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Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg History of Immunology.

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1 Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg History of Immunology Part 3: IMMUNOCHEMISTRY – The Antibody Problem Core Module Immunology Doctoral Training Group GK1660 Erlangen 2011

2 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 2 TIME LINE - History of Immunology Discovery of cells and germs (1683 - 1876) Prevention of Infection (1840 – today) Start of Immunology (1796-1910) The antibody problem: Immunochemistry (1910 - 1975) Self-/non-self discrimination (1940 – today) Models to explain antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

3 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 3 1.Preventive Immunization o Jenner (1789)-1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic humoral immunity and complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Widal (1896) – Widal agglutination test for typhoid fever o Bordet (1901) - Complement fixation test o Wassermann (1905) – Syphilis test o Landsteiner (1901) – Blood goups in human 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930 TOPCIS: Start of Immunology C. Bogdan (July) S. Finotto (July)

4 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 4 Klebs discovers bacteria on material from diceased diphtheria patient Behring- werke in Marburg Behring& Ehrlich (Berlin) 1 st serum therapy in humans Behring& Ehrlich (Berlin) 1 st serum therapy in humans TIMELINE: Serum Therapy of Diphtheria Löffler identifies C. diphtheriae as the cause of diphtheria Roux and Yersin idenify soluble diphtheria toxin Hoechst (Behring) Industrial production of antisera in sheep Roux develops antisera in horses Behring 1st Serum therapy (diphtheria) in guinea pigs 1883 1894 1893 1904 1892 1884 1891 1888 1890 Behring & Kitasato 1 st serum therapy (tetanus) in mice Roux & Chaillon (Paris) Serum therapy in humans Park & Williams (NYC) Production of antisera in 1924 Safe Active Vacci- nation Ramon

5 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 5 Summary: Humoral immunity (1905) Eichmann, Klaus (2000). The network collective: rise and fall of a scientific paradigm http://en.wikipedia.org/wiki/Humoral_immunity#cite_note-G.E-3 JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen Scand. J. Immunol., 19, 281-285 In 1905 it was not clear that all these humoral activities can be traced back to the same class of inducible compounds (i.e., the antibody molecule) Today, Antikörper (Antibody) is a neutral term for the common component in all the different biological activities of immune sera

6 Discovery of a inducible, soluble and specific activity in the blood (later termed antibodies) in 1890 The first paradigm in immunology Specific immunity induced by antigens is associated with the formation of antibodies The first paradigm in immunology Specific immunity induced by antigens is associated with the formation of antibodies

7 1908 Paul Ehrlich: Ehrlich (1908). Über Antigene und Antikörper. Einleitung in Handbuch der Immunitätsforschung. P.1 -10 Very nice overview about the knowledge of antibody and antigen in 1908. Another Paradigm in Immunology Infections are cleared by cellular and humoral immunity Another Paradigm in Immunology Infections are cleared by cellular and humoral immunity

8 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 8 IMMUNOLOGY: Own Discipline PAUL-EHRLICH-INSTITUT (devoted to serum therapy Paul-Ehrlich Institute für Serum-forschung und Serumprüfung (1896) bis Paul-Ehrlich Institute Bundesamt für Sera und Impfstoffe (1990) Das Paul-Ehrlich- Institut für Serumprüfung und Serumforschung Jahre1896 in Steglitz bei Berlin Paul-Ehrlich-Institut im Jahre 1990 als Bundesamt für Sera und Impfstoffe in Langen bei Frankfurt/Main Königliches Institut für experimentelle Therapie + Georg- Speyer-Haus 1922. Frankfurt Ab 1947 Paul- Ehrlich-Institut für Exp. Therapie Georg-Speyer-Haus, 1906, Frankfurt Königliches Institut für experimentelle Therapie, Frankfurt 1899

9 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 9 JOURNALS o Zeitschrift für Immunitätsforschung (1908) o J. of Immunology (1913) o Eur. J. Immunology (1970) PROFESSIONAL SOCIETIES o American Associaten of Immunologist (1913) o Deutsche Gesellschaft für Immunologie = DGfI (1953) IMMUNOLOGY: Own Discipline

10 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 10 TIME LINE - History of Immunology Discovery of cells and germs (1683 - 1876) Prevention of Infection (1840 – today) Start of Immunology (1796-1910) The antibody problem - Immunochemistry (1910 - 1975) Self-/non-self discrimination (1940 – today) Models to explain antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

11 THE ANTIBODY PROBLEM (1910 - 1975 )

12 Immunochemistry of Antibodies o Antigens o Antibody-Antigen Interaction o Purification o Detection o Identification o Quantification o Structure

13 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 13 Immunochemistry Antigens - Features and Origin of Term Antibodies are proteins Antibody quantitation Antibody structure Variable and Hypervariable regions (paratop) Crystal structure Monoclonal antibodies TOPICS: THE ANTIBODY PROBLEM

14 THE ANTIBODY PROBLEM Antigens

15 Side Visit - Antigens

16 Antigen – Orgin of the Term László Detre a.k.a. Ladislas Deutsch, Ladislaus Deutsch (1874-1939, Hungary) Since he believed in Buchners theory, he called in his first publication (1899, in French) the hypothetical substance that induces immunity Substances immunogenes ou antigenes i.e., a substance between a toxin and an antitoxin (just like zymogen is a precursor of an enzyme) In the German version of his article published in 1903, Deutsch accepted Ehrlichs theory and used the noun Antigen and states that this is a contraction of Antiisomatogen = Immunkörperbildner Oxford EnglishDictionary indicates that the logical construction should be anti(body)-gen for antibody generating JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen. Scand. J. Immunol., 19, 281-285 Eichmann, K. (2008). The network collective: rise and fall of a scientific paradigm. Birkhäuser Verlag,

17 Antigens - Definitions Antigen Any Substance that combines directly after the key-lock principle ( Emil Fischer, 1894) with B cell receptor or antibodies ( Paul Ehrlich) or T cell receptors or MHC Immunogen Substance that induces a humoral or T cell-mediated immune response Hapten Antigen that binds to immune receptors but does not induce an immune response Allergen A substance that provokes an allergic reaction Tolerogen A substance that invokes a specific immune non-responsiveness Superantigen A class of antigens that cause non-specific activation of T-cells, resulting in polyclonal T cell activation and massive cytokine release.

