DE68926508T2 - Erzeugung von antikörpern aus transgenen tieren - Google Patents

Erzeugung von antikörpern aus transgenen tieren

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DE68926508T2
DE68926508T2 DE68926508T DE68926508T DE68926508T2 DE 68926508 T2 DE68926508 T2 DE 68926508T2 DE 68926508 T DE68926508 T DE 68926508T DE 68926508 T DE68926508 T DE 68926508T DE 68926508 T2 DE68926508 T2 DE 68926508T2
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human
cells
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Marianne Bruggemann
Michael Neuberger
Azim Surani
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Babraham Institute
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C07K2319/00—Fusion polypeptide

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Description

    Bereich der Erfindung
  • Die Erfindung betrifft ein Verfahren zur Erzeugung von Antikörpern (oder Immunglobulinen).
    • Hintergrund der Erfindung
  • Die Produktion von transgenen Tieren, die Fremdproteine,, beispielsweise Immunglobuline, in Körperflüssigkeiten, beispielsweise Milch, produzieren können, ist bekannt (vgl. Clark et al., Tibtech 5 (1987), 20-24, und Church et al., Tibtech 5 (1987), 13-19). Scangos et al., Advances in Genetics 24 (1987), 285-316, und Alt et al., Trends in Genetics 1(1985), 231-236, geben in ihren Publikationen eine Übersicht, die Hinweise auf Arbeiten enthält, in denen funktionell rearrangierte Immunglobulingene in Mäuse eingeführt werden. Jaenisch, Science 240 (1988), 1468- 1474, gibt einen Überblick über transgene Tiere und ihre Verwendung. Der Abschnitt auf Seite 1471 mit dem Titel "Immune System" bezieht sich unter anderem auf Veröffentlichungen von Goodhardt et al. (Proc. Natl. Acad. Sci. USA 84 (1987), 4229- 4233) und Bucchini et al. (Nature 326 (1987), 409-411), die Rearrangements von in die Keimbahn von Mäusen eingeführten Kaninchen- oder Hübner-Immunglobulin- Leichtketten-Genen betreffen.
    • Zusammenfassung der Erfindung
  • Ein erfindungsgemäßer Gesichtspunkt betrifft die Bereitstellung eines Verfahrens zur Herstellung eines Immunglobulins, das die Insertion von DNA in die Keimbahn eines nicht-menschlichen Tiers umfaßt, die ein mindestens einen Teil eines Immunglobulins menschlichen Ursprungs codierendes Nucleinsäuresegment enthält, das ein Gensegment einschließt, das einen Bereich der schweren Kette eines menschlichen Immunglobulins codiert, wobei das Gensegment nicht in vollständig rearrangierter Form vorliegt, wobei die DNA in dem Tier rearrangiert wird, so daß sie eine Anzahl von Immunglobulinen mit einem Teil oder Teilen, die von der inserierten DNA stammen, codiert, und in den Zellen oder der Körperflüssigkeit des Tieres exprimiert wird, und die Gewinnung von Immunglobulin aus Zellen oder geeigneter Körperflüssigkeit des Tieres.
  • Ein weiterer erfindungsgemäßer Gesichtspunkt betrifft die Bereitstellung eines Verfahrens zur Herstellung eines Immunglobulins gegen ein bestimmtes Antigen, das die Produktion eines transgenen Tieres durch Insertion von DNA fremden Ursprungs in die Keimbahn des Tieres umfaßt, die mindestens einen Teil eines Immunglobulins menschlichen Ursprungs codiert, das ein einen Bereich der schweren Kette eines menschlichen Immunglobulins codierendes Gensegment einschließt, wobei die DNA fremden Ursprungs einem Rearrangement oder einer Mutation im Lymphoidgewebe des transgenen Tieres unterliegt, so daß eine Vielzahl von rearrangierten Genen, die Immunglobuline, Immunglobulinfragmente oder chimere Immunglobuline codieren, produziert werden, so daß, nach Exposition des transgenen Tieres mit einem bestimmten Antigen, das Immunglobulin gegen das Antigen, das durch die rearrangierte oder mutierte DNA codiert wird, in Zellen oder Körperflüssigkeit des Tieres exprimiert wird, und Gewinnung von Immunglobulin aus Zellen oder geeigneter Körperflüssigkeit des Tieres.
