelphia, 1998), 165–81, 175; A. Cambrosio and P. Keating, “Between Fact and Technique: The Beginnings of Hybridoma Technology,” J Hist Biol 25, no. 2 (1982): 175–230, 208–12.

(41.) N. K. Jerne and A. A. Nordin, “Plaque-Formation in Agar by Single Antibody Producing Cells,” Science 140, no. 3565 (26 Apr. 1963): 405.

(42.) T. Staehelin, “Pittsburgh 1962, no. 63 Revisited: Too Many Antibodies, Too Few Ribosomes?” Scandinavian Journal of Immunology 62, supp. 1 (2005): 23–26.

(43.) Interviews with Ivan Lefkovits. Other plaque tests were successfully developed soon after the one devised by Jerne and Nordin. See Söderqvist, Science, 236–37; Eichmann, Köhler’s, 21.

(44.) G. Chedd, “Nobel Prizes for Antibody Structure,” New Sci (19 Oct. 1972): 142–43; L. A. Steiner, “Rodney Robert Porter (1917–1985),” Nature 317, no. 6036 (1985): 383; G. M. Edelman, “The Evolution of Somatic Selection: The Antibody Tale” (Dec. 1994), in J. F. Crow, ed., Perspectives on Genetics: Anecdotal, Historical, and Critical Commentaries (Madison, Wis., 2000), 426–32.

(p.244) (46.) BII, Annual Report (1972), introduction by N. K. Jerne, reprinted in I. Lefkovits, ed., A Portrait of the Immune System: Scientific Publications of NK Jerne (London, 1996), 745–52.

(47.) Interviews with Lefkovits; Söderqvist, Science, 258–60, 268.

(48.) N. K. Jerne, “The Immune System,” Sci Am (July 1973): 52–60; N. K. Jerne, “Towards a Network Theory of the Immune System,” Annales d’Immunologie 125C (1974): 373–89; K. Eichmann, The Network Collective: Rise and Fall of a Scientific Paradigm (Basel, 2008), ch. 9; Söderqvist, Science, 269–73.

(50.) M. Cohn, “Natural History of the Myeloma,” Cold Spring Harbor Symposium on Quantitative Biology 32 (1967): 211–12; W. Gerhard, T. J. Braciale, and N. R. Klinman, “The Analysis of the Monoclonal Immune Response to Influenza Virus. I. Production of Monoclonal Anti-Viral Antibodies in Vitro,” EJI 5 (1975): 720–25; G. Köhler and C. Milstein, “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256, no. 5517 (1975): 495–97; C. Milstein, “Monoclonal Antibodies,” Sci Am 243 (1980), 66–75; Eichmann, Köhler’s, 8–9, 40; Cambrosio and Keating, Exquisite, 28.

(51.) G. Barski, S. Sorieul, and F. Cornefert, “Production of Cells of a “Hybrid” Nature in Cultures in Vitro of Two Cellular Strains in Combination,” Comptes rendus hebdomadaires des seances de l’Academie des sciences 251 (1960): 1825–27Eichmann, Köhler’s, 49–53R. Cotton, “The Road to Monoclonal Antibodies,”

(52.) A hapten is partial antigen that can act with a previously existing antibody, but it cannot stimulate more antibody production unless combined with other molecules.

(53.) J. G. Sinkovics et al., “A System of Tissue Cultures for the Study of a Mouse Leukemia Virus,” J Infect Dis 119 (1969): 19–38; J. G. Sinkovics, “Early History of Specific Antibody-Producing Lymphocyte Hybridomas,” Cancer Research 41 (1981): 1246–47; J. G. Sinkovics, “An Interesting Observation Concerning Specific Antibody-Producing Hybridomas,” J Infect Dis 145 (1982): 135; A. R. Williamson and B. E. G. Wright, “Selection of a Single Antibody-Forming Cell Clone and Its Propagation in Syngeneic Mice,” PNAS 67 (1970): 1398; A. R. Williamson and B. A. Askonas, “Senescence of an Antibody-Forming Cell Clone,” Nature 238 (11 Aug. 1972): 337–39; B. A. Askonas and A. R. Williamson, “Factors Affecting the Propagation of a B Cell Clone Forming Antibody to the 2,4-Dinitrophenyl Group,” EJI 9 (1972): 487; B. A. Askonas, “From Protein Synthesis to Antibody Formation and Cellular Immunity,” Ann Rev Immunol 8 (1990): 1–21; Interview with Andrew McMichael; Askonas evidence in T. Tansey and P. Caterall, “Technology Transfer in Britain: The Case of Monoclonal Antibodies,” in T. Tansey et al., eds., History of Twentieth Century Medicine Witness Seminars, 1993–1997, vol. 1 (London, 1997), 1–31, 6–17.