18 Superantigen

19 Nach Herkunft Natürlich (Proteine, Kohlenhydrate, Nukleinsäuren, bakt. Toxine, Zellen) Synthetisch (Haptene, Polypeptide) Nach chemischen Gesichtspunkten Proteine Kohlenhydrate Nukleinsäuren Konjugierte Proteine (Hapten-Protein) Polypeptide Lipide Nach genetischer Beziehung zwischen Spender und Empfänger Autoantigen - aus dem zu immunisierenden Individuum Isoantigen- aus einem genetisch identischen (syngenen) Spender (Inzucht) Alloantigen - aus einem nichtverwandeten Spender derselben Spezies Xenoantigen - aus einem Spender einer anderen Spezies Antigens

20 Antigens – Recognition by B- and T cells Humorale Immunität Zelluläre Immunität BT (CD8)T (CD4) Naive Lympho- zyten B-Zell- Rezeptor (BZR) Ag T-Zell- Rezeptor (TZR) T Helfer T Killer Effektor- zellen Plasma T Regs Botenstoffe (z.B, Zytokine, Chemokine, Lymphotoxine Effektor- Moleküle AK MHC II MHC I Ag-Prozessierung & Präsentation Dendritische Zelle

21 Epitop (Determinante) Bereich auf dem Antigen, der an den Ag-Rezeptor bindet Paratop Bereich auf dem Antikörper, der mit dem Epitop des Antigens interagiert Ag VHVH VLVL Epitope und Paratope (Schlüssel-Schloß) Loops (fingers) that form the paratop are also called hyper- varibale regions (HV) or complementary determining regions (CDR3)

22 Almost allways processed peptides Rarely lipids and phospopeptides Must be presented by MHC molecules to TCR Co-receptors are required to stablize binding T Cell Epitopes Erklärt wieso CD8 + T-Killerzellen nur Zellen mit MHC-Klasse I CD4 + T-Helferzellen nur Zellen mit MHC-Klasse II erkennen MHC II (I) + Peptid TZR CD4 B CD8 ( α) THTH B Ziel TKTK

23 FR = framework (Gerüstregion); HV = hypervariabel Wu-Kabat-Plot: AA comparison of VH regions Variability Die loops that form the paratop are also called hypervaribale regions (HV) or complementary determining regions (CDR3) Ag VHVH VLVL L-CDR1 L-CDR2 L-CDR3 H-CDR1 H-CDR2 H-CDR3 AA position CDR1 CDR2 CDR3 Hypervariable Regions or CDRs Kuby, 4th edition

24 Induction of Antibodies (1924) Cells o Erythrocytes (Belfanti & Carbone) o Bacteria (Pfeiffer) o Spermien (Landsteiner, Metschnikow, Moxter) o Flimmerepithel (von Dungern) o Nierenzellen (Metschnikow) Proteins o e.g., Toxins (Ehrlich, Behring, …. o Albumin Organic compounds coupled to carrier protein (Landsteiner, 1920) Carbohydrates (Heidelberger & Avery, 1924)

25 Ig-Rezeptoren erkennen Proteine Lipide Nukleinsäuren Kohlenhydrate Organische Moleküle oder Haptene (Halb-Ag) Metalle Plastik Aber nur Proteine sind gute T-Zell-abhängige Antigene Die Welt der Antigene (Antikörper generierend) Kurzlebige Plasmazelle Gedächtnis- B-Zelle Ag IgM IgD Naive B-Zelle IgG, IgA, IgE IgM +/-T H +T H Langlebige Plasmazelle B Cell Epitopes - Composition

26 B Cell Epitopes - Conformations

27 polypeptide chain B Cell Epitopes - Conformations Conformational epitope Linear epitope Neo- Epitope Denaturation Epitope is lost Epitope remains intact Epitope is new

28 Antitoxin - Mode of Action

29 Mechanisms of antitoxic effect of Behrings serum therapy? Hypothesis 1 : Antitoxins destroys toxin. Disproved since toxins could be detected on toxin/anti-toxin mixtures Hypothesis 2 (e.g., Roux und Buchner): Antitoxin soll keine aktive Wirkung auf das Toxin ausüben, sondern in erster Reihe auf die Zellen einwirken und dieselben gewissermassen gegen die Giftwirkung immunisieren. Hypothesis 3 (Ehrlich): Gift und Gegengift paaren in den Gewebsflüssigkeiten zu einer Art Doppelverbindung, welche nicht mehr in bestimmten Geweben fixiert wird und welche daher keine Krankheitserscheinungen mehr auslöst. P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p. 41-43 Antitoxins: Mechanism of action (Ehrlich, 1897)

30 Antitoxin - Mode of Action - Buchner …. -

31 1893 Hans Buchner, Emil Roux, Emmerich & Loew: o Toxins are transformed in the body into their corres- ponding antitoxin o Antitoxin, instead of acting directly on the toxin, act direct ly on the living elements (cells) of the organism, preserving them from intoxication. BUCHNER (893). Münchener med. Wochenschr. Ueber Bacteriengifte und Gegengifte. p. 480. EMMERICH, R., LOEW, O. (1901). Über biochemischen Antagonismus. Zentralbl. Bakteriol. Mikrobiol. Hyg. (A) 30:552 EHRLICH (1901). Üeber Toxine und Antitoxine. Die Therapie der Gegenwart. Mai, p.193 1 st Theory to Explain Antitoxin (Buchner 1893)

32 Antitoxin - Mode of Action - Ehrlich -

33 Experiment: Antitoxins: Mechanism of action (Ehrlich, 1897) P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p. 41-43 Conclusion Cellular explanation of Roux and Buchner (hypothesis 2) disproved First evidence for direct (from mix in vitro) and chemical interaction (from titration) of antitoxin with toxin Ricin Mix of anti-ricin Serum and ricin before addition to RBC Tubes with blood from un-immunized rabbits - + Oberservation Anti-ricin toxin prevents ricin (lectin)-mediated clumping of red blood cells in a concentration dependent manner Ricin mediates clumbing of RBC + Ricin + + +