  • Mit DNA von fremdem Ursprung ist DNA gemeint, die von einem anderen Tier stammt. Wenn beispielsweise das transgene Tier eine Maus ist, stammt die inserierte DNA nicht von der Maus, sondern beispielsweise vom Menschen.
  • Die inserierte DNA kann aus einem Tier gewonnen oder synthetisch hergestellt werden. Die DNA kann mindestens einen Teil eines bekannten Immunglobulins oder nach einer Modifikation mindestens einen Teil eines veränderten Immunglobulins codieren. Geeignete Verfahren dafür sind gut bekannt.
  • Die inserierte DNA kann im transgenen Tier exprimiert werden, wobei ein mindestens teilweise vom inserierten Material stammendes Immunglobulin produziert wird. Es ist bekannt, daß die inserierte DNA im transgenen Tier rearrangiert wird, so daß eine Vielzahl von Immunglobulinen, die einen vom inserierten Genmaterial stammenden Teil oder Teile enthalten, produziert werden kann, selbst wenn das inserierte Material an der falschen Stelle oder in der falschen Geometrie in die Keimbahn eingebaut wurde. Je nach Art des inserierten Materials kann das Tier ein chimeres Immunglobulin produzieren, das z. B. aus einem Gemisch von Maus/Mensch- Immunglobulinanteilen zusammengesetzt ist, wobei das Material fremden Ursprungs nur einen Teil des Immunglobulins codiert, oder das Tier kann ein vollständig fremdes Immunglobulin produzieren, z. B. vollständig menschlichen Ursprungs, wobei die DNA fremden Ursprungs ein vollständiges Immunglobulin codiert. So können zur Verwendung beim Menschen geeignete, therapeutisch potentiell nützliche Immunglobuline mittels der Erfindung produziert werden.
  • Polyclonale Antiseren können nach einer Immunisierung des transgenen Tiers produziert werden. Alternativ können monoclonale Antikörper von dem transgenen Tier produziert werden, z. B. durch Fusion von Milzzellen des Tiers mit Myelomzellen und Screenen der erhaltenen Hybridome, um die den gewünschten Antikörper produzierenden Hybridome zu selektieren. Geeignete Verfahren dafür sind dem Fachmann vertraut.
  • In einer alternativen Vorgehensweise kann die DNA so in das Tier eingebaut werden, daß der gewünschte Antikörper in Körperflüssigkeiten, beispielsweise Serum oder nach außen abgegebene Flüssigkeiten des Tiers, beispielsweise Milch, Vormilch oder Speichel, produziert wird. Durch in vitro-Insertion von DNA, die mindestens teilweise ein Immunglobulin fremden Ursprungs codiert, in ein Milchprotein-codierendes Säugergen und durch anschließende Einführung des Gens in ein befruchtetes Ei des Säugers, z. B. durch Injektion, kann sich das Ei beispielsweise in einen ausgewachsenen weiblichen Säuger entwickeln, der Milch produziert, die mindestens teilweise von der inserierten DNA stammendes Immunglobulin enthält. Der gewünschte Antikörper kann anschließend aus der Milch gewonnen werden. Geeignete Verfahren dafür sind dem Fachmann bekannt.
  • Alternativ können die durch das Tier nach Insertion des genetischen Materials produzierten Immunglobulin-produzierenden Zellen entfernt werden, wonach in vitro auf Zellen selektiert wird, die ein gewünschtes Immunglobulin produzieren. Das Immunglobulin kann anschließend in vitro in üblicher Weise aus den selektierten Zellen gewonnen werden.
  • Es wurde festgestellt, daß ein transgenes Tier ein chimeres oder fremdes Immunglobulin (das von der inserierten DNA stammt) als Antwort auf ein später in das Tier eingeführtes Immunogen produzieren kann. Daher kann durch Einführung fremder, z. B. menschlicher, DNA, die im wesentlichen die vollständigen Art-spezifischen Bereiche eines Immunglobulins codiert, das Tier zur Produktion fremden Immunglobulins gegen alle in das Tier eingeführte Antigene stimuliert werden. Das transgene Tier kann so eine sehr nützliche, praktische und wertvolle Quelle für menschliche Immunglobuline gegen ein weites Spektrum von Antigenen liefern.
