Immunology in the Twentieth Century

From Basic Science to Clinical Application

Domenico Ribatti

• Publisher: Academic Press
• Year: 2018
• Print ISBN:
  9780128161456
• eBook ISBN:
  9780128161463
• Topic: Medicine
• Subtopic: Immunology

Immunology in the Twentieth Century: From Basic Science to Clinical Application grew out of common knowledge that those who survived many of the common infectious diseases rarely contracted the same disease again. This book charts the historical development of this vital branch of medicine in a concise volume, covering both the basic science involved and the clinical applications. Immunology as a distinctive subject developed in the mid-twentieth century as researchers started to understand how the adaptive immune system aids the defense against pathogens. The subject has grown in importance and diversified into specialist fields, such as immunohistochemistry, immunogenetics and immunopathology. - Provides a concise overhead of the history of immunology and its applications in medicine - Includes a discussion of the scientists who were pioneers in landmark discoveries in immunology - Summarizes the clinical applications of major discoveries

Chapter 1

The Antibodies

Abstract

This chapter provides a history of the discovery of the antibody production mechanism from the 19th Century, when the Louis Pasteur theory was accepted, to the present day. The chapter briefly outlines some of the various theories on antibody production that were put forward and the people who elucidated their veracity by experimental observation.

The chapter then discusses the history of the discovery of the antibody’s structure and the molecular basis for the diversity of antibodies, and how the understanding of how the huge antibody repertoire was possible. The chapter also outlines how antigens influence the structure of antibodies and the history of how this was discovered, covering direct template theory, indirect template theory, and clonal selection theory. The concepts of idiotype and antiidiotype are also introduced.

Keywords

Antibodies; side chains theory; monoclonal antibodies; antibody-poiesis; direct template theory; indirect template theory; clonal selection theory; idiotype; antiidiotype

1.1 The Antibody Production

The antibody production mechanism is a central problem of immunology. Research on this mechanism started at the end of 19th century, when the Louis Pasteur theory, according to which the disease causative agents are germs, was officially accepted. Several groups began to study the reactions between bacterial toxins and “antitoxins” that appear in the serum after the infection. The diphteria bacillus was isolated in 1884 and a few years later it was demonstrated that its culture contained a toxin produced by the same bacillus. The injection into a guinea pig of a fraction of the filtered culture fluid caused his death, while animals injected with nonletal doses of the toxin gradually became resistant to its action.

German bacteriologist Emil A. von Behring (Nobel Prize in 1901 for his discoveries on antidiphterial and antitetanic serums) demonstrated together with his Japanese colleague, Shibasaburo Kitasato, that the serum of vaccinated animals contained serum antitoxin and coined for these substances the term Antikörper, or antibodies. In the following years, Belgian physician Jules Bordet, professor of bacteriology and virology at the University of Brussels (Nobel Prize in 1919) demonstrated that antibodies-mediated bactericidal activity required the presence of a thermo-stimulant factor (complement) also present in normal serum. In 1899, Bordet demonstrated that if rabbit red blood cells were injected into the guinea pig, the serum of this one becomes capable of inducing hemolysis in the rabbit red blood cells; hemolytic capacity was destroyed by heating at 56°C for 30 min and reintegrated by the addition of fresh serum of guinea pig not treated with rabbit red blood cells. In 1889, Paul Ehrlich and the pathologist Julius Morgenroth (1871–1924) confirmed these observations and defined as a complement supplementing the nonspecific factor present in normal serum, which was able to completing the hemolytic action of immune sera.¹

In 1890, von Behring and Ehrlich² published an article describing a technique for measuring antidiphtheria antibodies in the same preparations used by Behring, allowing standardization and thus making serum safe for clinical practice. Ehrlich's quantitative observations demonstrated that the immune response was equivalent to the proliferation of antibodies following contact with an infectious agent.³

In 1893 Hans Büchner, a German biochemist, hypothesized that the organism was able to rework the antigen converting it into the specific antibody. Ehrlich was the first to demonstrate that the biological meaning of immune response went beyond the protection from infections and to obtain from rabbits antibody antimilk and egg albumen proteins. He coined the term horror autotoxicus to indicate that vertebrates do not produce antibodies directed against one or more of the individual's own proteins, laying the groundwork to explain the difference between self and not-self. In 1900, Ehrlich proposed the side chains theory into the Cronian Lecture titled “On immunity, with special reference to cell life” to explain antibodies production. The theory hypothesized that a white blood cell had membrane receptors with side chains able to chemically bind the foreign substances. The bond induced the cells to produce many copies of the bound receptor which as antitoxins (antibodies) were transferred into the blood circulation and neutralized toxins.