34 1890 -1899 Ehrlich o The antotoxic substance inhibits the morbigenic action of the toxin by neutralizing the toxin, combining with the latter to form a compound of a chemical nature which is devoid of toxicity and without action on the organism. o According to this theory, the influence of the antitoxin on the toxin is direct, and does not require the intervention of the living cellular protoplasm. Such was also the belief of Prof. Ehrlich. 1 st Theory to Explain Antitoxin (Buchner 1893)

35 Antitoxin – Toxin Interaction - Chemical reactions -

36 P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p. 41-43 Antitoxin/Toxin: Chemical Interaction 1. Direct chemical interaction of antitoxin and toxin in solution

37 Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 Antitoxin/Toxin: Chemical Interaction 2.Strength of interaction of antitoxin and toxin is affected by concentration and temperature 3.Interaction is a chemcal reaction

38 Start of Immunochemistry Servate Arrhenius (1907) I have given to these lectures the title "Immunochemistry" and wish with this word to indicate that the chemical reactions of the substances that are produced by the injection of foreign substances into the blood of animals, i.e. by immunisation [sic], are under discussion in these pages. From this it follows also that the substances with which these products react, as proteins and ferments, are to be here considered with respect to their chemical composition. Arrhenius, S. Immunochemistry: The Application of the Principles of Physical Chemistry to the Study of the Biological Antibodies; The Macmillan Company: New York, 1907, vii. P. 31

39 The Arrhenius equation: Formuates emperature dependence of the reaction rate constant, and therefore, rate of a chemical reaction. Svante Arrhenius 1859 - 1927 Sweden Nobel Prize Chemistry 1903 Developed theoretical basis for electro- lytic dissociation and chemical reaction Nobel Prize Chemisty in 1903 Savante Arrhenius

40 Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 Winau F, Westphal O, Winau R (2004). "Paul Ehrlich--in search of the magic bullet". Microbes Infect. 6 (8): 786–9.

41 EHRLICH Regarded the relationship between toxins and antitoxins as a chemical neutralisation, that is to say, as a definite one-way reaction (irreversible) ARRHENIUS Reversible process with equilibration (A + B AB) In a mixture of antitoxin and toxin, there is a certain quantity of free toxin and antitoxin. Although both believed that the interaction between toxin (antigen) and antitoxin (antibody) is a chemical reaction, they disagreed on the degree of binding Controversy was negative for Ehrlich since Arrhenius was member of the Noble Prize committee in Stockholm Savante Arrhenius Paul Ehrlich

42 Ehrlich and Angelo Knorr demonstrated that neutralization is less rapid in dilute solutions than in concentrated ones. Svante Arrhenius demonstrated the occurrence of limited reactions between antitoxin and toxin analogous to the esterification of an alcohol by an acid, and in such a manner that there always exists, in a mixture of these two substances, a certain quantity of free toxin and antitoxin. the antitoxin inhibits the noxious action of the toxin, even outside the living organism, by uniting with it to form a compound in identically the same manner as when a strong base and a strong acid are brought together. Antitoxin – Toxin is a Chemical Reaction Toxins and Venoms and Their Antibodies. By EM. Pozzi-Eso-T. Authorized Translation by Alfred I. Cohn, Phar.D. i2mo, vii + 101 pages.

43 1890 -1899 Ehrlich o The antotoxic substance inhibits the morbigenic action of the toxin by neutralizing the toxin, combining with the latter to form a compound of a chemical nature which is devoid of toxicity and without action on the organism. o According to this theory, the influence of the antitoxin on the toxin is direct, and does not require the intervention of the living cellular protoplasm. Such was also the belief of Prof. Ehrlich. 1 st Theory to Explain Antitoxin (Buchner 1893)

44 P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p. 41-43 Antitoxin/Toxin: Chemical Interaction 1. Direct chemical interaction of antitoxin and toxin in solution

45 Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 Antitoxin/Toxin: Chemical Interaction 2.Strength of interaction of antitoxin and toxin is affected by concentration and temperature 3.Interaction is a chemcal reaction

46 Start of Immunochemistry Servate Arrhenius (1907) I have given to these lectures the title "Immunochemistry" and wish with this word to indicate that the chemical reactions of the substances that are produced by the injection of foreign substances into the blood of animals, i.e. by immunisation [sic], are under discussion in these pages. From this it follows also that the substances with which these products react, as proteins and ferments, are to be here considered with respect to their chemical composition. Arrhenius, S. Immunochemistry: The Application of the Principles of Physical Chemistry to the Study of the Biological Antibodies; The Macmillan Company: New York, 1907, vii. P. 31

47 The Arrhenius equation: Formuates emperature dependence of the reaction rate constant, and therefore, rate of a chemical reaction. Svante Arrhenius 1859 - 1927 Sweden Nobel Prize Chemistry 1903 Developed theoretical basis for electro- lytic dissociation and chemical reaction Nobel Prize Chemisty in 1903 Savante Arrhenius

48 Antitoxin – Toxin Interaction - Chemical reactions -

49 Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 Winau F, Westphal O, Winau R (2004). "Paul Ehrlich--in search of the magic bullet". Microbes Infect. 6 (8): 786–9.

50 EHRLICH Regarded the relationship between toxins and antitoxins as a chemical neutralisation, that is to say, as a definite one-way reaction (irreversible) ARRHENIUS Reversible process with equilibration (A + B AB) In a mixture of antitoxin and toxin, there is a certain quantity of free toxin and antitoxin. Although both believed that the interaction between toxin (antigen) and antitoxin (antibody) is a chemical reaction, they disagreed on the degree of binding Controversy was negative for Ehrlich since Arrhenius was member of the Noble Prize committee in Stockholm Savante Arrhenius Paul Ehrlich

51 Ehrlich and Angelo Knorr demonstrated that neutralization is less rapid in dilute solutions than in concentrated ones. Svante Arrhenius demonstrated the occurrence of limited reactions between antitoxin and toxin analogous to the esterification of an alcohol by an acid, and in such a manner that there always exists, in a mixture of these two substances, a certain quantity of free toxin and antitoxin. the antitoxin inhibits the noxious action of the toxin, even outside the living organism, by uniting with it to form a compound in identically the same manner as when a strong base and a strong acid are brought together. Antitoxin – Toxin is a Chemical Reaction Toxins and Venoms and Their Antibodies. By EM. Pozzi-Eso-T. Authorized Translation by Alfred I. Cohn, Phar.D. i2mo, vii + 101 pages.