  • Es wird angenommen, daß eine Integration in der Nähe des Genoms für ein erfolgreiches Rearrangement der inserierten DNA wesentlich ist. Die inserierte DNA kann daher als DNA vorliegen, die in prokaryotische Vektoren, beispielsweise Plasmide und Cosmide, cloniert ist. Multiple Plasmide oder Cosmide können ebenfalls verwendet werden, es ist dann jedoch wahrscheinlich erforderlich, daß sie in der Nähe des Genoms integrieren. Möglicherweise können größere DNA-Fraginente unter Verwendung von Hefevektoren als künstliche Chromosomen (vgl. D. T. Burke, G. F. Carle und M. V. Olson, "Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors", Science 236 (1987), 806-812) oder durch Einführung von Chromosomenfragmenten (vgl. J. Richer und C. W. Lo, "Introduction of human DNA into mouse eggs by injection of dissected human chromosome fragments", Science 245 (1989), 175-177) inseriert werden. Chromosomen- oder DNA-Fragmente von Wirbeltieren können ebenfalls als Quelle der inserierten DNA verwendet werden.
  • Die inserierte DNA kann auf übliche Weise in den Wirt eingeführt werden, beispielsweise durch Injektion oder andere Verfahren in befruchtete Eizellen oder embryonale Stammzellen. Es ist sicher zweckmäßig, ein Wirtstier zu verwenden, daß ursprünglich keine konstanten Bereiche von Immunglobulinen codierendes genetisches Material trägt, so daß das erhaltene transgene Tier zur Produktion von Immunglobulinen nur das inserierte fremde genetische Material verwendet. Dazu kann entweder eine natürlich vorkommende Wirtsmutante, der das relevante genetische Material fehlt, verwendet werden, oder es werden Mutanten, z. B. Zellinien, künstlich hergestellt, wobei letztendlich ein Wirt erhalten wird, aus dem das relevante genetische Material entfernt wurde.
  • Wenn das Wirtstier genetisches Material trägt, das konstante Bereiche von Immunglobulinen codiert, trägt das transgene Tier das natürlich vorkommende genetische Material und das inserierte genetische Material und produziert Immunglobuline, die vom natürlich vorkommenden genetischen Material, dem inserierten genetischen Material und Gemischen beider Arten von genetischem Material stammen. In diesem Fall kann das gewünschte Immunglobulin durch Screenen von vom transgenen Tier stammenden Hybridomen erhalten werden, z. B. durch Ausnutzung des Phänomens des allelen Ausschlusses der Antikörpergenexpression oder des differentiellen Chromosomenverlustes.
  • Ein weiterer erfindungsgemäßer Gesichtspunkt betrifft die Produktion eines transgenen Tiers, besonders eines nicht-menschlichen Tiers, in dessen Keimbahn DNA inseriert ist, die ein mindestens einen Teil eines Immunglobulins menschlichen Ursprungs codierendes Nucleinsäuresegment umfaßt, das ein Gensegment einschließt, das einen Bereich der schweren Kette eines menschlichen Immunglobulins codiert, wobei dieses Gensegment nicht in vollständig rearrangierter Form vorliegt, wobei die DNA in dem Tier reärrangiert wird, so daß sie eine Anzahl von Immunglobulinen mit einem Teil oder Teilen, die von der inserierten DNA stammen, codiert, und in Zellen oder geeigneter Körperflüssigkeit des Tieres exprimiert wird.
  • Der Schutzbereich der Erfindung umfaßt ferner ein Immunglobulin, das aus einem transgenen Tier erfindungsgemäß erhalten oder durch das erfindungsgemäße Verfahren produziert werden kann.
  • In einem Beispiel wurden Linien von transgenen Mäusen etabliert, die ein in ihre Keimbahn eingeführtes DNA-Segment trugen, das Immunglobulin-VH-Gene, einige D-Segmente und alle JH- und Cµ-Gensegmente in der Keimbahn-Konfiguration enthielt. Ein VH-Gen, alle JH-Segmente und die Exons, die den sezernierten konstanten Bereich der schweren Kette des IgM-Antikörpers codierten, stammten vom Menschen und das übrige Material von der Maus. Die Gensegmente unterlagen einer produktiven VH-D- JH-Rekombination im Lymphgewebe der transgenen Mäuse, wobei als Ergebnis ein chimerer Mensch/Maus-IgM-Antikörper im Serum synthetisiert wurde.
