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Core Module Immunology Doctoral Training Group GK1660 Erlangen 2011 History of Immunology Part 3: IMMUNOCHEMISTRY – The Antibody Problem Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg 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) Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 2 TIMELINE: Serum Therapy of Diphtheria Klebs discovers bacteria on material from diceased diphtheria patient 1883 Roux and Yersin idenify soluble diphtheria toxin 1884 1888 Löffler identifies C. diphtheriae as the cause of diphtheria Behring 1st Serum therapy (diphtheria) in guinea pigs Roux develops antisera in horses 1891 1892 1890 Behring & Kitasato 1st serum therapy (tetanus) in mice Hoechst (Behring) Industrial production of antisera in sheep Safe Active Vaccination Ramon Behring& Ehrlich (Berlin) 1st serum therapy in humans 1893 Behringwerke in Marburg 1894 1904 1924 Roux & Chaillon (Paris) Serum therapy in humans Park & Williams (NYC) Production of antisera in Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 4 Summary: Humoral immunity (1905) SOLUBLE, INDUCIBLE & SPECIFIC IMMUNITY (Antitoxins, Immunkörper, Amboceptor, Zwischenkörper, Immunisin, substance sensibilisatrice, copula, Desmon, philocytase, fixateur) Antitoxins Behring (1890) Bacteriolysins Pfeiffer (1895) Agglutinins von Gruber (1896) Precipitins Kraus (1897) Hemolysins Belfanti & Crabone (1898) Opsonins Wright (1903) 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 • 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 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 5 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“ Another Paradigm in Immunology „Infections are cleared by cellular and humoral immunity“ 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. 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-EhrlichInstitut für Serumprüfung und Serumforschung Jahre1896 in Steglitz bei Berlin Königliches Institut für experimentelle Therapie, Frankfurt 1899 Königliches Institut für experimentelle Therapie + GeorgSpeyer-Haus 1922. Frankfurt Ab 1947 PaulEhrlich-Institut für Exp. Therapie Paul-Ehrlich-Institut im Jahre 1990 als Bundesamt für Sera und Impfstoffe in Langen bei Frankfurt/Main Georg-Speyer-Haus, 1906, Frankfurt Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 8 IMMUNOLOGY: Own Discipline 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) Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 9 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) Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 10 THE ANTIBODY PROBLEM (1910 - 1975 ) Immunochemistry of Antibodies o o o o o o o Antigens Antibody-Antigen Interaction Purification Detection Identification Quantification Structure TOPICS: THE ANTIBODY PROBLEM Immunochemistry Antigens - Features and Origin of Term Antibodies are proteins Antibody quantitation Antibody structure Variable and Hypervariable regions (paratop) Crystal structure Monoclonal antibodies Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 13 THE ANTIBODY PROBLEM Antigens Side Visit - Antigens Antigen – Orgin of the Term László Detre a.k.a. Ladislas Deutsch, Ladislaus Deutsch (1874-1939, Hungary) • Since he believed in Buchner‘s 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 Ehrlich‘s 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, 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. Antigens • 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 Isoantigen Alloantigen Xenoantigen - aus dem zu immunisierenden Individuum aus einem genetisch identischen (syngenen) Spender (Inzucht) aus einem nichtverwandeten Spender derselben Spezies aus einem Spender einer anderen Spezies Antigens – Recognition by B- and T cells B-ZellRezeptor (BZR) Naive Lymphozyten B Effektorzellen EffektorMoleküle Plasma T (CD4) T (CD8) THelfer TRegs TKiller Ag Dendritische Zelle Botenstoffe AK Humorale Immunität (z.B, Zytokine, Chemokine, Lymphotoxine Zelluläre Immunität T-ZellRezeptor (TZR) MHC II MHC I Ag-Prozessierung & Präsentation Epitope und Paratope (Schlüssel-Schloß) Epitop (Determinante) Ag → Bereich auf dem Antigen, der an den Ag-Rezeptor bindet VH VL Paratop → Bereich auf dem Antikörper, der mit dem Epitop des Antigens interagiert Loops („fingers“) that form the paratop are also called hypervaribale regions (HV) or complementary determining regions (CDR3) T Cell Epitopes Almost allways processed peptides Rarely lipids and phospopeptides Must be presented by MHC molecules to TCR Co-receptors are required to stablize binding TH TK TZR CD4 CD8 (α) MHC II (I) + Peptid B B Ziel Erklärt wieso CD8+ T-Killerzellen nur Zellen mit MHC-Klasse I CD4+ T-Helferzellen nur Zellen mit MHC-Klasse II erkennen Induction of Antibodies (1924) Cells o o o o o Erythrocytes (Belfanti & Carbone) Bacteria (Pfeiffer) Spermien (Landsteiner, Metschnikow, Moxter) Flimmerepithel (von Dungern) Nierenzellen (Metschnikow) Proteins o e.g., Toxins (Ehrlich, Behring, …. o Albumin Organic compounds coupled to carrier protein (Landsteiner, 1920) Carbohydrates (Heidelberger & Avery, 1924) B Cell Epitopes - Composition Die Welt der Antigene (Antikörper generierend) IgM Ig-Rezeptoren erkennen • Proteine • Lipide • Nukleinsäuren Ag • Kohlenhydrate • Organische Moleküle oder Haptene (Halb-Ag) • Metalle +/-TH IgM Kurzlebige Plasmazelle IgG, IgA, IgE IgD Naive B-Zelle +TH Langlebige Plasmazelle • Plastik Aber nur Proteine sind gute T-Zell-abhängige Antigene GedächtnisB-Zelle B Cell Epitopes - Conformations Conformational epitope Linear epitope NeoEpitope polypeptide chain Denaturation Epitope is lost Epitope remains intact Epitope is new Antitoxin - Mode of Action Antitoxins: Mechanism of action (Ehrlich, 1897) Mechanisms of antitoxic effect of Behring‘s 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 Antitoxin - Mode of Action - Buchner …. - 1st