52 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 52 Start of Immunochemistry Servate Arrhenius (1907) I have given to these lectures the title "Immunochemistry" and wish with this word to indicate that the chemical reactions of the substances that are produced by the injection of foreign substances into the blood of animals, i.e. by immunisation [sic], are under discussion in these pages. From this it follows also that the substances with which these products react, as proteins and ferments, are to be here considered with respect to their chemical composition. Arrhenius, S. Immunochemistry: The Application of the Principles of Physical Chemistry to the Study of the Biological Antibodies; The Macmillan Company: New York, 1907, vii. P. 31

53 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 53 regarding the chemical description of the immune response, and Many researchers perceived the interaction between toxin (antigen) and antitoxin (antibody) is a chemical reaction Ehrlich: the toxin-antitoxin reaction is not bound to living matter, but can also take place in vitro. 9 Controversy between Arrhenius and Ehrlich, as well as the larger part of the debate within the Karolinska Institute over a Nobel prize to Ehrlich, Ehrlich: Regarded the relationship between toxins and antitoxins as a chemical neutralisation, that is to say, as a definite one-way reaction, Arrhenius looked at it as a reversible process of equilibration, which followed general physical laws. Immunochemistry

54 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 54 Ehrlich was extremely absent-minded, aloof, brusque and, in his later years, eccentric [27]. As head of the Frankfurt Institute for Experimental Therapy he developed a highly demanding and autocratic style of leadership.

55 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 55 The crux of the dispute, then, was the nature of the immune response. Focusing on the molecular structure of the immune reactants, Ehrlich claimed that the interaction of toxin and antitoxin implied a high degree of specificity and, thus, resulted in definite chemical bonds. In principle, he presumed the process to be irreversible, although he admitted that, under certain in vitro conditions, it could be reversible. Apart from some remarkable exceptions which will be discussed later, Arrhenius agreed with Ehflich about the pertinent facts; his criticism lay in their appropriate interpretation. Concentrating on the reactants' physical properties, Arrhenius studied the kinetics and stoichiometry of the process and concluded that the reactions in question were similar to those taking place in highly dissociated electrolytes, so that the result of the process were loose, reversible chemical bindings. According to him, the physical laws which underlay chemical reactions in general should also be applied in immunochemistry. As later developments in biochemistry have shown, neither of these standpoints was totally incorrect, and during the following decades the differences between the two positions gradually disappeared. At that time, however, they seemed unsurmountable.

56 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 56 Start of Immunochemistry The Arrhenius equation is a simple, but remarkably accurate, formula for the temperature dependence of the reaction rate constant, and therefore, rate of a chemical reaction. Svante Arrhenius 18591859 - 19271927 Sweden Nobel Prize Chemistry 1903 in Stockholm) war ein schwedischer Physiker und Chemiker und Nobelpreisträger für Chemie.StockholmPhysikerChemikerNobelpreisträger für Chemie Als seine bedeutendste Leistung ist die Ausarbeitung der Grundlagen der elektrolytischen Dissoziation anzusehen.

57 THE ANTIBODY PROBLEM Antigen-Antibody Reactions

58 Two most important advances in the attack on the problem of the nature of immunological reactions were the discovery that the specific precipitate contains both antigen and antibody (7) and the discovery that antibodies, which give antisera their characteristic properties, are proteins. The verification of these facts was provided by the work of many investigators over a score of years. This work, which is summarized in Marracks monograph (6, chap. II), culminated in the preparation of purified antibody by Felton and Bailey (S), Heidelberger and collaborators (9), and others, and the determination of its properties, including amino-acid composition and molecular weight, which show that it is very closely related to normal serum globulin (6, chap. II). Pauling Review

59 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 59 LANDSTEINER The most strikung feature of the humoral immune response is the specific nature with antigen Even a single amino acid change can e distuingished by antibodies Can also distinge distinguish between D and L amino acids

60 By the mid-1930s, Pauling was beginning to understand that simply knowing the structures of individual proteins was not enough. The essence of life resulted not from individual molecules, but from the interactions between them. How did organisms make offspring that carried their specific characteristics? How did enzymes recognize and bind precisely to specific substrate molecules? How did the body produce antibodies that recognized and bound to specific foreign, invading antigens? How did proteins, these flexible, delicate, complex molecules, have the uncanny ability to recognize and interact with specific molecules? These questions all fell under the heading of biological specificity. To this topic Pauling directed much of his attention during the late 1930s and 1940s. To understand biological specificity, Pauling decided to work first with antibodies and antigens, the understanding of which immunologists such as Karl Landsteiner were beginning to perfect. Pauling met and spoke with Landsteiner on several occasions, and he began his own research program in the late 1930s combining Landsteiner's methods with his own most recent chemical techniques. During a decade of antibody experiments, carried out through the late 1940s, Pauling built a detailed picture of the binding of antibody and antigen at the molecular level. His findings were surprising. Pauling demonstrated that the precise binding of antigen to antibody was accomplished not by typical chemical means, but rather through the shapes of molecules. He discovered that an antibody fits an antigen as a glove fits a hand. Their shapes were complementary. When the fit was tight, the surfaces of antibody and antigen came into very close contact, making possible the formation of many weak bonds that operated at close quarters and were relatively unimportant in traditional chemistry--van der Waals' forces, hydrogen bonds, and so forth. To work, the fit had to be incredibly precise. Even a single atom out of place could significantly affect the binding. Having demonstrated the importance of complementary structure with antibodies, Pauling extended his idea to other biological systems, including the interaction of enzymes with substrates, odors with olfactory receptors, and even the possibility of genes composed of two complementary molecules. Pauling's idea that biological specificity was due in great part to complementary "fitting" of large molecules to one another was essential in the development of molecular biology. The path of his research now formed a broad arc, from early work on the chemical bond as a determinant of molecular structure; through finding out the structures of large molecules, first inorganic substances, then biomolecules; and, finally, to elucidating the interactions between large biomolecules. By the early 1950s, Pauling felt that he had discovered the essentials of life at the molecular level. He was ready for something new. http://www.webcitation.org/5uK0FQBmR

