Today's Medicine, Tomorrow's Science
Essays on Paths of Discovery in the Biomedical Sciences
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Over the past century, beginning with efforts of medical bacteriologists to find ways of protecting man and his domestic animals fron the ravages of infectious diseases, the field of immunobiology has involved a steadily broadening scope of inquiry. From many disciplines, approaches and interestes, clinical and laboratory investigators have gradually defined and are seeking to understand the three key features of the phenomenon called immunity: recognition, specificity, and memory. That is, first, how does the immune system "recognize" potentially hazardous foreign antigens, and of equal import, recognize the body's own constituents as harmless? Or, in the words of Burnet, how does the immune system distinguish between "not self" and "self?" The immune system, secondly, operates with remarkable specificity against millions of chemically different antigenic structures. Thus, closely related to understanding how recognition occurs, immunobiologists want to know how the immune system produces a specfic antibody against any one of millions of antigens. The third key characteristic of the immune system is "memory:" how, days or years after its first exposure to an antigen, is an organism able to "remember" that exposure and react more rapidly and strongly to a second encounter?

Fundung answers to these questions, investigators have long realized, has obvious medical significance. And, as has become increasingly evident in recent years, the quest to understand how immune defense mechanisms operate also has led us into the "central regions" of biology. "The current explosion in immunological research," as Lewin pinted out in 1974,

throws out waves of discoveries that are giving insights into the science undreamed of a few years ago. Immunobiology is now one of the most rewarding and promising areas of biological research. It is rewarding because it spreads into many areas of acute social misery giving prospects of long-hoped-for cures: the successes of vaccinations against many bacterial and viral infections may soon be followed by assaults on cancer and diseases such as rheumatoid arthritis and the presently unassailable parasitic infections; immunology also holds the key to safe organ transplantations. And it is promising because the drive toward understanding the molecular mechanisms behind the immune responses is certain to reveal features fundamental to the whole of biology. (Lewin 1974, p. 2)

During the relatively brief span of its existence, the field of immunobiology has become so vast, and the lines of disease-oriented and basic research pursued within its compass so complex and intertwined, that no one book could pretend to be a definitive history. Within the even narrower scope of a single essay, we will look at only a single but critical strand in the history of immunology, a strand that exemplifies how "major thrusts toward fundamental progress in immunobiology have been guided by questions formulated from experiences in the clinic" (Bach and Good 1972, p. xiii). These clinical problems, in the main, have centered around what immunologist Robert Good has termed "the crucial experiments of nature," a series of diseases which have "contributed maximally to the development of our modern concepts of immunology" (Good 1972, p. 23).

The particular disease problem that we will examine is multiple myeloma, a relatively rare form of cancer that accounts for only some 0.5 percent of all malignancies and about two deaths per 100,000 persons per year. But although a rare form of cancer, multiple myeloma "probably represents the most important of all experiments of nature" for immunobiologists (Good 1972, p. 25). For, as a trail of medical and scientific research has revealed since the first case of multiple myeloma was documented in 1846, this cancer is a tumor of the plasma cells which are responsible for the production of antibodies. In patients with multiple myeloma, as we shall learn, the immune system's protein metabolism is so altered that the excretion of certain abnormal proteins is a hallmark of the disease. The study of these abnormal proteins, in turn, has generated extensive and significant knowledge not only about multiple eases, and the structure, biosynthesis, and genetic control of antibodies.

The particular strand among this network of research and discovery that we will follow is that which runs from the first clinical descriptions and study of multiple myeloma in the mid-19th century to the analysis of antibody structure in the 1950s and 1960s, an analysis that rested on defining the nature of one of the abnormal proteins of multiple myeloma patients, known as Bence Jones protein. The elucidation of an antibody's structure marked the birth of molecular immunology, an event that future historians may well judge to have been a "revolution" in the science of immunology. One of the first results of that revolution, as we shall see, has been major strides in understanding how an antibody's molecular structure specifies two of its functions: to recognize a specific type of antigen, and to then perform a particular type of defense function, termed the antibody's effector function.

In part because both the history of immunology and our present knowledge of immune phenomena are so complex, we are going to reverse our historical narrative and begin with the end of the story: a brief account of knowledge about the immune system, particularly the structure and function of antibodies, as of the early 1970s. Then we will go back to the mid-19th century, and learn why multiple myeloma has proved to be the critical experiment of nature for unraveling the molecular structure of antibodies and, in turn, for beginning to understand how that structure specifies antibody functions. Separate paths of inquiry about the nature of myeloma proteins and about the nature of antibodies were pursued until the late 1950s, and, mirroring this historical disjunction, we will look separately at attempts to answer two questions: what is Bence Jones protein? , and what is an antibody? Finally, we will see how these two questions converged during the 1960s, when the study of myeloma proteins entered and profoundly affected the mainstream of immunological research.

The Immune System's Architecture

At any given moment, there are some trillion cells in the human body called lymphocytes, a type of white cell which exercises the recognition, specificity, and memory functions of the immune system. In the late 1960's, principally through clinical research on immunodeficiency diseases,(1) immunologists learned that lymphocytes are composed of two cell types: B cells and T cells. The identification of B and T cells resolved earlier decades of often intensive debate about whether immunity is primarily a humoral or cellular phenomenon, for these cells represent a dual humoral and cellular system of immunity. In this dual system, the T lymphocytes are responsible for the cellular immune response, which functions against certain microorganisms and parasites, viruses, cancer cells, and organ transplants.(2) The B cells, in turn are responsible for the production of antibodies, and thus mediate the humoral response of the immune system. In the past few years, researchers have found that this duality is not a complete or simple one. There is an overlap in the responses of B and T cells to common antigens, and the production of antibodies involves not just the response of B cells to certain antigens, but a complex cooperative response involving T and B cells and another type of white cell, the macrophage.

Antibodies have been identified as large proteins, containing about 1300 amino acids in their chains, that are associated with the globulin class of proteins and thus also are termed immunoglobulins. The antibody or immunoglobulin molecules are arranged on the surface of B cells, with any given B cell having only one type of antibody molecule. We have noted that there are millions of possible antigens, and, correspondingly, there are millions of different antibodies, each structured to fit or bind to an antigen like the matching of a lock and key.

The huge variety of antibodies have been grouped into five classes, according to their molecular structure. It is IgG, the major mammalian antibody class, that we shall refer to most often as we trace the role of multiple myeloma research in elucidating the structure of antibodies.

The first major, fundamental information revealed by the molecular analysis of antibody structure, as we have noted briefly, is how the antibody's two principal functions are determined by its structure. The antibody's recognition function - its ability to "identify" a specific antigen - is governed by the shape of the antigenbinding site in the molecule, and is unique to a given antibody. The antibody's effector function - its ability to execute a given type of defensive action against an antigen - is determined by another part of the antibody molecule, and is common to an antibody class.(3) [END OF PAGE 94]

The details of antibody structure that emerged in the late 1950s and 1960s from the union of immunology and molecular biology have shown us that an antibody or immunoglobulin molecule is composed of four separate protein chains, linked together with special types of chemical bonds. The arrangement of the four chains the the antibody molecule as a whole the shape of the letter Y.

An antibody's four protein chains are composed of two pairs: a shorter pair called the light chains, and a called the heavy chains. The light chains, and a longer pair called the heavy. The light chains and the heavy chains, in turn, have two common structural features that determine the antibody's recognition and effector functions: one part of each chain is structurally "constant," while the rest of the chain is "variable."

"Constant region" designates that area of the heavy or light protein chain in which the amino acid sequence is identical, within a given immunoglobulin class. That is, for example, the constant regions of heavy and light chains in all IgG antibodies have identical amino acid sequences. Thus, molecular studies have shown that it is the constant region of the chains which determines an antibody's class. And, as we have said, the antibody class is characterized by the effector or defense function particular to that class.

As its name implies, the variable region is that relatively short area of the protein chain in which the amino acid sequence differs from antibody to antibody. It is this variable region that provides the antibody molecule's recognition function, or, stated another way, it is the site of the antibody's antigen binding capability. The hugh number of possible amino acid sequences rpovided by both the heavy and light chains' variable regions(4) give each antibody its unique ability to "recognize" a particular type of antigen, forming a specifically shaped "lock" or binding site that matches the antigen "key."