  • Nach der Immunisierung wurden durch Fusion von Milzzellen der transgenen Mäuse und der NSO-Myelome die Hybridome etabliert. Viele Hybride sezernieren menschliche chimere monoclonale IgM-Antikörper. Diese transgenen Mäuselinien können daher zur Herstellung von chimeren menschlichen Antiseren oder monoclonalen Antikörpern verwendet werden.
  • Die Mäuse antworten ferner auf eine Immunisierung mit menschlichen Antigenen mit der Produktion chimerer Antikörper gegen das eingeführte Antigen, und dieses Verfahren kann daher auch zur Herstellung einer Anzahl von üblichen menschlichen oder chimeren menschlichen Antikörpern, die gegen menschliche sowie heterologe Antigene gerichtet sind, verwendet werden, da die transgenen Mäuse gegen menschliche antigene Determinanten keine Toleranz zeigen.
  • In einem weiteren Beispiel wurden transgene Mäuse, die ausschließlich menschliche VH-, D-, JH- und Cµ-Sequenzen trugen, durch Injektion von Cosmiden, die menschliche VH-Gene, D-Segmente, J-Segmente und den konstanten Cµ-Bereich enthielten, in befruchtete Mäuseeizellen hergestellt. Die erhaltenen Mäuse produzierten zwischen 10 und 100 µg/ml menschliche µ-Ketten enthaltende Antikörper in ihrem Serum, wobei Maus-IgM mit etwa 200 µg/ml vorhanden war.
  • Die Erfindung wird in den nachstehenden Beispielen, die sich auf die beigefügten Figuren beziehen, näher erläutert.
  • Fig. 1 zeigt die Struktur von Plasmid-DNA;
  • Fig. 2 zeigt eine Southern-Blot-Analyse der DNA von Geweben und Hybridomen, die von transgenen Mäusen stammen, wobei die DNA von Fig. 1 in die Keimbahn inseriert ist;
  • Fig. 3 zeigt eine Northern-Blot-Analyse von cytoplasmatischer RNA von Hybridomen der transgenen Mäuse;
  • Fig. 4 zeigt eine Analyse von Immunglobulin, das von zwei von transgenen Mäusen etablierten Hybridomen sezerniert wird;
  • Fig. 5 zeigt die Struktur von IgA-lambda-Plasmid-DNA;
  • Fig. 6 zeigt eine Reihe von FACS-Histogrammen, die die Fluoreszenzintensität einer bestimmten Anzahl von Zellen in Abhängigkeit von der Zellzahl zeigen;
  • Fig. 7 zeigt eine Reihe von FACS-Profilen, die die Fluoreszenzintensität einer bestimmten Anzahl von Zellen in Abhängigkeit von der Streuung zeigen, wobei jeder Punkt eine Zelle repräsentiert; und
  • Fig. 8 zeigt eine weitere Reihe von FACS-Histogrammen, die der Fig. 6 entsprechen.
    • Beispiel 1 Das Transgen
  • Plasmid-DNA mit der in Fig. 1 dargestellten Struktur wurde in Mauseizellen injiziert. In dieser Figur werden menschliche Sequenzen durch ausgefüllte Balken, Maussequenzen durch offene Balken, Vektoren durch gekreuzte Balken und D-Elemente durch schraffierte Balken repräsentiert. Restriktionsstellen werden wie folgt abgekürzt: Bg, BglII; P, PvuI; R, EcoRI; K, KpnI; B, BamHI; X, XbaI; und H, HindIII.
  • Die Herkunft der einzelnen DNA-Segmente ist wie nachstehend beschrieben:
    • Der Vektor
  • Der vollständige "Mini-IgH-Locus" wurde zwischen die EcoRI- und BglII-Stellen eines pUC12-Derivats, das in eine aufgefüllte NarI-Stelle clonierte BglII-Linker enthielt, cloniert.