61 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 61 Introduction of Haptens (Halb- Antigen) Landsteiner realized that while proteins were immunogenic (capable of inducing an immune response) the lack of structural knowledge of proteins made using them to study the chemical specificity of "serological reactions" impossible. He turned to the use of small molecules which had a defined chemical composition to study antibody specificity. Landsteiner was the first to systematically develop and chart the use of small molecules as probes of antibody recognition. One important observation to come out of Landsteiner's investigations was that small molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies.

62 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 62 One important observation to come out of Landsteiner's investigations was that small molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies. Antibodies againstInduction of Antibodies (1924)

63 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 63 Immunity againts Haptens (Landsteiner 1920-21) Antisera againts small organic molecules, in of themselves, are not capable of inducing an immune response. To circumvent this, he found that small molecules could be covalently attached to a carrier protein to engender an immune response. These small molecules, called haptens, contained a diazonium group, a functionality that readily forms a covalent bond with surface-bound lysines on a carrier protein. While this diazonium group strategy has been replaced with other methods of covalent linkage, [(a) Erlanger, B. F. In Methods in Enzymology; Academic Press: San Diego, 1980; Vol. 70, pp 85-104. (b) Brinkley, M. Bioconj. Chem. 1992, 3, 2-13. 22] immunization with hapten-carrier protein conjugates is still practiced today to obtain hapten-specific antibodies. Landsteiner demonstrated that antibodies exhibited regioselective binding behavior R designates the acid groups (COOH or SO 3 H or AsO 3 H 2 ) Figure 3. Reproduction of Table 21 taken from reference 21 showing the cross-reactivity between serum derived via immunization with a 3- aminobenzenesulfonic acid hapten and various small molecule (antigen)-protein conjugates.

64 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 64 R designates the acid groups (COOH or SO 3 H or AsO 3 H 2 ) Figure 3. Reproduction of Table 21 taken from reference 21 showing the cross-reactivity between serum derived via immunization with a 3- aminobenzenesulfonic acid hapten and various small molecule (antigen)-protein conjugates. serum derived from immunization with 3-aminobenzenesulfonic acid exhibits very limited cross-reactivity with the corresponding ortho and para regioisomers. However, as would be expected, considerable precipitation is observed when the hapten itself is used. These results indicated that shape-selectivity plays a major role in antibody recognition. Figure 3. Reproduction of Table 21 taken from reference 21 (Landsteiner, K. The Specificity of Serological Reactions; Harvard University Press: Cambridge, Massachusetts, 1945, p 156. ) showing the cross-reactivity between serum derived via immunization with a 3-aminobenzenesulfonic acid hapten and various small molecule (antigen)-protein conjugates.

65 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 65 p.169 serum derived from immunization with 3- aminobenzenesulfonic acid exhibits

66 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 66 p. 173

67 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 67 As early as 1898 with his assistant Tito Carbone he commenced a careful biochemical study of the nature of antitoxins, and after numerous experiments on the fractionation of antitoxic sera reached the conclusion that antitoxins in horse serum are associated with globulins precipitated by half saturation with ammonium sulphate. Michael Heidelberger was the first to apply mathematics to the reaction of antibodies and their antigens (the "precipitin reaction"). Heidelberger's calculations launched decades of research that helped reveal the specificity, function, and origin of antibodies.

68 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 68 Antibodies are proteins Heidelberger and Averys early experiments hinged on a simple element: nitrogen. Based on its presence or absence, the duo showed that the antibody- reactive substances on pneumococcal bacterial capsules were nitrogen-free carbohydrates, and that the reacting antibodies were nitrogen containing proteins (see How Heidelberger and Avery sweetened immunology JEM 202:1306). The discovery that antibodies are proteins was a notable achievement, particularly at a time when the nature of antibodiessubstances known only to reside in proteinaceous serum remained a mystery

69 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 69 Early StudiesANTIBODIES ARE PROTEINS As early as 1898 with his assistant Tito Carbone he commenced a careful biochemical study of the nature of antitoxins, and after numerous experiments on the fractionation of antitoxic sera reached the conclusion that antitoxins in horse serum are associated with globulins precipitated by half saturation with ammonium sulphate.

70 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 70 Immunbodies againts carbohydrates (1924 serum derived from immunization with 3- aminobenzenesulfonic acid exhibits

71 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 71

72 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 72 Aus: Kuby, Immunology Ovalbumin Absorption an Ovalbumin unbehandelt Serum Stärke-Elektrophoresese -Globuline sind Antikörper Antibodies are -Globulins (1939)

73 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 73 QUANTITATION of Ag/Ab reactions Heidelberger, M., and KendaIl, F. E. (1929). A QUANTITATIVE STUDY OF THE PRECIPITIN REACTION BETWEEN TYPE III PNEUMOCOCCUS POLYSACCHARIDE AND PURIFIED HOMOLOGOUS ANTIBODY J. Exp. Med., 1929,60, 809. [Antibody] [Antigen = SSSIII]

74 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 74 Menge der Ak-Ag-Komplexe Antigen-Konzentration Antikörper-Konzentration Aus Janeway: Immunobiology QUANTITATION of Ag/Ab reactions

75 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 75 QUANTITATION of Ag/Ab reactions