In brief summation, we know today that B lymphocytes have on their surfaces a particular type of large protein molecule, called antibody or immunoglobulin. This molecule consists of two heavy and two light protein chains arranged in a Y-shape. Each end of the "Y" has an antigen-binding site, in the variable regions of the heavy and light chains. These two sites are the same in any given antibody molecule, and they enable the antibody to "recognize" and bind with a particular antigen. Linked to the two light chains in each antibody are two heavy chains, and it is the long constant regions of these chains that primarily determine an antibody's class and hence its effector function.

The knowledge of the structure of an immunoglobulin molecule that we have summarized is a very recent achievement in the history of immunology. It is knowledge, as we have suggested, that has opened a new, sophisticated, and conceptually powerful era of molecular immunology. And, at the same time that it has opened a new era of research, the recent detailing of an antibody's molecular structure is the end of a trail of investigation that began more than a century ago, in 1845, when Dr. Thomas Watson asked Dr. Bence Jones, "What is it?"

"What is It?" Multiple Myeloma
and Bence Jones' Proteins

The Puzzle Begins: The First Case of Multiple Myeloma. Dr. Watson's query to Dr. Bence Jones concerned a urine sample from a seriously ill patient, who died two months after certain peculiar characteristics of his urine had first been noticed by another physician involved in his care, Dr. William Macintyre. The sample, Dr. Watson wrote in his brief note, "contains urine of very high specific gravity. When boiled it becomes slightly opaque. On the addition of nitric acid, it effervesces, assumes a reddish hue, and becomes quite clear; but as it cools, assumes the consistency and appearance which you see. Heat relinquishes it. What is it? " (Bence Jones 1847, p. 52).

This question, about the urinary substance that later became known as Bence Jones protein, would occupy the attention of clinical and basic researchers for more than 100 years. The patient who prompted the inquiry had the first recorded case of multiple myeloma, which his physicians recognized as a malignant bone disease and named "mollities ossium."

Three astute physicians became involved in exploring the nature of this previously unrecognized disease, and, in a series of papers published between 1846 and 1850, they described their findings concerning the clinical features of the disease, the gross and microscopic postmortem findings, and the characteristics of the strange urinary protein. Dr. William Macintyre, a London physician, entered the case in October 1845, when he was asked by Dr. Watson to see a patient whom the latter had been treating for several months. From his careful history and physical examination, Maclntyre discovered several important clinical features of multiple myeloma, including severe bone pains and an unusual protein in the urine. Although history has linked Bence Jones' name with the characteristic proteins of multiple myeloma patients, it was Macintyre who first thought to examine the patient's urine. In so doing, he observed [END OF PAGE 95] that heating the sample caused an unknown protein to precipate at temperatures much lower than those of other proteins, and that this protein would redissolve as the urine's temperature was raised, and then reprecipitate as the urine cooled.

Drs. Macintyre and Watson independently sent samples of their patient's urine to another physician for further study. They both chose Dr. Henry Bence Jones, a physician to St. George's Hospital who was noted as a "chemical pathologist." Bence Jones confirmed Macintyre's findings concerning the urinary protein, and went on to study it in greater detail. Macintyre's case report paper in 1850 dealt mainly with the clinical features of the disease, which is doubtless why he failed to receive historical credit for discovering the protein (MacIntyre 1850; Clamp 1967).

The third member of this physician trio became involved in the case after the patient's death. John Dalrymple, a surgeon to the Royal Ophthalmic Hospital and a member of the Microscopical Society, was asked to do a histological study of material from the patient's lumbar vertebrae and a rib. His paper, accompanied by two woodcut drawings of the cells he had examined , today would be read as a description of malignant plasma cells (Dalrymple 1846).

The immediate cause of death listed for this first recorded victim of multiple myeloma was -[kidney] atrophy from albuminuria." At the time, "albuminuria" was a term used non-specifically to designate proteinuria - the excretion of large amounts of protein in the urine. Dr. Macintyre's and Bence Jones' recognition and analysis of their patient's abnormal urinary protein was, in the context of their day, a significant achievement, for the terms "protein" and "albumin" had just entered the lexicon of chemistry in 1838, originally with a meaning different from its modern usage.(5)

Bence Jones discussed his study of the protein that would bear his name in 1847 and 1848 papers published in three of England's most prestigious medical and scientific journals: Lancet, the Proceedings of the Royal Society, and the Philosophical Transactions of the Royal Society.

The first paper, in Lancet, was the text of a lecture to the Royal College of Physicians - testimony to the young physician's eminence as a chemical pathologist. The theme of Bence Jones' lecture was the chemical effects of oxygen in the human body, a topic he addressed principally with reference to his own experiments on the urinary products of normal subjects and patients suffering from various diseases. Although the professional role of clinical researcher did nor emerge until the early decades of the 20th century, Bence Jones was, in effect, working as a clinical researcher in the 1840s, seeking to define metabolic processes in normal and diseased human subjects.

It was in this context that Bence Jones introduced the case he had worked on with Drs. Watson and MacIntyre, stating that "I have found another oxide of albumen in the urine, in a case of mollities ossium" (Bence Jones 1847, pp. 91-92). Bence Jones then described the clinical symptoms and read the note from Dr. Watson that accompanied the urine sample, before reporting on his physical and chemical analysis of the "enormous quantities" of the substance in the urine. Reflecting the newness of protein chemistry in 1845, Bence Jones argued that his analysis showed the substance to be an "oxide of albumen," not an "oxide of protein" (see note 5).

"It will immediately be asked," Bence Jones correctly realized, "what is the connection between Mollities Ossium and this state of the urine?" "To such a question," he acknowledged, "I am as yet unable to give a positive answer" (Bence Jones 1847, p. 92). He did, however, have an hypothesis to advance: chlorine, formed by the decomposition of sodium chloride in cells of the bones and kidney, "may have been the cause of the solution of the earthy matter of the bone" (i.e., of the pathological fractures of the patient's bones-another characteristic of multiple myeloma). This hypothesis, in turn, generated the question of "why do the cells take on this peculiar action?" Bence Jones had, no answer, but could only note that "on this question,"' the whole of secretion and nutrition are involved" (Bence Jones 1847, p. 92).

To Dr. Watson's query, "What is it? ", Bence Jones thus could give only a partial answer, albeit as good characterization of the urinary substance's properties, as the analytic techniques of his day permitted. He could not, he realized, do more than guess at the exact origin and nature of the substance, and at its relationship to the disease, multiple myeloma. For he, together with Drs. Watson, Macintyre, and Dalrymple, had uncovered, a medical puzzle that would intrigue scores of disease detectives for generations to come.

A Hundred Years Later: "Much Remains to Learned." As one scans the literature on multiple myeloma that developed from the late 1840s to the early 1950s, one initially striking impression is how [END OF PAGE 96] much attention was devoted to this relatively rare disease by physicians and their research colleagues in "physiological chemistry" or biochemistry. "Mollities" renamed myeloma by Rustizky in 1873, became a generally recognized disease entity when a German physician alerted physicians in 1889 to the fact that a certain constellation of symptoms usually meant that a patient had multiple myeloma. Those symptoms, Maclntyre and his colleagues had first noted in 1845, included deformity and abnormal fragility of bones, bone pain, cachexia (a general wasting that occurs in chronic diseases), and the presence of Bence Jones protein in the urine (Kahler 1889).

As clinicians encountered and defined the characteristic signs of multiple myeloma, they discovered that the excretion of large amounts of Bence Jones protein in the urnie was not the only protein abnormality associated with the disease. Two other changes in protein metabolism also were found: an increased level of certain abnormal globulins in the serum, and the deposition of protein in various body tissues. Of these three protein abnormalities, Bence Jones proteinuria received particular attention, because, as physicians searched for its presence in other disease states, they found that it rarely occurred except in muitiple myeloma. Thus, the presence of Bence Jones protein in a patient's urine, as Kahler emphasized, was an almost sure sign of multiple myeloma.