(p.245) (54.) R. Klinman, “Antibody with Homogeneous Antigen Binding Produced by Splenic Foci in Organ Culture,” Immunochemistry 6, no. 5 (1969): 757–59.

(55.) Interview with Walter Gerhard.

(56.) W. Gerhard, “The Analysis of the Monoclonal Immune Response to Influenza Virus, II: The Antigenicity of the Viral Hemagglutinin,” JEM 144 (1976): 985–95; Gerhard, Braciale, and Klinman, “Analysis of the Monoclonal Immune Response”; T. J. Braciale, W. Gerhard, and N. R. Klinman, “Analysis of the Humoral Immune Response to Influenza Virus in Vitro,” JI 116 (1976): 827–34; H. Koprowski, W. Gerhard, and C. M. Croce, “Production of Antibodies against Influenza Virus by Somatic Cell Hybrids between Mouse Myeloma and Primed Spleen Cells,” PNAS 74, no. 7 (1977): 2985–88; P. C. Doherty, “Challenged by Complexity: My Twentieth Century in Immunology,” Ann Rev Immunol 25 (2007): 1–19; Cambrosio and Keating, Exquisite, 15–16; R. Vaughan, Listen to the Music: The Life of Hilary Koprowski (New York, 2000), 172–73.

(57.) R. Levy and J. Dilley, “The In Vitro Antibody Response to Cell Surface Antigens, I: The Xenogeneic Response to Human Leukemia Cells,” JI 119, no. 2 (1977): 387–93; R. Levy and J. Dilley, “The In Vitro Antibody Response to Cell Surface Antigens, II: Monoclonal Antibodies to Human Leukemia Cells,” JI 119, no. 2 (1977): 394–400; R. Levy, R. Wartnke, R. Dorfman, and J. Haimovich, “The Monoclonality of Human B-Cell Lymphomas,” JEM 145 (1977): 1014–28; B. Azar, “Profile of Ronald Levy,” PNAS 107, no. 29 (2010): 12745–46; interviews with Ron Levy and Zenon Stepleswki.

(59.) Milstein and his team learned tissue culturing from Abraham Karpas, who was then based in a laboratory next to Milstein at the LMB. Milstein and Cotton had become involved in the fusion of cells primarily to study the activation or expression of antibody genes. By fusing two cells it was hoped that they could find out what controlled the expression of genes in antibodies. By creating a hybrid cell from two different parental antibody cells, Milstein and Cotton wanted to know if the fused antibody would express the two sets of genes from the parental cells, or if only one set of genes would be expressed from the parental cell. The fused antibody was found to express both sets of genes. See A. Karpas, “César Milstein (1927–2002): A Somewhat Personal Reflection,” Trends in Immunology 23 (2002): 321–22; R. G. H. Cotton and C. Milstein, “Fusion of Two Immunoglobulin-Producing Myeloma Cells,” Nature 244 (1973): 42–43; Cotton, “Road”; C. Milstein, “Inspiration from Diversity in the Immune System,” New Sci (21 May 1987): 54–58; Interview with David Secher; J. Schwaber and E. P. Cohen, “Human X Mouse Somatic Cell Hybrid Clone Secreting Immunoglobulins of Both Parental Types,” Nature 244, no. 5416 (1973): 444–47; J. Schwaber and E. Cohen, “Pattern of Immunoglobulin Synthesis and Assembly in a Human-Mouse Somatic Cell Hybrid Clone,” PNAS 71, no. 6 (1974): 2203–207; Cambrosio and Keating, “Between.”

(61.) H. Waldmann, H. Pope, and I. Lefkovits, “Limiting Dilution Analysis of Helper T-Cell Function,” Immunology 31 (1976): 343–52.Cambrosio and Keating, “Between,” 209, 211, 214Eichmann, Köhler’s, 52–53

(62.) M. Goldberg, Cell Wars: The Immune System’s Newest Weapons against Cancer (New York, 1989), 15.

(63.) N. Wade, “Hybridomas: The Making of a Revolution,” Science 215 (1982): 1073–75, 1074Eichmann, Köhler’s, 67