76 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 76

77 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 77 The study of antibodies began in 1890 when Emil von Behring and Kitasato Shibasaburō described antibody activity against diphtheria and tetanus toxins. Behring and Kitasato put forward the theory of humoral immunity, proposing that a mediator in serum could react with a foreign antigen. [71][72] Their idea prompted Paul Ehrlich to propose the side chain theory for antibody and antigen interaction in 1897, when he hypothesized that receptors (described as side chains) on the surface of cells could bind specifically to toxins – in a "lock-and-key" interaction – and that this binding reaction was the trigger for the production of antibodies. [73] Other researchers believed that antibodies existed freely in the blood and, in 1904, Almroth Wright suggested that soluble antibodies coated bacteria to label them for phagocytosis and killing; a process that he named opsoninization. [74]Emil von BehringKitasato Shibasaburōdiphtheriatetanus toxinshumoral immunity [71][72]side chain theorytoxins [73]Almroth Wrightbacteriaphagocytosisopsoninization [74]

78 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 78 The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that "if two substances give rise to two different antikörper, then they themselves must be different". [67] However, the term was not accepted immediately and several other terms for antibody were proposed; these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin. [67] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper. [67]Paul Ehrlich [67] antitoxin [67]

79 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 79 In the 1920s, Michael Heidelberger and Oswald Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies were made of protein. [75] The biochemical properties of antigen-antibody binding interactions were examined in more detail in the late 1930s by John Marrack. [76] The next major advance was in the 1940s, when Linus Pauling confirmed the lock-and-key theory proposed by Ehrlich by showing that the interactions between antibodies and antigens depended more on their shape than their chemical composition. [77] In 1948, Astrid Fagreaus discovered that B cells, in the form of plasma cells, were responsible for generating antibodies. [78]Michael Heidelberger Oswald Avery [75] John Marrack [76]Linus Pauling [77]Astrid Fagreausplasma cells [78]

80 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 80 Further work concentrated on characterizing the structures of the antibody proteins. A major advance in these structural studies was the discovery in the early 1960s by Gerald Edelman and Joseph Gally of the antibody light chain, [79] and their realization that this protein was the same as the Bence-Jones protein described in 1845 by Henry Bence Jones. [80] Edelman went on to discover that antibodies are composed of disulfide bond-linked heavy and light chains. Around the same time, antibody- binding (Fab) and antibody tail (Fc) regions of IgG were characterized by Rodney Porter. [81] Together, these scientists deduced the structure and complete amino acid sequence of IgG, a feat for which they were jointly awarded the 1972 Nobel Prize in Physiology or Medicine. [81] The Fv fragment was prepared and characterized by David Givol. [82] While most of these early studies focused on IgM and IgG, other immunoglobulin isotypes were identified in the 1960s: Thomas Tomasi discovered secretory antibody (IgA) [83] and David S. Rowe and John L. Fahey identified IgD, [84] and IgE was identified by Kikishige Ishizaka and Teruki Ishizaka as a class of antibodies involved in allergic reactions. [85] Gerald EdelmanJoseph Gallylight chain [79]Bence-Jones protein Henry Bence Jones [80] disulfide bondRodney Porter [81]amino acidNobel Prize in Physiology or Medicine [81] [82]Thomas TomasiIgA [83]David S. RoweJohn L. Fahey [84]IgEKikishige IshizakaTeruki Ishizaka [85]

81 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 81 Genetic studies identifying the basis of the vast diversity of these antibody proteins when somatic recombination of immunoglobulin genes was by Susumu Tonegawa in 1976. [86]Susumu Tonegawa [86]

82 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 82 http://what-when-how.com/molecular- biology/idiotypes-molecular-biology/ The concept of the idiotype of immunoglobulins emerged in the early 1960s from two different approaches, one by Oudin with rabbit antibodies, the other by Kunkel and analysis of the immunochemical characteristics of human myeloma proteins. At the time, Oudin had described the allotypic specificities as defining antigenic characteristics of a group of individuals within a given animal species. This was observed for rabbit immunoglobulins and was the consequence of allelic variants of both the heavy and light chains. It was then found that when rabbit antibodies prepared against Salmonella typhi were injected into another rabbit expressing the same (known) allotypes, they induced the synthesis of antibodies that specifically recognized the anti-Salmonella antibodies raised in the first rabbit. It was also shown that they did not react with normal rabbit serum taken before the Salmonella injection, thereby excluding that they identified a new allotypic specificity. Idiotypic specificities were thus defined as antigenic specificities characteristic of one antibody (the idiotype) produced by one animal and specific for one given antigen. Antibodies produced by the second animal were termed anti-idiotypic antibodies. Idiotypes are also referred to as Ab1 while anti- idiotypes are referred to as Ab2, in a simplified nomenclature.anti-idiotypicIdiotypes At the same time, Kunkel was comparing the immunochemical characteristics of human myeloma proteins with those of normal immunoglobulins. He found that antibodies raised against one given myeloma protein, which cross-reacted against normal immunoglobulins, became specific for the immunizing protein once extensively adsorbed on normal immunoglobulins. This was taken as indicative of determinants specific for any given monoclonal product, although it was not entirely clear whether it was also linked to the pathological "abnormal" nature of the myeloma protein. Structural analysis confirmed, as anticipated, that idiotypic determinants, or idiotopes, were present on the Fab fragments and, more precisely, on the variable regions of H and/or L chains (see Immunoglobulin Structure), depending on the idiotype. Extensive analysis of idiotype structure performed by a combination of immunochemical and structural analysis of anti-hapten antibodies revealed several types of idiotopes. Some Ab1-Ab2 interactions could be inhibited by a hapten of Ab1, indicating that the corresponding idiotopes were part of the antibody combining site, whereas other were not. Genetic analysis also revealed that some idiotypic specificities were common to several idiotypes, whereas others were strictly specific for one given Ab1, leading to the distinction of public and private idiotypes, a notion that was directly related to the dual origin of antibody diversity, germline encoded and somatically generated. Idiotypy was studied extensively when it was discovered that the cascade Ag(X) ^ Ab1 ^ Ab2 could be continued and amplified in a large idiotypic network of interactions, providing a basis for autoregulation of the immune system. An especially interesting observation was that Ab2 could induce certain Ab3 molecules that resembled Ab1 in that they could bind the original Ag(X) antigen. This led to the definition of the "internal image" of the antigen, proposed by Jerne, containing the idea that the collection of normal immunoglobulins of an individual could represent a huge population of internal images of the outside antigen world. It also stimulated approaches of idiotypic vaccines that could have been used in place of classical antigens, an interesting idea whenever antigens were either difficult to identify or poorly immunogenic.idiotopes idiotypesantigens