Physicians became interested in Bence Jones protein, as well as the abnormal serum protein and amyloidosis, not only because they were characteristic indicators of multiple myeloma. From the time it was first observed in 1845, the etiology or cause of multiple myeloma was a puzzle. And, as had Dr. Bence Jones, physicians hoped that study of the characteristic changes in their patients' protein metabolism might reveal what caused the disease.

Through the early 1950s, the study of Bence Jones protein, was pursued predominantly as a problem in clinical medicine. Physicians and other clinically-oriented researchers (mainly physiological chemists) were attempting to solve questions about the abnormal protein's origin and its chemical constitution primarily in reference to questions about multiple [END OF PAGE 93] myeloma. Research reports and discussions of various hypotheses about Bence Jones protein appeared as separate articles, and as components of extensive clinical review articles that detailed hundreds of case reports of multiple myeloma.

The state of knowledge about multiple myeloma and Bence Jones protein a century after the first case report is exemplified by a 1953 clinical monograph by Snapper, Turner, and Moscovitz, physicians at Mt. Sinai Hospital in New York. Having extensively reviewed the literature on multiple myeloma, and having personally dealt with ninety-seven cases over a seven-year period, the physicians wrote at the beginning of their book: "it is remarkable that today, after more than a century of study, much remains to be learned about the disease in general, and the source, chemistry, and constitution of Bence Jones protein in particular" (Snapper et al, 1953, p. 1).

Over the course of a century, Snapper and his colleagues went on to note, new ideas about the disease's etiology gradually had developed. Most authorities no longer regarded multiple myeloma as "merely" a tumor derived from bone marrow. But having decided what it was merely not, the question of what multiple myeloma was remained a matter of several unproven hypotheses, generated by clinical studies and, after 1935, by experiments with mice in which myeloma-like tumors could be produced. "For the time being," Snapper and his colleagues had to conclude in 1953, "the nature and pathogenesis of multiple myeloma remains completely unknown. In the absence of a proven etiologic agent, our knowledge can only be furthered by repeated and careful clinical observation and study" (Snapper et al. 1953, p. 4).

The status of knowledge about Bence Jones proteinuria in the early 1950s was much akin to that of the etiology of multiple myeloma: many questions, a variety of competing hypotheses, and little definitive knowledge. To the extent permitted by physicochemical analytic techniques as they developed over a century, investigators had pursued three major questions about this abnormal urinary product: what is the metabolic origin of Bence Jones protein, what is its chemical nature, and what is its relationship to normal serum proteins and to the abnormal serum proteins found in multiple myeloma patients?

Not surprisingly, ideas about the origin of Bence Jones protein tended to be linked with the development of myeloma itself. Thus, many favored a hypothesis put forward in the 1930s, that Bence Jones protein is formed by the malignant myeloma cells in the bone marrow (Magnus-Levy 1938). One extension of this idea, cited often in the literature after it was proposed by Dent and Rose in 1949, also sought to account for the [END OF PAGE 97] cause of myeloma itself. Dent and rose reported, on the basis of their analysis of one patient's urine, that Bence Jones protein was one of a small group of proteins, including the tobacco mosaic virus, that lacked the amino acid methionine in its composition. This finding, in light of the current knowledge about the structure and function of viruses, suggested that multiple myeloma "is due to invasion of the body by a virus which lives and multiplies in the plasma cells of the bone marrow . . . It is further suggested that the Bence Jones protein, when combined with nucleic acid in the plasma cells, is the virus itself" (Dent and Rose 1949, pp. 616-617).

Another view was that Bence Jones protein was more indirectly the product of the bone marrow's malignant myeloma cells. In part reasoning from the fact that the protein had been found to have a relatively low molecular weight, Rundles and his colleagues cautiously proposed that "the serum components. . are produced in all likelihood by the abnormal plasma cells. . Bence Jones proteins could possibly be derived from the abnormal serum constituents, since the latter have a molecular weight about three to six times as great, if the latter were to disintegrate or be split into protein moities filterable through the glomeruli [of the kidneys] " (R undles, Cooper and Willett, p. 1125).

Yet another school of thought held that Bence Jones protein is not an abnormal product somehow triggered by myeloma. Rather, as Meyler suggested, Bence Jones protein is produced by normal bone marrow, but in quantities too small to be detected in a normal person's urine or blood serum. When myeloma develops, however, the protein's production increases so greatly that it is excreted in readily detectable amounts in the urine (Meyler 1936).

These and other ideas about the source of Bence Jones protein were, by and large, as speculative as Dr. Bence Jones' original idea. Lack of knowledge about the etiology of myeloma itself, the fact that a given investigator usually had only one or at best a few patients' samples to study, and technical difficulties in isolating and analyzing Bence Jones protein, all contributed to the high degree of uncertainty about where this urinary protein came from. These same factors also bore upon the decades of conflicting ideas and data about the protein's chemical nature. Up to this point in our narrative we have used the singular, Bence Jones protein, to reflect the historical fact that for many decades it was generally regarded and discussed as a homogenous substance, identical in all multiple myeloma patients. Two French investigators, Ville and Derrieu, had suggested in 1907 that the protein might not be the same in all cases, but the early literature generally cited a 1911 paper by Hopkins and Savoy. The latter, after analyzing the amino acid composition of Bence Jones protein obtained from two patients, concluded that the chemical composition was identical (Hopkins and Savoy 1911).

The plural term, Bence Jones proteins, reflecting knowledge that the substance was not chemically identical in all patients, did not enter the literature until the 1920s, and one finds even today that the singular term continues to be used. Given the ultimate significance of Bence Jones proteins vis-a-vis our understanding of antibody structure, it is interesting that attempts to identify and characterize the abnormal proteins myeloma patients relied heavily on immunologic methods. But, and this is an important historical "but," these immunological methods were used for decades simply as useful analytic techniques, quite independent of the work on antibodies by immunologists and biochemists that eventually would involve Bence Jones proteins.

By the early 1900s, knowledge of antigenic reactions gained by the medical bacteriologists who pioneered the early development of immunology had begun to indicate that proteins have individual antigenic specificity (see Bulloch 1938, Lechevalier and Solotorovsky 1974). Thus, immunological reactions were recognized by physiologists and chemists as a useful way of detecting differences in proteins that, by other criteria, appeared alike. Two chronologically scattered reports on the study of Bence Jones proteins by immunologic methods appeared in 1911 and 1921; both showed, contrary to earlier studies, that the protein was not the same as normal serum protein (Massini 1911; Hektoen 1921).

Then, in 1922, these reports were confirmed and extended by Bayne-Jones and Wilson from Johns Hopkins, who used immunologic tests to study normal serum proteins and twelve samples of Bence,Jones proteins obtained from five patients. They found, first, that "the Bence Jones proteins are immunologically different from the proteins of normal human serum," and these results, they noted, supported a newly emerging major concept in protein research - "that the specificity of proteins is not dependent upon their biological origin, but due to their chemical composition" (Bayne-Jones and Wilson 1922a, p. 43). Bayne-Jones and [END OF PAGE 98] Wilson's work also challenged the "tendency to assume that all preparations of Bence-Jones protein are identical in structure and composition." For they had found that, immunologically, their preparations could be categorized into "two and possibly three groups." Thus, they concluded, Bence Jones "protein" actually is a group of similar but not identical protein substances (Bayne-Jones and Wilson 1922b).

Bayne-Jones' and Wilson's work, as well as earlier studies of the constitution and characteristics of Bence-Jones proteins, was subject to a major criticism. Most preparations of the protein were impure, containing other urinary or serum substances, and thus the results of a given experiment could not unequivocally be attributed to Bence Jones protein. By the 1940s, however, researchers were able to obtain crystallized protein extracts more readily and reliably. Repeating earlier immunological studies with these purer samples, investigators confirmed the existence of two immunologically groups of Bence Jones proteins, and found, further, that a given patient may excrete both types (Hektoen and Welker 1940).