The side chains theory was based on the assumption that the specificity of the interaction between antigen and antibody was discrete and absolute, in other words the interaction between the antibody molecular structure and the antigen would depend on a complementary stereo adaptation, precise and exclusive, and the chemical reaction dynamic was governed by strong chemical bonds (covalent) (Corbellini, 1996, pp. 256–7).

Ehrlich theory postulated that the antibodies were produced before antigens exposure. This theory was no longer acceptable when it was possible to demonstrate that antibodies against any lab chemically synthesized substance could be formed although no previous exposure had occurred, and it was assumed that the antibodies would be synthesized using the antigen as a template. Franco Celada (1992, p. 13) highlighted that the Ehrlich theory suggests the natural appearance and without any external informational intervention, of different antibody structures in the same cell and the selection of those able to bind to the antigen. The selected structure would then be reproduced in many copies. His contemporary immunologists did not accept the Ehrlich’s suggested idea and for over half a century prevailed the innate preference for an explanation that attributed to the antigen the ability to initiate the lymphocytes to antibody-specificity being produced from themselves and from the daughter cells. This attitude had reduced immunology to the last bastion of Lamarckian inheritance.

In 1906, in Germany, Ernest P. Pick, an experimental Czechoslovakian pathologist, together with Friedrich P. Obermayer, an Austrian physician and chemist, demonstrated that when chemical groups containing iodine and nitrate are attached to a protein, its antigenic properties were deeply modified. The Austrian Karl Landsteiner, when he was working at the Rockefeller Institute for Medical Research, put the antigenic proteins at his disposal into contact with a wide variety of chemical groups derived from both pathogenic microbes or synthesized in test tubes (synthetic dyes or haptens)⁴ and demonstrated that every molecule induced the formation of a different antibody, so confirming the observations of Pick and Obermayer.

Since almost nothing was known about the antibody biochemical features but it was possible to induce the organism to react against any foreign chemical structure as long as it was adequately presented, it was intuitive for the immunochemists that the production of a specific antibody occurred under the direct or indirect guidance of the antigen, i.e., the antigen would play an instructive role, acting as a template for the building of antibody structure of recognition (Corbellini, 1996, p. 258).

The clear result of this was that an animal was able to synthesize a wide (unlimited) set of antibodies but at the same time it was difficult to think that each white blood cell could produce such a large number of side chains. The antibodies present in the serum of an animal immunized by a specific antigen are polyclonal since they constitute the secretion product of different clones of B-lymphocyte each able to produce immunoglobulins (Ig) with peculiar characteristics as to the iso- and idiotype. Polyclonal antibodies have presented numerous limitations mainly related to the heterogeneity of antibody preparation obtained at different times.

These problems have been overcome by the hybridoma technology by which homogeneous populations of antibodies with a defined specificity can be obtained. These antibodies have been termed monoclonal as they are produced by a single cell clone. The revolutionary procedure for obtaining monoclonal antibodies was described by Georges Köhler and Cesar Milstein,⁵ Nobel Prize in Medicine in 1984. These authors demonstrated that the fusion between malignant plasma cell and lymphoid cell generated as a result some cells called hybridoma cells that acquired both the properties of a myeloma cell⁶ to undergo infinite replication cycles and the characteristic of the immune lymphoid cell to secrete antibodies.⁷

Köhler and Milstein carried out the fusion between myeloma cells and spleen lymphocytes of a mouse after its immunization with a particular antigen. Individual hybrid cells can be cloned and each clone can produce strong quantities of identical antibodies directed against a single antigenic determinant. Clones can be kept alive for ever and injected into animals to obtain large-scale monoclonal antibodies. Monoclonal antibodies were used for the analyses of somatic mutations and it was possible to demonstrate that mutations are very rare in IgMs, which correspond to primary response, while they are common in antibodies of different classes that correspond to secondary response.

The application of murine monoclonal antibodies in humans leads to at least three principal limitations: (1) they are immunogenic; (2) show a relatively short half-life; and (3) have a poor murine antibodies Fc region recognition by the human effector mechanisms. The development of a series of experimental approaches allowed the transforming of a murine immunoglobulin into a chimeric form (part from human and part from mice), and ultimately totally human, allowing its therapeutic use.

With the so-called humanization, it is possible to selectively replace as much as possible of the murine antibody molecule, including the antigen binding regions, with human proteins. The completely human antibodies production has been possible through the development of phage-display platforms and recently by means of genetically modified mice. The validity of this innovative pharmacological approach is confirmed since more than 30 monoclonal antibodies approved by the United States (FDA, Food and Drug Administration) and European (EMA, European Medical Agency) regulatory authorities are present today and about 500 more are being developed. At present the monoclonal antibodies are used in many clinical fields including oncology, hematology, rheumatology, immunology, and most recently the cardiovascular field.