83 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 83 Side visit: Edman degradation Isoltion of myeloma protein Reduction of disulfide bonds Cleavage of isolated H and L chains by CNBr and pepsin, trypsin etc Separation of fragments by ion exchnage and gel chromatography Determination of N- (by Edman clevage) and C-terminal AA Stepwise release of N-terminal aa by the danysl Edman degradation method Deetecion of cleaved off aa by paper chromatography

84 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 84 The Complete Sequence of hu IgG1 (1969) Edelman et al. (1969). THE COVALENT STRUCTURE OF AN ENTIRE TG IMMUNOGLOBULIN MOLECULE. PNAS VOL. 63, 79, 1969 Isoltion of myeloma protein Reduction of disulfide bonds Cleavage of isolated H and L chains by CNBr and pepsin, trypsin etc Separation of fragments by ion exchnage and gel chromatography Determination of N- (by Edman clevage) and C-terminal AA Stepwise release of N-terminal aa by the danysl Edman degradation method Deetecion of cleaved off aa by paper chromatography

85 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 85

86 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 86 Reproduced from The Journal of Experimental Medicine, 1970, 132: 211–250. Copyright 1970 Hypervariable regions are discovered (1970) Number of different amino acids at a given position Variability = Frequency of the most common amino acid at that position Thus at position 7 63 proteins were studied, serine occurred 41 times and 4 different amino acids, Pro, Thr, Ser, and Asp, have been reported. The frequency of the most common is 41/63 = 0.65 and the variability is then 4/0.65 = 6.15. Wu-Kaba Plot

87 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 87 Hypervariable regions form paratop

88 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 88 Merkmale einer Ig-Faltdomäne Anzahl der Aminosäuren Zylindrische, globuläre Form aus 100 - 110 Aminosäuren Anzahl und Orientierung der -Stränge 7 (C-Region) bzw. 8 (V-Region) anti-parallele Ketten in - Struktur β-Stränge (Sekundärstruktur) Anzahl der Faltblätter Zwei Lagen anti-paralleler -Stränge bilden zwei -Faltblätter, die durch eine Disulfidbrücke miteinander verbunden sind Ig-Superfamilie Ig-Faltdomänen kommen in vielen anderen Proteinen vor (CD4, CD8, MHC Klasse I und II, T-Zellrezeptor, ICAMs…. Tertärstruktur einer Ig-Faltdomäne V C -Strang Disulfid- rücke Janeway Immunobiology Antigen- Bindnungs stelle

89 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 89 Je drei Schlaufen (Finger) der V H - und V L -Domäne (Hände) bilden die Antigenbindungstasche (Schloss) oder Paratop des Antikörpers Paratop ist der Teil des Antikörpers, der mit dem Epitop (Schlüssel) des Antigens interagiert In 1960, Niels Jerne coined the term epitope when he proposed that an antigen particle carries several epitopes (Jerne, N., Ann. Rev. Microbiol., 1960. 14: p. 341-358) Die Antigenbindungsstelle (Paratop) Antigenbindungstelle = Paratop H L CLCL VLVL VHVH

90 The hybrioma technique and monoclonal antibodies Georges Köhler & Cesar Milstein Nobelprize 1984

91 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 91 Polyclonal - monoclonal 91 Polyklonale Antiseren Heterogenes Gemisch von Antikörper, die ver- schiedene Epitope auf dem Immunogen erkennen Immunisieren von Tieren mit Antigen in komplettem Freunds Adjuvans [besteht aus Mineralöl ( Depot-wirkung) und abgetöteten Tuberkelbazil-len(unspezifische Aktivierung von DZ u. M )] Immunisierungen werden mehrmals wiederholt (Boosts) Monoklonale Antikörper homogener Antikörper, der nur ein Epitop auf dem Immunogen erkennt Immunisierung von Maus, Ratte, Hamster, Kaninchen oder (Mensch) Generierung von Hybridomen Immunisierung von Labortieren mit Antigenen

92 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 92 Polyclonal - monoclonal PolyclonalPolyclonal antibodies (or antisera) are antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope.antibodies B cellantigen epitope These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammals serum. immunizationmammalserumantigenlymphocytesIgG immunoglobulinspolyclonalIgGserum

93 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 93 The road to monoclonal antibodies Kunkel shows that myelomas produce Ig Milstein and Cotton succesfully fused a mouse and rat myeloma cell Littlefield introduces HAT selection to select for fused somatic cell hybrids xxxx 1964 1973 1962 Michael Potter estabishes Ig- producing mouse myelomas 2000 First mamamilan cell hybrids xxxxx xxx 2001 2002 Köhler and Milstein Hybridoma Köhler and Milstein Hybridoma

94 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 94 Overview immortalising B cells via somatic cell hybridisation they built on several advances Somatic cell hybdriisatin technique Selection for hybrid cells Usage of meyloma as a fusion partner

95 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 95 Selection for purine synthesis (HAT selection) Littlefield, J. W. 1964. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145:709-710.