In the 1940s, researchers also began to use other new techniques such as ultracentrifugation and electrophoresis in addition to immunological tests, that permitted more precise qualitative and quantitative characterizations of the metabolic abnormalities in multiple myeloma. By using a "broad and flexible analytic approach," as Moore and his colleagues noted in 1943, investigators hoped to sort out "the multiplicity of Bence Jones proteins and ... their correspondingly varied properties. . .[and] the varied serum protein patterns of patients with Bence Jones proteinuria" (Moore, Kabat ,1943, p. 74). By the early 1950s, the use of a combination of techniques had produced a mass of detailed information about the properties of Bence Jones proteins and the abnormal serum proteins of myeloma patients. But the findings of different researchers did not always accord, and so the exact nature of and relationship between the various protein abnormalities with myeloma remained "still a matter for conjecture" (Snapper et al. 1953, p. 58).

First Answers: Biochemical and Immunologic Studies in the 1950s. When Snapper and his colleagues observed in 1953 that "the exact nature of Bence Jones protein has never been determined," a series of studies had begun that would yield the first tentative answers to the question originally posed by Dr. Watson in 1845. These investigations of Bence Jones and other myeloma proteins in the 1950s fall into two major groups in terms of methods. First, as represented by the work of Frank W. Putnam and his associates at the University of Chicago, new knowledge about the structure and origin of Bence Jones proteins began to emerge from quantitative and qualitative biochemical analyses of multiple myeloma proteins. Secondly, investigators continued to examine the immunological relationships among the various abnormal proteins found in myeloma patients, and in so doing also uncovered new information about the nature of Bence Jones proteins. Representative of this work in the 1950s was the research by Korngold and Lipari at New York's Sloan Kettering Institute for Cancer Research and Cornell Medical College's Department of Biochemistry, and that by Slater and his associates at the Hospital of the Rockefeller Institute for Medical Research.

In examining the work on myeloma proteins in the 1950s, one finds a significant shift in the objectives of the researches. Through the early 1950s the abnormal proteins of multiple myeloma had been investigated primarily - although not exclusively - in relation to the light they might shed on the disease itself. Some investigators, particularly those like F. G. Hopkins who were based in university laboratories rather than hospitals, also studied Bence Jones protein because "a proper understanding of the disturbances involved could hardly fail to throw light on normal protein metabolism" (Hopkins and Savoy 1911, p. 190). But, in the main, the literature on abnormal myeloma proteins through the early 1950s indicates that these proteins were objects of clinical research, studied in reference to questions about multiple myeloma.

From the early 1950s on, however, one finds an increasing focus of interest in the myeloma proteins as "experiments of nature," as abnormal substances whose study might yield knowledge about the normal synthesis and structure of proteins in general, and about the globulin class of proteins in particular. In the remainder of this chapter, the trails of investigation that we will follow are those that led from Bence Jones proteins to the birth of molecular immunology, Once again, of course, this has not been the exclusive focus of research since the 1950s, for, as in earlier decades, the abnormal proteins of multiple myeloma have continued to be studied in reference to this and other diseases. And, as one looks at sources of support since the early 1950s for the increasingly basic lines of research that utilized Bence Jones proteins, one finds that it has come princi-[END OF PAGE 99]pally from government and private sources that recognize the joint medical and basic science import of the work. Thus, for example, Putnam's work in Tne 1950s was supported in part by the National Cancer Institute (NCI) and a private University of Chicago Fund, Korngold's researches by the Atomic Energy Commission and the NCI, and Slater's work by the Rockefeller Institute for Medical Research. Finally, in noting a shift of emphasis in the study of Bence Jones protein in the early 1950s, it is worth re-emphasizing that the work hinged on the existence of patients who, whatever the objectives of the various researchers, served as research subjects because they were afflicted with a critical experiment of nature.(6)

One of the major figures in the recent intertwined history of research on Bence Jones proteins and the structure of antibodies is biochemist Frank Putnam. The first of his many papers on multiple myeloma proteins appeared in 1953 when Putnam was at the University of Chicago. In his first foray into myeloma proteins, Putnam used sera from twenty-five patients to do quantitative physicochemical studies of the abnormal serum globulins, seeking, as had others before him, to determine their relationship to normal globulins (Putnam and Udin 1953). He had also launched studies of Bence Jones proteins, seeking to define their chemical structure and their metabolic origin. These serum and urinary proteins, Putnam explained, were of great interest to him as a protein chemist because "the profuse synthesis and diverse nature of the proteins elaborated in multiple myeloma constitute the most profound alteration in protein metabolism in any disease. It may be hoped that the biochemical study of this striking phenomenon may have import in the analysis of the mechanism of protein synthesis" (Putnam and Stelos 1953,p.357).

In his first paper on Bence Jones proteins, Putnam dealt with the still-debated question of whether they were chemically identical substances. His comparative physical and chemical analysis of Bence Jones protein from eighteen myeloma patients, Putnam reported, "has shown that in no instance are the proteins identical in physicochemical properties" (Putnam and Stelos 1953, p. 356; see also Putnam and Miyake 1954). Physicochemical evidence for the conclusion that different patients excrete different Bence Jones proteins, Putnam noted, had been repeatedly supported by immunological studies showing that "there are at least two antigenically specific groups of these proteins." But just what the diverse substances are was still a puzzle, one that Putnam sought to solve by studying their metabolic origin.

To tackle the question of the origin of Bence Jones proteins, Putnam and Sarah Hardy conducted isotopic studies on two patients, to trace the rate of synthesis and possible chemical precursor relationships of both myeloma globulins and Bence Jones proteins (Hardy and Putnam 1953; Putnam and Hardy 1955; Hardy and Putnam 1955). Several important findings, bearing on many debated questions about the nature of and relationship between serum and urinary proteins, emerged from these technically complex studies.

Putnam and Hardy found, first, that the serum globulin and Bence Jones proteins appeared to be synthesized independently in the patient's body. This and other data overturned a long-held hypothesis about the origin of Bence Jones proteins: that they were breakdown or degradative products of normal tissue proteins or of serum proteins. Rather, Putnam and Hardy found Bence Jones proteins to be synthesized "de novo" by the myeloma patient, and to be derived directly from the body's pool of metabolic nitrogen rather than from any tissue or plasma protein precursor.

Up to this point in their researches, Putnam and his colleagues had established two important "nots" about Bence Jones proteins. They are not physicochemically identical, and they are not formed as breakdown products of protein metabolism. What the proteins are - in terms of their structure and the site of their synthesis - remained uncertain; but the boundaries of the puzzle had been greatly clarified by these "nots."

We will resume our account of Putnam's work on Bence Jones proteins' structure and origin in a later section, when we move to the studies in the 1960s that elucidated antibody structure. Clues to this latter line of research, that would link together the efforts to determine the nature of Bence Jones proteins and the attempts of immunologists to decipher the structure of antibodies, emerged in the 1950s, through further immunological studies of myeloma proteins. The work of two research groups, in particular, pointed to the identification of Bence Jones proteins, and how that identification would lead to the molecular analysis of antibody structure.

At the Rockefeller Institute for Medical Research a group of investigators in 1950 were seeking to define the [END OF PAGE 100] immunological properties of normal human gamma globulin (protein antibodies), which had just been successfully purified. Their work, they found, was hampered because the various gamma globulin fractions were not single proteins. To overcome this problem, they seized upon "the special readily purified" serum proteins found in "tremendous quantity" in myeloma patients, hoping that study of these abnormal serum proteins would help to "clarify the relationship between the fractions of (normal) gamma globulin" (Kunkel, Slater, and Good 1951, p. 190).

The physicians who undertook this project in 1950 included Dr. H. G. Kunkel, Dr. A. J. Slater, and a young fellow at the Institute, Dr. Robert A. Good.(7) After demonstrating an immunological relationship between myeloma serum globulin and fractions of normal gamma globulin, Kunkel and Slater went on to study the immunological relationships between the myeloma serum globulins from a series of twenty-one patients. Three British investigators had shown in 1950 that myeloma serum globulin has two immunologically distinct components, gamm and beta (Wuhrmann, Wunderly, and Hassig 1950). As had this and previous studies, including Putnam's, the Rockefeller group's work indicated that every one of the myeloma proteins was different and individually specific to a given patient. But there also were commonalities, as the British study had found: the myeloma gamma globulin proteins were related immunologically to each other and to normal gamma globulin; the myeloma beta globulins similarly were interrelated, but were mostly distinct from the gamma globulins.