    • Die VH-Gene
  • Es gibt zwei VH-Gene im Mini-Locus. VH26 stammt vom Menschen und wurde als ein BglII-EcoRI-Fragment von 0,85 kb vom Phagen Lambda VH26 erhalten [Matthyssens & Rabbitts PNAS 77 (1980), 6561-6565]. VH-186.2 ist ein Keimbahn-VH-Gen von C57BL/6-Mäusen und wurde als SacI-KpnI-Fragment von 2,5 kb erhalten [Bothwell, Paskind, Reth, Imanishi-Kari, Rajewsky & Baltimore, Cell 24 (1981), 625-637].
    • Die D-Segmente
  • Das BALB/c-Maus-D-Q52-Element wurde als ein 221 nt- XhoI-SacI-Fragment erhalten. Nach der Clonierung zwischen die SacI- und SalI-Stellen von M13tg131 wurde eine ortsspezifische Mutagenese mit den Oligonucleotiden
  • durchgeführt, um M13tg131-Clone zu erzeugen, die D-Elemente trugen, die mit denen der Maus SP2- bzw. FL16-Familien verwandt waren. Diese D-Familien werden sowohl in der Maus als auch im Menschen gefunden. Ein weiteres D-Element das - menschliche D-Q52 - wurde in das menschliche JH-Cluster eingeschlossen (vgl. nachstehende Beschreibung).
    • Das JH-Cluster
  • Ein 3,5 kb-BglII-Fragment von menschlicher DNA, die die -sechs funktionellen menschlichen JH-Segmente, drei Pseudo-JH-Segmente sowie das menschliche D-Q52-Element einschloß, wurde verwendet [Ravetch et al., Cell 27 (1981), 583-591].
    • Die IgH-Enhancer
  • Ein Teil des menschlichen IgH-Enhancers ist im JH- Cluster-enthaltenden BglII-Fragment enthalten. Eine vollständige Kopie des Mausenhancers ist im 1 kb-XbaI-Fragment enthalten.
    • Der "Switch"-Bereich und der Cu-Bereich
  • Das 7,5 kb-XbaI-Fragment von menschlicher DNA schließt den µ-"switch"-Bereich und die Exons 1 bis 4 des konstanten Bereichs der schweren µ-Kette ein. Die µ-Membran-Exons und der Hauptteil des Introns zwischen dem Cµ4-Exon und dem CµM1-Membran-Exon werden durch ein 2,5 kb-HindIII-SphI-Fragment des Maus-µ-CH-Gens erhalten, in dem die SphI-Stelle unter Verwendung von Linkern in eine BglII-Stelle umgewandelt wurde.
    • Die transgenen Mäuse
  • Plasmid-DNA wurde mit BglII linearisiert, nach einer Elektrophorese in einem Agarosegel gereinigt und, wie bereits beschrieben, in den männlichen Pronucleus von befruchteten Eizellen von C57BL/6J · CBA/Ca-Mäusen injiziert [Reik et al., Eur. J. Immunol. 17 (1987), 465-469]. Eine Southern-Blot-Analyse von Schwanz-DNA zeigte, daß 12 von 32 geborenen Mäusen den Mini-Locus trugen. Der größte Teil der nachfolgenden Arbeit wurde mit den Nachkommen von drei Stammäusen durchgeführt - HIg 17, 19 und 29, von denen alle eine geringe Zahl (2 bis 5) von Kopien des Mini- Locus trugen.
    • Serumtests
  • Serum der Stammäuse wurde durch ELISA auf die Gegenwart von Antikörpern, die für menschliches IgM charakteristische antigene Determinanten enthielten, getestet. Es stellte sich heraus, daß die nicht-immunisierten transgenen Mäuse zwischen 10 und 100 µg/ml chimere menschliche IgM in ihrem Serum enthielten. Eine Immunfluoreszenzanalyse von Lymphocyten im peripheren Blut zeigte ferner die Gegenwart von Zellen, die mit biotinylierten Spezies-spezifischen anti-Mensch-IgM- Antikörpern und Fluorescein-konjugiertem Streptavidin gefärbt werden konnten.
