Transcript History_hmj_antibodies - Erlangen (Hans

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)
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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
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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
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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
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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)
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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)
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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
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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
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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.
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QUANTITATION of Ag/Ab reactions
Antikörper-Konzentration
Antigen-Konzentration
Menge der Ak-Ag-Komplexe
Aus Janeway: Immunobiology
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QUANTITATION of Ag/Ab reactions
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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
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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
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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
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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
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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
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Fusion of myeloma pairs (Milstein 1973)
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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
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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 … )
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Monoclonals as Biologics
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Monoclonals as Biologics
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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
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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).
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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
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