These findings on the immunological relationships among normal and myeloma globulins, Slater and Kunkel realized, had important implications for understanding the origin of myeloma proteins.

Insufficient knowledge of the properties of normal serum proteins had made it difficult to determine conclusively whether multiple myeloma proteins are truly abnormal entities or simply represent a great elevation of a single one of the many normal globulin components. In general the data obtained in the present study favor the hypothesis that these proteins are not normal but related to normal constituents.

It is tempting to consider the myeloma proteins as intermediates in the synthesis of y-globulin and antibodies with the more closely related myeloma proteins having most of the determinant groups of y-globulin. However, definite evidence on this point is lacking. (Slater, Ward, and Kunkel 1955, p. 106)

During the same time period, a more specific focus on the immunologic relationships between Bence Jones protein and normal and myeloma serum proteins was being pursued at Sloan Kettering Institute by Korngold and Lipari.(8) Citing both the Rockerfeller group's work and Putnam's researches, and thanking Putnam for his "valuable suggestions and criticisms," Korngold and Lipari reported on their own immunological studies in two papers in Cancer during 1956. In their first paper, on an antigenic analysis of myeloma serum globulins, Korngold and Lipari reported that their data were "compatible with the theory that MM (Multiple Myeloma) globulins are altered gamma-globulins (Korngold and Lipari 1956a, p. 191).

Then, in their second paper, Korngold and Lipari addressed the relationship between Bence Jones (BJ) proteins and serum proteins (globulins). Experiments from the 1920s on, they observed, "have created the impression that BJ proteins are antigenetically distinct and unrelated to eith MM globulin or normal gamma-globulin." But newer techniques had "made a properly controlled study of such antigenic relationships possible." In doing such a controlled antigenic study, Korngold and Lipari demonstrated, in contrast to previously accepted views, that Bence Jones proteins "are related to both normal gamma-globulins and the MM globulins" (Korngold and Lipari 1956b, p. 268). They also found, as had many prior invesitgators, that there are antigenic differences among Bence Jones proteins. And, going a step further, they showed that the urine of a given myeloma patient contains two major antigenic types of Bence Jones proteins (subsequently designated as K and L).

As had other investigators for many decades, Korngold and Lipari brought their findings to bear on the question of the origin of Bence Jones proteins. "The immunologic data presented here," they wrote in 1956,

show that BJ protein is antigenically related to norma gamma-globulin and the patient's abnormal MM globulin. Since BJ protein contains determinants present in MM globulin but not in normal gamma-globulin, it must be assumed that it is more closely related to the former . . . In the light of the [END OF PAGE 101] foregoing considerations, it may be speculated that BJ proteins are produced by cells that are no longer capable of synthesizing the complete MM globulin. The incompletely synthesized proteins, which are smaller and more deficient in antigenic groupings than the serum proteins, are excreted into the urine as BJ proteins. (Korngold and Lipari 1956b, p. 217)

In this "speculation," as the next decade of research would show, lay both the answer to Dr. Watson's question about Bence Jones protein, and the means for immunologists to uncover the structure of the antibody molecule.

What is it?: Ideas About Antibody and Immunity

To appreciate the impact that the identification and structural analysis of Bence Jones proteins had upon immunology in the 1960s, we need to look briefly at another "What is it? " question, one asked quite independently of work on myeloma proteins, by those who sought to determine the nature of antibodies and how they interacted with antigens. This path of inquiry, long and complex, can be dealt with here only briefly and selectively, to suggest the nature of theories about antibodies through the 1950s and thus set the stage for seeing how the study of Bence Jones proteins entered into immunology.

The science of immunology began to develop in the last decades of the 19th century as one of the major yields of the new field of bacteriology. "The whole initial concept of immunity," as Burnet has pointed out, "was in relation to infectious disease in man or his domestic animals. . .once [Pasteur] had shown the potentiality of immunization with attenuated pathogens (1880) the central objectives of immunological research were defined for the next sixty or seventy years. Immunology was one of the practically important aspects of medical bacteriology and almost all those concerned with its advance were medically trained" (Burnet 1969, p. 5).

By the late 1880s, building on Pasteur's epochal achievements between 1880-1885 with attenuated vaccines against diseases such as fowl cholera, anthrax, and rabies, the new field of immunology began developing in two major, often intersecting directions (Pasteur 1880a,b, 1884, 1885; Pasteur, Chamberland, Roux 1885). The enormous practical implications of vaccines, first, generated continuing efforts to extend the range of diseases for which prophylactic innoculations could be developed, and to find new ways of preparing such vaccines (see Parish 1965). Secondly, medical bacteriologists began attempting to explain the mechanisms or processes responsible for immunity. The identification of specific pathogenic bacteria focused attention on the question of why animals, including man, are normally resistant to most bacteria. At the same time, the development of vaccines framed questions about how immunity is produced and about the process of recovery from an infectious disease.

From the mid-1880s through the early years of the 20th century, much of the effort to explain the nature of immunity revolved around what were seen as two opposing explanatory models. The cellular theory held that immunity depends upon the ability of certain white cells in the body, phagocytes, to engulf infective materials and destroy them by a process of intracellular digestion (Metchnikoff 1888, 1901, 1908). The other major contender was the humoral theory, which proposed that certain substances or properties in the body fluids, principally the blood, were responsible for immunity (Nuttall 1888; Buchner 1890, 1900). The debate that flourished between proponents of cellular and humoral theories of immunity was one of the classic controversies that dot the history of science, generating fruitful research and ideas as well as polemical attacks and counterattacks. As often happens, this controversy abated when researchers recognized that their work pointed to the involvement of both humoral and cellular factors in immunity, a view that eventually would be confirmed in the 1960s with the discovery of the dual system of immunity residing in the T and B lymphocytes.

In the midst of the cellular vs. humoral theory debate, a major landmark in the history of immunology was established on December 4, 1890, when the discovery of antitoxin was announced by Emil von Behring and Shibasaburo Kitasato, researchers in Robert Koch's Berlin laboratory. Their discovery posed a major challenge both to the cellular theory and to relatively simple humoral explanations of immunity, opened up the field of serology, and, most importantly for our purposes, focused attention on what later would be termed the antigen -antibody relationship (von Behring and Kitasato 1890, transl. in Brock 1962; von Behring 1901).

Von Behring and Kitasato's experiments provided the first evidence that substances which are able to neutralize foreign materials are formed in blood serum [END OF PAGE 102] in response to infection. And, as they showed in the case of tetanus toxin, these antitoxic substances are highly specific. In the wake of this discovery, researchers soon demonstrated that animals can produce antitoxins against a wide range of poisonous substances. The extensive experiments and theories of Paul Ehrlich proved particularly important, for his work both broadened the scope of immunology beyond its early focus on diseases, and began to direct the attention of immunologists to what they later would call antibodies (Ehrlich 1891, 1897, 1900, 1908; Marquardt 1951).

By the turn of the century, a series of crucial questions had been framed about the nature of antibodies and antigens and how they interact, questions that only have begun to be answered in fine detail since the 1960s. The antitoxin experiments quickly revealed one of the most striking properties of antibodies, their specificity, and in turn posed the question of the source or mechanism of this specificity. Initial ideas centered on the toxin source of specificity, but by 1900 a variety of experiments had shown that antibodies were something other than modified toxins, some sort of special substances in the body that acted in specific response to the presence of an antigen.

One of the first major general theories of immunity that attempted to account for the origin and specificity of antibodies was the "side chain" theory developed by Paul Ehrlich in 1897. Ehrlich's theory, a blend of late 19th century biolological and chemical fact and fancy, proposed that certain cells in the body have special performed side-chains or groups of atoms that perfectly fit a grouping of atoms in a toxin - like a match between a lock and key. Once a side chain or antitoxin has become locked into a toxin, and its parent cell thus made inactive, Ehrlich supposed that the body began to manufacture moe of the side chains, thus accounting for antitoxin production (Ehrlich 1900).