    • Hybridome von transgenen Mäusen
  • Transgene Mäuse wurden entweder mit roten Blutkörperchen vom Menschen oder vom Schaf intraperitoneal immunisiert. Milzen wurden zu verschiedenen Zeiten nach der Immunisierung entfernt, mit NSO-Myelomen fusioniert und die Hybride in HAT-Medium selektiert. Viele dieser Hybride produzierten chimere menschliche IgM, wie durch einen ELISA-Test gezeigt werden konnte.
    • DNA-Rearrangement des Mini-Locus
  • Eine Southern-Blot-Analyse von DNA aus Geweben der transgenen Mäuse sowie aus Hybridomen zeigte, daß es eine hohe Häufigkeit von DNA-Rearrangements im Mini-Ig-Locus im Lymphgewebe der transgenen Mäuse gab.
  • DNA von Geweben oder von aus transgenen Mäusen etablierten Hybridomen wurde mit EcoRI gespalten und mit einer menschlichen IgH-Enhancersonde (Ball-BglII- Fragment) hybridisiert, die an einen Bereich zwischen dem menschlichen J6-Element und dem Maus-IgH-Enhancer im Mini-Locus hybridisierte. Die Ergebnisse der Southern-Blot-Analyse der DNA sind in Fig. 2 dargestellt. Die Größe der Markerfragmente ist in der Figur in kb dargestellt.
    • Transkription des Mini-Locus
  • Cytoplasmatische RNA (5 µg) aus dem NSO-Fusionspartner oder den Hybridomen von transgenen Mäusen wurde mit Sonden aus menschlicher Cµ, menschlicher VH26 oder Maus-VH186 abgesucht. Die Ergebnisse der Northern-Blot-Analyse der cytoplasmatischen RNA sind in Fig. 3 dargestellt, die zeigt, daß die Hybridome mRNA enthielten, die mit Sonden für menschliche µ-Ketten sowie für die V-Gene VH26 und/oder VH186 hybridisierte. Daher können sowohl das menschliche VH26 als auch VH186 der Maus rearrangieren, und somit kann eine Zellinie etabliert werden, die einen chimeren menschlichen IgM-Antikörper sezerniert.
    • Antikörper-Sekretion durch Hybridome von transgenen Mäusen
  • Die Proteinproduktion durch clonierte Hybridomzellinien wurde durch eine biosynthetische Markierung mit L-[³&sup5;S]-Methionin und anschließende Reinigung mit Antiserum gegen die menschliche µ-Kette analysiert. Insbesondere wurden Zellen über Nacht in Medium, das L-[³&sup5;S]-Methionin, den durch Immunpräzipitation aus dem Kulturüberstand gereinigten Antikörper und ein Antiserum gegen die menschliche µ- Kette enthielt, inkubiert. Die Reinigung der Überstände der transgenen Hybridome 35.5 und 24a wurde in Gegenwart eines großen Überschusses (50 µg) an nicht-radioaktivem, gereinigtem monoclonalem Maus-IgM-Antikörper (B1-8), wie es durch "+" in Fig. 4 angezeigt ist, durchgeführt. Wie es bei der Maus-IgM-sezernierenden Zellinie beobachtet werden kann, zeigt das Antiserum gegen die menschliche µ-Kette eine Kreuzreaktion mit der Maus-µ-Kette, diese Kreuzreaktion kann jedoch mit dem nicht-radioaktivem Maus-B1-8-IgM-Antikörper konkurrieren - vgl. die vier Bahnen auf der linken Seite von Fig. 4.
  • Dies zeigt, daß Hybridome dieser transgenen Mäuse, die Antikörper mit unterschiedlichen menschlichen µ-Ketten sezernieren, etabliert werden können.