1.2 The Antibodies Structure

In 1926, Lloyd D. Felton (1885–1953) an American immunologist together with his colleague George H. Bailey determined the protein structure of antibodies, which was confirmed through quantitative and qualitative studies of antigen–antibody complex precipitation reactions carried out by Michael Heidelberg and Forrest E. Kendall.

In 1938, Arne Tiselius, a Swedish Biochemist and Nobel Prize in Chemistry in 1948, and US Elvin Kabat, one of the founding fathers of quantitative immunocytochemistry, demonstrated that the serum electrophoresis allowed the separation of the globulins into three bands (α, β, γ), and they established that antibodies were contained in the γ fraction, the one that migrated more slowly. Following this observation, the term gammaglobulins was used as an antibody synonym. Subsequently, since it resulted that the antibody activity was contained also in α and β fractions the World Health Organization (WHO) proposed the term of Ig for all the proteins provided with specific antibody activity. It was also possible to establish that immune serum contained different types of antibodies which were subdivided into five classes, Ig G, M, A, D, E on the basis of the different functional and structural characteristics. The basic distinction is based on the existence of class antigens, located in heavy chains (H). These classes depend on different portions of the heavy chain polypeptide, portions that possess antigenic activity by constituting an antibody molecule own epitopes.⁸ Some Ig also have additional polypeptides and some of them form five unit oligomeric associations each consisted of heavy and light coupled chains.

In 1956, at the Pasteur Institute in Paris, the French biologist Jacques Oudin (1916–83) discovered the allotypic markers that are the individual specificity of antibody populations within the species, and the Swedish immunogenicist Rune Grubb (1920–98) described the human allotypes. It was possible to demonstrate that the intraspecific antigenic determinants located into the antibodies were genetically determined, i.e., the antigenic differences reflected the primary structural differences of antibodies.

In 1959, the British biochemist Rodney Porter (1917–85), together with the US biologist Gerald E. Edelman (1929–2014), Nobel prize in Medicine in 1972, whilst treating the rabbit antibodies with papaine, broke a monomeric immunoglobulin into three fragments, two of them with the same molecular weight of about 50.000 Da, defined as fragment antigen binding (fab), each containing a site for the combination with the antigen, and the other as crystallizable fragment (fc), with an analogue molecular weight, responsible for numerous effective functions such as complement activation, distribution of Ig in the various compartments of the body, cross-linking of the placental barrier, and catabolism of the whole molecule. The resolution of Ig structure was obtained by establishing the relationship between the polypeptide chains identified by Edelman and the papaine digestion products obtained by Porter.

In 1959, Edelman demonstrated that the reduction with mercaptoethanol in the presence of urea induced the break of 15 disulfide bounds in a monomeric Ig molecule with the separation of four polypeptide chains, two described as heavy, each made of 440 amino acids, and two as light (L) chains, each made of 220 amino acids, with different molecular weights of 55.000 and 23.000 Da, respectively. In 1962, the English biochemist J.B. Fleischman, utilizing mercaptoethanol as a reduction reagent in association with acetamide iodine as agent alchilant, obtained the subdivision of Ig into four polypeptide soluble chains, separable into two fractions by Sephadex-G75 gel-filtation.

In 1963, the US immunologist Henry Kunkel (1916–83) recognized the existence of an Ig marker located in the recognition regions that indicated the antigen specificity of the single antibody, and in 1966 Oudin and M. Michel defined this serological feature as “Idiotypic specificity.”

In 1965, Norbert Hilschmann and Lyman C. Craig of Rockfeller University and Frank W. Putman of the University of Florida for the first time sequenced the light chains of a Bence Jones⁹ myeloma protein, demonstrating that it consisted of 214 amino acids, and that the polypeptide chains of an antibody were constituted by constant regions (C) and variable regions (V) corresponding to the antigen combination site. It resulted that the Bence Jones proteins obtained from several multiple myeloma patients presented different amino acid sequences and the differences were with regard to the first half of polypeptide chain. In this way it was ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Preface
• Acknowledgement
• Chapter 1. The Antibodies
• Chapter 2. The Cells of Immunity
• Chapter 3. The Genetic Basis of Immune Response
• Chapter 4. Immune System Pathologies
• Chapter 5. Immunity and Tumors: The Surveillance Theory
• Chapter 6. Conclusions
• References