Ehrlich's theory generated vast amounts of research by both its supporters and critics. A major challenge to his theory, and another significant broadening of immunology's scope of inquiry, was set into motion in 1898 when Pasteur's protegé, Jules Bordet, demonstrated the immune lysis (destruction) of red blood cells (Bordet 1898, 1899, 1903). Bordet's work triggered an increasing interest in the immunological behavior of both cells and body fluids. Then, Karl Landsteiner showed that any molecule, either natural or artificial could stimulate an immune response under certain conditions, a finding that argued against the validity of Ehrlich's theory (Landsteiner 1930, 1946). For it was hard to conceive, as the side-chain theory demanded, that the body had an infinite number of preformed side-chains or receptors on its cells, capable of fitting specifically to a structurally infinite variety of antigens.

Rather, Landsteiner's work with artificial antigens seemed to indicate that antigens govern the specificity of antibodies by somehow directing or "instructing" the cell's activities. Instructive theories of antibody formation gained in sophistication and status in the 1940s with the entry of Linus Pauling into immunology, for his work began an "effective association of immunology with the developing principles of biochemistry (Burnet 1969, p. 5). Stimulated by conversations with Karl Landsteiner, Pauling had begun to work on the molecular bases of serological reactions in the mid-1930s. From this work, in turn, he developed, in as precise form as the time permitted, what is now recognized as the classical instructive theory of antibody formation (Pauling 1940).

Between 1940 and the mid-1950s, modifications of Pauling's instructive theory were proposed by several researchers, consonant with new methods and knowledge in protein chemistry and immunology (see Burnet 1963). At the same time, it also became evident that a number of important immunological phenomena could not be explained by instructive theories. In particular, these theories could not account for the persistence of modified immunological reactivity, or for the fact that antibodies usually are not produced against chemical configurations normally present in the body (the phenomenon of immunological "recognition") (Burnet and Fenner 1949). Such considerations led to the development of various "selective" theories of antibody formation, a development linked most closely with the work of Australian virologist and immunologist F. MacFarlane Burnet.

Beginning with his 1941 book, The Production of Antibodies, Burnet had carefully studied and critiqued instructive theories, and sought to interpret antibody formation primarily in biological rather than in chemical terms.(9) But, as Burnet himself recognized, the first major alternative to the generally accepted instructive theory came in 1955, when Danish immunologist Niels Jerne proposed a "natural selection theory" (Jerne 1955, 1967). Jerne's theory served to explain why the body does not make antibodies against its own constit-[END OF PAGE 103]uents; why, in Burnet's work, it recognizes "self" as opposed to "not self." But there were explanatory deficiencies in Jerne's theory which were met in 1957 by new forms of a selection theory, developed independently by David Talmage at the University of Colorado and by Burnet (Talmage 1957, Burnet 1957).

Both Talmage and Burnet recognized the variety of globulins that can be present in the blood, and felt that a satisfactory theory of antibody production should assume that the antibody -replicating elements were cells (like various globulin cells or their precursors), rather than, as in Jerne's view, extracellular protein able to replicate only if incorporated into a particular cell type like the phagocyte. Burnet developed this conviction in terms of a "clonal" selection theory (the word "clone" designates a group of cells that originate from the same parent cell). "In its simplest form," Burnet has explained:

the "clonal selection" theory postulates that, in the course of embryonic differentiation, a very large number of clones of mesenchymal cells arise, each carrying a specific immunological potentiality to react with a single antigenic determinant. Depending on various circumstances, the reaction following contact with antigen may be manifested in one of three different forms: (a) the cell may be damaged so that its capacity to multiply is lost, or it is actually destroyed; (b) it may be stimulated to proliferate; or (c) it may undergo conversion to plasma cell character and produce and liberate antibody. Antibody production follows the normal rules of protein synthesis, the information needed for its specificity being stored in the cell genome. (Burnet 1963, pp. 91-92)

Through the late 1950s and into the 1960s, Jerne, Talmage, Burnet and other proponents of a selective theory of antibody formation and action argued lengthily and persuasively for the logical correctness of their views. But, as they knew, instructive theories had long held sway, and at the time there was no direct experimental evidence to confirm one or the other school of thought. By the early 1970s, however, new lines of evidence pointed to the essential validity of selective theories and, in the words of Gerald Edelman, "the fundamental idea of these theories is now the central dogma of modern immunology: molecular recognition of antigens occurs by selection among clones of cells already committed to producing the appropriate antibodies, each of different specificity" (Edelman 1973, p. 830).

Looming large among the evidence that established clonal selection as the central dogma of modern immunology was the molecular analysis of antibody structure. And, as we resume our chronicle of Dr. Bence Jones' strange protein, we will learn how it served in the 1960s as the crucial experiment of nature for that analysis.

Bence Jones Proteins, Light Chains,
and the Structure of Antibodies

By the late 1950s, the study of myeloma proteins had generated a number of provocative clues to the identity of Bence Jones Droteins. These myeloma proteins now were known to be antigenically related to both normal and myeloma immunoglobulins, and hence it seemed reasonable to speculate that BJ proteins might represent intermediary or incompletely synthesized immunoglobulins.

During this same period, in the late 1950s, immunologists and molecular biologists began an intensive attack on the structure of antibodies. As we saw in the preceding section, there was a substantial body of information and theories about the functions and origins of these proteins, but little was known about their detailed chemical structure. Determining the structure of antibodies, researchers knew, should provide understanding of their unique specificity for antigens, and perhaps a basis for choosing between instructive and selective theories of antibody production. But researchers had been confounded by the experimental problems created by two aspects of antibodies: they are very large proteins (with a molecular weight of 150,000 or greater), and they are very heterogeneous chemically (a problem, too, for those who had been working on Bence Jones proteins).

The ultimately successful quest to define the molecular structure of an antibody was launched during 1958, in researches centered at the Rockefeller Institute in New York, and in the Department of Immunology at St. Mary's Hospital Medical School in London. The leaders of the two research teams were physician-immunologist Gerald Edelman and biochemist-immunologist Rodney Porter, who in 1972 would be honored will the Nobel Prize in Physiology or Medicine for the.. structural studies of immunoglobulines.

Rodney Porter, who had trained as a biochemist under Frederick Sanger, developed a method of breaking an immunoglobulin molecule into analyzable fragments [END OF PAGE 104] by using protein-dissolving enzymes. One of his major findings from this work, which occupied years of effort, immunoglobulin G (IgG) molecule contained three globular fragments: an "Fc" fragment common to all molecules, and two identical "Fab" fragments each of which carried a specific antigen-binder 1959, 1973).

In New York, Edelman developed another approach to cleaving IgG molecules that involved breaking their disulfide bonds and then exposing the molecule to dissociating solvents. By these methods, Edelman found that the IgG molecule was not, as had been thought, composed of a single polypeptide chain, but rather was composed of several discrete chains linked by disulfide bonds. This first phase of his research also indicated that the IgG molecule had two kinds of polypeptide chains, later named heavy and light chains, and that these chains were not the same as the fragments Porter with his enzyme cleavage method (Edleman 1959; Edelman and Poulik 1961). Understanding the gross structure of antibodies, then, would involve the relationship between the polypeptide chains isolated by Edelman's methods and the fragments found by Porter.

The second major obstacle to a structural analysis, however,still confronted researchers in 1960: the enormous chemical diversity of antibodies. As Edelman recalled in his Nobel address, this chemical diversity posed "two challenging questions."

First did the observed heterogeneity of antibodies reside only in the conformation of their polypeptide chains, as was then widely assumed, or did this heterogeneity reflect differences in the primary structures of these chains, as required implicitly by the clonal selection theory? Second, if the heterogeneity did imply a large population molecules with different primary structures, how could one obtain the homogeneous material needed for carrying out a detailed analysis? (Edelman 1973, p.831)

An "accident nature rather than direct physico-chemical assault" proved to be the means by which his colleagues were able to deal simultaneously with both of these challenges. Drawing upon the prior work by Putnam's group and by Slater and Kunkel at the Rockefeller, Edelman realized that Bence Jones proteins would provide him with a readily available, relatively homogeneous and low molecular weight substance, known to be antigenically related to immunoglobins.