    • Beispiel 2
  • Es können transgene Tiere hergestellt werden, die sowohl in ihren Körperflüssigkeiten als auch in ihren abgesonderten Sekreten spezifische Antikörper produzieren. Dieses Beispiel betrifft transgene Mäuse, in deren Keimbahn Gene, die die schweren und leichten Ketten eines Antigen-spezifischen chimeren menschlichen IgA2- Antikörpers codieren, integriert wurden. Neun transgene Mauslinien, in deren Keimbahn die in Fig. 5 dargestellte DNA integriert wurde, wurden etabliert. In Fig. 5 stellen die dicken ausgefüllten Kästchen Maus-Ig-DNA, die gestreiften Kästchen menschliche DNA, leere Kästchen das Maus-IgH und den SV40-Enhancer und die dünnen ausgefüllten Linien den pSV2gpt-Vektor dar. Die Abkürzungen der Restriktionsstellen entsprechen der Fig. 1. Das Plasmid ist ein Derivat vom bereits beschriebenen pSV- VNPH-alpha 2 [Bruggemann et al., J. Exp. Med. 166 (1987), 1351-1361]. Es enthält ein 7,4 kb-EcoRI-Fragment, das das rearrangierte lambda 1-Gen des Maus-HOPC2020- Plasmacytoms enthielt [Bernard et al., Cell 15 (1978), 1133-1140]. Die Plasmid-DNA wurde im Vektor an der PvuI-Stelle linearisiert und die transgenen Mäusen wurden wie bereits beschrieben hergestellt [Reik et al., Eur. F. Immunol., 17 (1987), 465-469].
  • Der transgene IgA-2-lambda-Antikörper weist eine Spezifität für das Hapten 4-Hydroxy-3-nitrophenacetyl (NP) auf. Die Expression des transgenen Antikörpers wurde durch einen ELISA-Test unter Verwendung von an Plastikplatten gekuppeltem NP-Rinderserumalbumin und durch Entwicklung mit einem biotinylierten anti-Menschalpha-Antiserum gemessen. Gesamt-Ig wurde unter Verwendung von anti-Maus-Ig-beschichtetem Kunststoff und durch Entwicklung mit biotinyliertem anti-Maus-kappa- Antiserum gemessen. Die Konzentration des chimeren anti-NP-IgA2 wurde in der Vormilch, im Serum und in der Milch von sieben transgenen Mäusen und einer Kontrollmaus ermittelt. Die Ergebnisse sind in Tabelle 1 dargestellt. Vormilch wurde dem Muttertier nach einer hormonalen Injektion innerhalb von 24 Stunden nach der Geburt und die Milch 13 bis 15 Tage nach der Geburt des Kindes entnommen. Serum wurde gleichzeitig mit der Milch entnommen.
  • Diese Daten zeigen, daß transgene Tiere zur spezifischen Antikörperproduktion in großem Umfang aus Milch, Vormilch, Seren, Speichel etc. verwendet werden können sowie Tiere, die eine spezifische nützliche Antikörper enthaltende Milch liefern, gezüchtet werden können. Es ist ferner deutlich, daß die Konzentration des transgenen Antikörpers in der Vormilch im Vergleich zur Milch höher ist. Ferner beeinflußt das Transgen nicht die Fähigkeit des Tiers zur Produktion einer großen Menge an endogenen Antikörpern. Die Tiere zeigen keine Anzeichen von signifikanter Immunschwäche oder Krankheit.
    • Beispiel 3
  • In diesem Beispiel wurden Antigen-spezifische Hybridome unter Verwendung von menschlichen µ-Ketten aus transgenen Mäusen produziert.
  • Um zu zeigen, daß die in Beispiel 1 beschriebenen transgenen Mäuse zur Produktion von Antigen-spezifischen Antikörpern, in denen der Anteil der schweren Kette an der Antigen-Bindungsstelle durch den transgenen menschlichen Minilocus der schweren Kette geliefert wird, verwendet werden können, wurden die transgenen Mäuse mit 10&sup7; Schaferythrocyten (SRC) immunisiert. Milzzellen wurden 6 Tage später mit den NSO-Plasmacytomen fusioniert (Köhler und Milstein, Nature 256 (1975), 495-497). Die Zellen wurden in 96 Vertiefungen aufweisenden "Costar"-Platten ausplattiert, so daß 1 Hybridom pro Vertiefung erhalten wurde. Hybride, die für die menschliche µ-Kette positiv waren, wurden in einem ELISA unter Verwendung von biotinyliertem anti-Mensch- IgM nachgewiesen. Antigen-spezifische Hybride wurden durch Hämagglutination unter Verwendung von Schaferythrocyten identifiziert. Es wurden Vertiefungen ausgewählt, die Antikörper enthielten, die gemäß einem ELISA-Test für Schaferythrocyten spezifisch waren und die menschliche µ-Kette, jedoch weder Maus-µ-Ketten noch schwere gamma- Ketten der Maus enthielten.