Theory to Explain Antitoxin (Buchner 1893) 1893 Hans Buchner, Emil Roux, Emmerich & Loew: o Toxins are transformed in the body into their corresponding 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 Antitoxin - Mode of Action - Ehrlich - Antitoxins: Mechanism of action (Ehrlich, 1897) Mix of anti-ricin Serum and ricin before addition to RBC Experiment: Tubes with blood from un-immunized rabbits Ricin mediates clumbing of RBC Ricin - + + + + + Ricin Oberservation → Anti-ricin toxin prevents ricin (lectin)-mediated clumping of red blood cells in a concentration dependent manner 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 P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p. 41-43 Antitoxin – Toxin Interaction - Chemical reactions - Antitoxin/Toxin: Chemical Interaction 1. Direct chemical interaction of antitoxin and toxin in solution P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p. 41-43 Antitoxin/Toxin: Chemical Interaction 2. Strength of interaction of antitoxin and toxin is affected by concentration and temperature 3. Interaction is a chemcal reaction • Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 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 Savante Arrhenius Svante Arrhenius 1859 - 1927 Sweden Nobel Prize Chemistry 1903 Developed theoretical basis for electrolytic dissociation and chemical reaction → Nobel Prize Chemisty in 1903 The Arrhenius equation: Formuates emperature dependence of the reaction rate constant, and therefore, rate of a chemical reaction. Savante Arrhenius ↔ Paul Ehrlich 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 Antibodies are g-Globulins (1939) unbehandelt Stärke-Elektrophoresese Serum Absorption an Ovalbumin Ovalbumin Aus: Kuby, Immunology g-Globuline sind Antikörper Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 72 QUANTITATION of Ag/Ab reactions [Antibody] [Antigen = SSSIII] 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. Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 73 QUANTITATION of Ag/Ab reactions Antikörper-Konzentration Antigen-Konzentration Menge der Ak-Ag-Komplexe Aus Janeway: Immunobiology Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 74 QUANTITATION of Ag/Ab reactions Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 75 Hypervariable regions are discovered (1970) Number of different amino acids at a given position Wu-Kaba Plot 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. Reproduced from The Journal of Experimental Medicine, 1970, 132: 211–250. Copyright 1970 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 86 Tertärstruktur einer Ig-Faltdomäne AntigenBindnungs stelle Merkmale einer Ig-Faltdomäne Anzahl der Aminosäuren Zylindrische, globuläre Form aus 100 - 110 Aminosäuren Anzahl und Orientierung der b-Stränge 7 (C-Region) bzw. 8 (V-Region) anti-parallele Ketten in bStruktur β-Stränge (Sekundärstruktur) V Anzahl der Faltblätter Zwei Lagen anti-paralleler b-Stränge bilden zwei b-Faltblätter, die durch eine Disulfidbrücke miteinander verbunden sind C Disulfidrücke b-Strang Ig-Superfamilie Ig-Faltdomänen kommen in vielen anderen Proteinen vor (CD4, CD8, MHC Klasse I und II, T-Zellrezeptor, ICAMs…. Janeway Immunobiology Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 88 Die Antigenbindungsstelle (Paratop) • Je drei Schlaufen (Finger) der VH- und VL-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) Antigenbindungstelle = Paratop VH VL H CL L Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 89 The hybrioma technique and monoclonal antibodies Georges Köhler & Cesar Milstein Nobelprize 1984 Polyclonal - monoclonal Immunisierung von Labortieren mit Antigenen Polyklonale Antiseren Heterogenes Gemisch von Antikörper, die verschiedene 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. MF)] 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 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 91 91 Hybridomatechnik: Gewinnung monoklonaler Antikörper (Köhler und Milstein, 1976, Nobelpreis 1984) Milz-B HGPRT + Ig+ sterblich Myelom PEG HGPRT Igunsterblic h Aminopterin blockiert De Novo Purin- und Pyrimidinsynthese De Novo Salvage Pathway Thymidin Hypoxanthin B-Zell/Myelom-Hybrid HAT-Medium: Hypoxanthin, Aminopterin, Thymidin Thymidinkinase (TK+) Aminopterin Ig+ HGPRT + unsterblic h HypoxanthinGuaninPhosphoribosyltransferase (HGPRT+) Nukleotide Aus Kuby DNA, RNA Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 96 96 Fusion of myeloma pairs (Milstein 1973) Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 99 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 Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 100 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 … ) Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 102 Monoclonals as Biologics Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 104 Monoclonals as Biologics Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 105 Catalytic Antibodies Richard Lerner, San Diego, 1986; Peter Schultz, Berkely 1986) Conventional Antibody Bindsantigenbut doesnot cleave Is "used" upduring reaction Catalytic Antibody Bindsantigenand cleavesit Is regenerated after reaction Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 109 Periodatoxidation von p-Nitro-Toluol-methyl-Sulfid zu Sulfoxid Instabiler Übergangszustand Hapten (Stabiles Analog zum Übergangszustand) Aminophosphonsäure L.C.Hsieh-Wilson, P.G.Schultz, and R.C.Stevens. Proc. Natl. Acad. Sci. USA, 93:5363-5367 (1996). Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 110 110 Enzymatische Spaltung und Inaktivierung von Kokain (Landy and coworkers, New York 1993) H C N 3 O O OCH 3 O H 3C N O O OCH 3 O H 3C O N HO OCH 3 OH O + C HO HO Cocaine (active) Unstable Transition State Cleavage Products (inactive) O H C 3 N OCH 3 O O P HO Stable Transition State Analog Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 111 111