By 1961, Edelman had begun to formulate "a unifying hypothesis . . . for the structure of proteins in the [immunoglobulin] family." IgG molecules, he and Poulik stated, "appear to consist of several polypeptide chains linked by disulfide bonds," and bivalent antibodies "may contain two chains that are similar or identical in structure." Thus, the heterogeneity and antigenic specificity of antibodies "may arise from various combinations of different chains as well as from differences in the sequence of amino acids within each type of chain" (Edelman and Poulik 1961, p. 880).

The discovery that immunoglobulin molecules consist of several polypeptide units, Edelman recognized, also had "a possible bearing upon the pathenogenesis of diseases of gammaglobulin production." "A primary defect in. . .multiple myeloma," he suggested, "may be a failure of specificity and control in production and linkage of various subunits to form larger molecules." Thus, he and Poulik were able to propose a structural basis for previous speculations that Bence Jones proteins were incompletely synthesized myeloma globulins. "Bence Jones proteins may be polypeptide chains that have not been incorporated into the myeloma globulins because of a failure in the linkage process" (Edelman and Poulik 1961, p. 881; see also Poulik and Edelman 1961).

Edelman's hypothesis about the nature of Bence Jones proteins was confirmed "one exciting afternoon" when he and a graduate student, Joseph Gaily, "heated solutions of light chains isolated from our own serum immunoglobulins in the classical test for Bence Jones proteinuria": the test used by Dr. Watson in 1845, that had generated a century of efforts to identify Bence Jones proteins. Dr. Watson's question, "What is it?", at last was answered. Edelman and Gaily, aware of the history underlying their own work, noted that "Dr. Jones concluded that it was the 'hydrated deutoxide of albumin.' [Our] studies would supply the question with another qualified answer: Bence Jones proteins appear to be polypepticle chains of the L [light chain] type that have not been incorporated into myeloma proteins" (Edelman and Gally 1962, p. 225). What the Rockefeller investigators had observed was that their serum immunoglobulin light chains "behaved as Bence Jones proteins, the solution first becoming turbid, then clearing upon [END OF PAGE 105] further heatings. A comparison of light chains of myeloma proteins with Bence Jones proteins. . .confirmed the hypothesis (Edelman 1973, p. 831; Edelman and Gally 1962, 1968; Mannik and Kunkel 1962).

The Rockefeller group's experiments, by 1961, had three major implications for a molecular structure-activity analysis of antibodies. First, "because different Bence Jones proteins had different amino acid compositions [as determined by Putnam and others in the 1950s], it was clear that immunoglobulins must vary in their primary structures." Second, this deduction in turn "lent strong support to selective theories of antibody formation." Third, because Bence Jones proteins now were known to be identical or analogous to light chains, with a relatively low molecular weight, it had become possible to begin "a direct analysis of the primary structure of an immunoglobulin moelcule" (Edelman 1973, p. 832).(10)

The first laborious analyses of the amino acid sequences in Bence Jones proteins were undertaken by Hi1schmann and Craig at the Rockefeller and by Frank Putnam, then heading a laboratory group at the University of Florida College of Medicine. By spring 1965, partial sequence analyses of several different Bence Jones proteins had revealed another key feature of antibody structure: the structural diversity between light chains was limited to what became known as the variable region of the chain (Hilschmann and Craig 1965; Titani and Putnam 1965; Titani, Whitley et al. 1965; Putnam and Easley 1965).(11)

Through the mid-1960s structural analysis was focused on the antibody molecule's light chain, because of the natural experiment afforded by Bence Jones proteins. Less work was being done on the other type of polypeptide chain identified by Edelmann's experiments, the heavy or H chain. But by 1964, even in the absence of much detailed knowledge, comparisons between heavy and light chain structure had shed light on another reason for the diversity of antibodies: the existence of antibody classes. Comparisons of chain structure in the first three classes to be identified showed that classes have similar kinds of light chains, and that it is structural differences in their heavy chains which give them their distinctive class characteristics or effector functions (Bull. WHO 1964).

From the mid-1960s on, the analysis of both the heavy and light chains of antibodies moved forward at an accelerating pace, generating an increasingly more detailed picture of an antibody molecule's structure and new glimpses into how that structure governs an antibody's functions. In l962, Rodney Porter and his colleagues in London had begun to link their analysis of the molecule's Fc and Fab fragments with the Edelman group's study of polypeptide chains. From this work Porter hazarded what proved to be a brilliant guess: the IgG molecule is composed of two large (heavy) polypeptide chains and two smaller (light) chains, making in total a four polypeptide chain structure (Fleischman, Pain, Porter 1962; Fleischman, Porter, and Press 1963).

Aided by this new understanding of the relationship between chain structure and Porter's enzyme fragments, plus the growing knowledge about the structure of Bence Jones proteins, Edelman and Gally in 1964 developed a topological model of the IgG molecule (Edelman and Gally 1964). Then, in 1965, Edelman judged that it was feasible to begin working out the complete molecular structure of an immunoglobulin molecule, and set to work on a sample of human myeloma IgG.

While Edelman's group was laboring in New York, Frank Putnam and his associates, now at the University of Indiana, were pursuing their painstaking mapping of the amino acid sequences in type K and L Bence Jones proteins (the two major antigenic types identified by Korngold and Lipari in 1955). In 1966, Putnam's group published a "tentatively" complete amino acid sequence for type K, and in 1967 reported a complete sequence analysis for type L Bence Jones protein (Putnam, Titani, Whitley 1966; Wikler, Titani et al. 1967; Putnam 1969). Thus, for the first time, researchers had accomplished the task of determining the structure of a Bence Jones protein, and hence of a light chain in the antibody molecule.(12)

Solving the complete primary structure of an antibody molecule, the task undertaken with myeloma IgG by Edelman's group in 1965, received a strong stimulus from Putnam's determination of the L-type light chain structure. Another major input came from Porter's group in London, when they reported on the heavy chain structure of IgG from a myeloma patient and from normal rabbit serum (Porter 1967). Porter and his colleagues focused their efforts on that half of the heavy chain where they believed the antibody's antigen combining site was located. The partial analysis he had completed by 1967 encouraged Porter to "claim that, if the [END OF PAGE 106] work. . .on the sequence of the heavy chains . . . can be carried to completion, it appears to offer a feasible experimental approach to obtaining an answer to the question: does the amino acid sequence alone control antibody specificity, and, if so, how is it achieved? (Porter 1967, p. 425).

The achievements of groups such as Putnam's and Porter's represented important pieces of the puzzle of antibody structure, but its total solution required more. If, as researchers now suspected, each immunoglobulin molecule consisted of two identical light chains and two identical heavy chains, a complete map of its primary structure would be obtained by a sequence analysis of one light and one heavy chain, and by locating all the disulfide bonds and the various carbohydrate prosthetic groups attached to the chains.

This was the task that Edelman and five colleagues at Rockefeller Institute had undertaken in 1965, a task that would occupy "a good portion of [their] waking hours" for four years (Edelman 1973, p. 883). Then, at a scientific meeting in 1969 Edelman announced the completion of their work. "We now report the amino acid sequence fo an entire human (IgG1 immunoglobulin (molecular weight 150,000), the location of all disulficle bonds, the arrangement of light and heavy chains, and the length of the heavy chain [variable] region" (Edelman, Cunningham et al. 1969, p. 78).

Within the scope of this essay we have come full circle, for now, in 1969, immunologists had gained an understanding of the primary structure of an antibody molecule, and of how the variable and constant regions in its heavy and light chains make possible an antibody's extraordinary specificity and its class or effector functions. With this understanding the science of immunology, that had begun in the 19th century with the work of medical bacteriologists such as Louis Pasteur and Robert Koch, underwent a major revolution. And, as we have seen, a "crucial experiment of nature," the long puzzling proteins of multiple myeloma patients, occupied center stage in the revolution. The quest to identify the nature and origin of these proteins, begun by Dr. Watson's query in 1845, had ended more than a century later in "the first of the projects of molecular immunology, the task of which is to interpret the properties of the immune system in terms of molecular structures" (Edelman 1973, P. 830).