96 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 96 Aus Kuby Hypoxanthin Hypoxanthin- Guanin- Phospho- ribosyl- transferase (HGPRT + ) Salvage Pathway Thymidin Thymidin- kinase (TK + ) De Novo Nukleotide DNA, RNA Aminopterin blockiert De Novo Purin- und Pyrimidinsynthese Myelom HGPRT + Ig + sterblich Milz-B HGPRT - Ig - unsterblic h Amin- opterin PEG Hybridomatechnik: Gewinnung monoklonaler Antikörper (Köhler und Milstein, 1976, Nobelpreis 1984) HAT-Medium: Hypoxanthin, Aminopterin, Thymidin B-Zell/Myelom-Hybrid Ig + HGPRT + unsterblic h

97 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 97 Mouse Plasmacytoma Lines (M. Potter xxx) Generation of HGPRT- or TK- myeloma lines

98 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 98 Somatic cell hybrids – Time line

99 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 99 Fusion of myeloma pairs (Milstein 1973)

100 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 100 The final experiment (Koehler & Milstein 1974) Continuous cultures of fused cells secreting antibody of predefined specificity. Kohler G and Milstein C. Nature 256: 495, 1975

101 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 101 Why hybridoma technology is so effective. The procedure of somatic cell hybridisation developed by Kohler and Milstein has revolutionised immunochemistry. Why is it so effective ? It is so effective for two reasons: The first is that the antibodies produced in this way employ B cells from hyper-immune animals. These cells therefore have undergone stringent antigen selection and affinity maturation and, when immortalised, will yield high-affinity, monoclonal antibodies.hybridoma technologyaffinity maturation The second reason is that the fusion procedure itself selects for blast cells. Therefore, if a final boost is carried out 2-3 days before fusion, the frequency of antigen- specific clones among the population of hybrid myelomas is very high.

102 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 102 Impact of hybridoma technique Tools to detect proteins in single cells Tools for practical application in biotechnology and medicine Diagnosis and therapy of diseases Final confirmation of the clonal selection theory (one B cell – one antibody) also others already provided strong evidence (see lecture in repertoire) Provided material for elucidating the mechanisms governing the genetic control of antibody synthesis and diversity (e.g., class switch, somatic hyper mutation, mRNA stability, genetic control elemenst, antibody assembly … )

103 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 103 One example: Discovery of BiP

104 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 104 Monoclonals as Biologics

105 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 105 Monoclonals as Biologics

106 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 106 History of Immunology http://www.servinghistory.com/topics/History_of_immunology Who named it? http://www.whonamedit.com/doctor.cfm/2511.html The antibody resuurce page http://www.antibodyresource.com Bacterology Online http://www.textbookofbacteriology.net/endotoxin.html Bacteriology & Immunology Textbook Chronology http://books.google.de/books?id=G7F20VzerbgC&printsec=frontcover&dq= A+chronology+of+microbiology+in+historical+context&hl=en&ei=64m6TdD CK4P1sgapza3- BQ&sa=X&oi=book_result&ct=result&resnum=1&ved=0CC8Q6AEwAA#v=o nepage&q&f=false Klinisches Wörterbuch http://www.textlog.de/11113.htmlhttp://www.textlog.de/11113.html http://lotarionline.unipv.it/moodle/file.php/1/immunology/lectures/list.html

107 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 107 One example: Discovery of BiP

108 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 108 Durch Immunisieren z.B. eines Kaninchens mit gereinigtem Maus-IgM aus einer weißen Maus = BALB/c wird die Produktion folgender Kaninchen-Antikörper induziert. Anti-isotypische AK (gegen Epitope in C-Regionen der H- und L-Kette) BALB/c Gegen verschiedene AK-Klassen (iso) C57Bl/6 Die konstante Region der H-Kette aus einer BALB/c-Maus (weiß) und einer C57Bl/6-Maus (schwarz) unterscheidet sich in einer Aminosäure verschiedene Allotypen codiert durch Varianten desselben Gens bzw. Allels) BALB/c Anti-allotypische AK (gegen ein bestimmtes Epitop In C-Region) Gegen verschiedene Formen eines bestimmten AK (allo) Anti-idiotypische AK (gegen Epitope in V-Regionen der H- und L-Kette) Gegen eine bestimmte (private= idio) Form eines AK Antikörper gegen Immunglobuline

109 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 109 Conventional Antibody CatalyticAntibody Bindsantigenbut doesnotcleave Is"used" upduring reaction Bindsantigenandcleavesit Isregeneratedafterreaction Catalytic Antibodies Richard Lerner, San Diego, 1986; Peter Schultz, Berkely 1986)

110 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 110 L.C.Hsieh-Wilson, P.G.Schultz, and R.C.Stevens. Proc. Natl. Acad. Sci. USA, 93:5363-5367 (1996). Periodatoxidation von p-Nitro-Toluol-methyl-Sulfid zu Sulfoxid Instabiler Übergangszustand Hapten (Stabiles Analog zum Übergangszustand) Amino- phosphonsäure

111 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 111 Enzymatische Spaltung und Inaktivierung von Kokain (Landy and coworkers, New York 1993) Cocaine Cleavage Products (inactive) (active) N O O O OCH 3 H C 3 HO N O O O OCH 3 H C 3 HO N O OH O OCH 3 H C 3 HO C + Unstable Transition State N O O O OCH 3 H C 3 HO P Stable Transition State Analog

112 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 112 In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p149

113 DNA is the substance of heridtiy

114 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 114 Miescher isolates nucleic acids from puss (Tübingen) Hersey-Case confirmed that DNA is the material of heridity Oswald Avery (Rockefeller) showed 1871 1964 1952 1928 Griffiths shows that a heat-resistance substance from pathologic S- pneumococcus transformed hamless R-fom into a pathologic strIN xxxxx xxx 2001 2002 Köhler and Milstein Hybridoma Köhler and Milstein Hybridoma

115 Falsifying famous dogmas

116 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 116 1958 Francis Crick The Central Dogma: Sequential information transfe ris unidriectional from DNA to RNA to protein Reverse trasnfe ris forbidden Exception 1971 david Baltimore and Howard Temin independtyl showed that certains RNA viruses use an enzym the trasnforms RNA back to DNA 1982 Pruisiner (UCSF): Prions (proteins) can tranfer structural information Is Cricks hypothesis falsified. No ist not since most processes of infomation retrival follow his hypotheis. What was falsified was the term DOGMA since dogma menas without exception, so it became a theory The Central Dogma

117 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 117 Started 1930 and lasted 20 years Instructional theory: All antibody have the same aa sequence and binding of antigen fixed binding pocketz and thus specificty. Once folded, the confoamtiaon was stable Clonal selection: one cell one antibody The latter became adogma, even so Milstein showed in 1994 that But this exeption did not falsify the clonal selction theory Contovery Formation of Antibody specificity

118 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 118

119 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 119

120 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 120


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