Those involved in the many facets of immunobiology recognize that the molecular analysis of antibody structure is only a beginning, albeit a profoundly important one, in understanding the nature of the immune system. The structural basis of antibody diversity has been revealed, but, as Gerald Edelman observed in his 1972 Nobel Prize address, "two great problems of molecular and cellular immunology remain to be solved." The first of these problems is "the origin of intrasubgroup diversity:" what are the genetic mechanisms that lie behind the enormous variability of amino acid sequences within portions of the heavy and light chains? (see note 11). The second problem, recognized since Burnet proposed his clonal selection theory, is to explain the induction of antibody synthesis: how is the clonal expansion of lymphocytes triggered after their receptor antibodies combine with antigens? Eventually, immunologists are confident, these major questions too will be answered, and immunology again will be transformed "both as a discipline and as an increasingly important branch of medicine" (Edelman 1973, p. 839). [END OF PAGE 107]


(1) Immunodeficiency diseases reter to Those conditions, frequently inherited, that are caused by various types of defects in immunological function. Because a majority of patients with inherited forms of immunodeficiency have been found to have quantitative deficiencies of T cells, B cells, and their products, the study of these diseases has had a major import for the development of modern immunological concepts (see Bach and Good 1972).

(2) Like the B cell, the T cell has receptors on its surface that can specifically recognize antigens. But, whereas the B cell's response to an antigen is to produce antibody, the T cell releases substances called lymphokines. The lymphokines, in turn, attract monocytes (a type of white cell) which engulf and digest invaders such as bacteria. The T cells also directly attack foreign proteins.

(3) The principal defensive or effector functions of each of the five antibody classes are, briefly, as follows. IgM, the first antibody to be produced in response to the presence of an antigen, is evolutionarily the most primitive of the five types, which perhaps accounts for its relatively weak binding ability. IgM is involved in certain autoimmune diseases, such as rheumatoid arthritis. The major antibody type in mammals is IgG. Both IgG and IgM act against foreign organisms and toxins, but IgG has a more specific antigen binding ability and remains in the blood much longer than IgM. IgG also is the only antibody type able to cross the placenta, to confer passive immunity from mother to fetus. IgA is present in large quantities in the intestine, where it acts as a barrier to prevent the escape of pathogenic organisms from the gut into the blood stream. The known functions of IgE are primarily negative ones, from man's perspective, as this antibody is responsible for triggering allergic and asthmatic responses. The functions of the fifth type of antibody, IgD, presently are unknown (Lewin 1974, pp. 28-31).

(4) An antibody's structurally specific antigen binding site results from how the amino acids are arranged in the variable regions of its heavy and light chains, and the vast number of possible binding site shapes are generated by permutations in the possible sequences of the 108 amino acids that make up the variable regions of both the heavy and light chains. Investigators also have found that some areas of the variable regions are more variable in amino acid sequences than others; these are termed hypervariable regions.

(5) The views of organic chemists in the 11840s on the nature of albumin and protein were developed, most influentially, by the work of Berzelius, Mulder, and Liebig on the elementary analysis of albuminoid substances. Berzelius proposed the word "protein" in 1838 to designate the "organic oxide of fibrin and albumin," and he felt that this "protein" was the "primitive or principal stubstance of animal nutrition that plants prepare for the herbivores, and which the latter then furnish to the carnivores." Fruton notes that Berzelius' and Mulders' conclusions about the identity of fundamental protein units were adopted by the influential Liebig in 1841 "on the basis of analyses performed by his associates Johann Joseph Scherer and Henry Bence Jones" (Fruton 1972, pp. 96-97).

(6) For those interested in the nature and development of clinical research, we note that multiple myeloma patients participated in two different types of experimental work. First, some patients had blood and urine samples collected for laboratory analysis, or in vitro research. Secondly, some patients were given substances, such as radioactively labelled glycine, and than had blood and urine samples collected for in vitro study.

(7) Good was at the Rockefeller Institute on a one-year fellowship, having become interested in immunology through his work as a pediatrician at the University of Minnesota. When Good returned to Minnesota in 1950, he brought with him a number of intriguing and puzzling clinical observations from his year in New York. Among these was his learning from myeloma patients that they had serious problems with infection. Why was this the case, Good wondered, since these patients had such high levels of plasma cells or antibodies in their blood, whose function is to combat infection. It was the pursuit of such clinical puzzles that, among other accomplishments, would lead Good and his colleagues at Minnesota in the 1950s and 1960s to decipher the dual humoral and cellular system of immunity (sea Good 1972; Lewin 1974, ch. 1).

(8) Other research groups besides those discussed in the chapter also were conducting important research with Bence Jones proteins, myeloma proteins and normal globulins in the 1950s, that would feed into the molecular analysis of antibody structure. Among these groups were H. F. Deutsch and his colleagues in the Department of Physiological Chemistry at the University of Wisconsin, who worked on physicochemical and immunochemical properties of myeloma and Bence Jones proteins and of the macroglobulins found in patients with Waldenstrom's macroglobulinemia (see Deutsch 1955; Deutsch et al. 1955, 1956; Morton and Deutsch 1958).

(9) As part of their indirect template theory, Burnet and Fenner in 1949 predicted the existence of immunological tolerance, a phenomena seen but not understood in 1946 when Owen observed that most twin cattle are born with and retain a mixture of each other's red blood cells. As part of their theory that the body's recognition of self develops slowly during fetal life, rather than being inherited, Burnet and Fenner predicted that an antigen experimentally administered to an embryo would not generate antibodies, but would be accepted as a normal part of the embryo's body. This state of "acquired tolerance" was demonstrated in 1953, in experiments on tissue grafting by Medawar and his colleagues. Medawar's immunological research, in turn, had developed out of his work for England's Medical Research Council in the early 1940s on the problem of skin grafts for W. E. 11 burn victims. In 1960, Burnet and Medawar received the Nobel Prize for their work on acquired immunity (Burnet and Fenner 1949; Medawar 1944, 1945, 1957).

(10) Edelman and Gaily recognized that their use of myeloma globulins to analyze immunoglobulin structure confronted them "with a peculiar dilemma in structure-activity determination. Either we commit a fundamental heresy in analyzing a myeloma globulin the activity of which is unknown, or a structural heresy in studying the amino acid sequences of an indeterminately [END OF PAGE 108] large mixture of specific antibodies." Despite this dilemma, they felt that the evidence for myeloma globulins being typical immunoglobins permitted them to proceed with a "confident analysis of the over-all structure of immunoglobins" (Edelman and Gally 1968, pp. 329-330).

(11) The discovery that the variable region of the antibody molecule is responsible for the enormous range of structurally specific antigen binding sites, as Porter observed in his Nobel address, "raised very difficult problems as to the genetic origin of these many different amino acid sequences" (Porter 1973, p. 716). A now widely accepted explanation for the sequence variability seemed at first a heresy, for it contradicted the one gene-one polypepticle dogma established, as we saw in chapter 5, largely through the analysis of abnormal hemoglobins. What geneticists and immunologists accept as the most likely explanation for the structure of antibody chains is a "two genes-one polypepticle chain" control mechanism, in which one gene codes for the constant region and one for the variable region. Two major explanations for a two-gene mechanism have been advanced, the germ-line and the somatic mutation theories. According to the latter theory, the multiple genes that code for the variable regions are the product of somatic mutation of a relatively small number of germ-line genes. (see Porter 1973; Putnam 1969, 1972).

(12) Following his work on the structure of light chains, Putnam and his colleagues went on to "tackle the largest immunoglobulin of all, the IgM macroblobulin produced in large amounts by patients with Waldenstrom's macroglobulinemia" (Putnam 1972, p. 370). The task was a formidable one, for the IgM is many times larger than IgG, with a molecular weight of about 1,000,000, and has several other properties that also complicated the task of analyzing its primary structure (see Porter 1972, pp. 371-378). [END OF PAGE 109]

Chapter 6

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