  • Um nachzuweisen, daß der durch die selektierten Hybridome sezernierte Antikörper tatsächlich ein anti-SRC-Immunglobulin menschlicher µ-Kette und leichter Maus-Kette war, wurde eine Analyse über Fluoreszenz-aktivierte Zellsortierung (FACS) durchgeführt. Für die FACS-Analyse wurden 107 Schaferythrocyten 30 min. mit 20 µl Kulturüberstand inkubiert, einmal mit phosphatgepufferter Kochsalzlösung gewaschen und weiter entweder mit biotinyliertem Antiserum gegen die menschliche schwere µ- Kette, biotinyliertem anti-Maus-µ-Antiserum oder biotinyliertem Antiserum gegen die leichte kappa-Kette der Maus inkubiert. Nach 30 Minuten wurden die Zellen wie zuvor gewaschen und mit FITC-gekuppeltem Streptavidin inkubiert. Die Zellen wurden nach 30 Minuten gewaschen und konnten nach schonendem Aufbrechen analysiert werden.
  • Ergebnisse für 3 verschiedene gegen Schaferythrocyten-Antigen gerichtete IgM-produzierende Hybride sind in den FACS-Histogrammen der Fig. 6, 7 bzw. 8 dargestellt.
  • Die Buchstaben A, B, C und D in diesen Figuren weisen auf ein unterschiedliches Färbungsverhalten hin. Die Fig. 6A, 7A und 8A sind Ergebnisse von negativen Kontrollen unter Verwendung von Schaferythrocyten alleine, von mit dem Antikörper inkubierten Schaferythrocyten oder von Schaferythrocyten, die mit einem Fluoreszenz-markierten (FITC) zweiten Antikörper (entweder anti-Mensch-µ-anti-Maus-µ oder anti-Maus-kappa) inkubiert wurden. Die Fig. 6B, 7B und 8B zeigen Ergebnisse, die unter Verwendung von mit dem Hybrid-Kulturüberstand und Fluoreszenz-markierten (FITC) anti-Mensch-µ inkubierten Schaferythrocyten erhalten wurden. Die Fig. 6C, 7C und 8C zeigen Ergebnisse, die unter Verwendung von mit dem Kulturüberstand der transgenen Hybride und Fluoreszenz-markierten (FITC) anti- Maus-inkubierten Schaferythrocyten erhalten wurden. Fig. 6D zeigt Ergebnisse, die unter Verwendung von mit dem Hybrid-Kulturüberstand und Fluoreszenz-markierten (FITC) anti-Maus-kappa inkubierten Schaferythrocyten erhalten wurden.
  • In den Fig. 6 und 8 ist die Fluoreszenzintensität einer fixierten Anzahl von Zellen gegen die Zellzahl aufgezeichnet. In Fig. 7 ist die Fluoreszenzintensität einer fixierten Anzahl von Zellen gegen die Streuung aufgezeichnet, wobei jeder Punkt eine Zelle darstellt.
  • Eine Verschiebung der Profile nach rechts (erhöhte Fluoreszenz) zeigt eine positive Färbung an, die nur bei Antikörpern beobachtet werden kann, die die menschlichen schweren µ-Ketten und die leichten kappa-Ketten der Maus aufweisen, jedoch nicht bei Antikörpern, die die schweren µ-Ketten der Maus oder schweren gamma-Ketten der Maus (nicht dargestellt) enthalten. Tabelle 1 IgA2-Antikörper in Körperflüssigkeiten von lambda-1-Mäusen anti-NP-IgA2-Ak Gesamtmenge an kappa-tragenden Ak Maus Serum Milch Vormilch Kontrolle ND = nicht ermittelt Alle Konzentrationen in µg/ml

Claims (13)

1. Verfahren zur Herstellung eines Immunglobulins, umfassend die Insertion von DNA in die Keimbahn eines nichtmenschlichen Tieres, die ein mindestens einen Teil eines Immunglobulins menschlichen Ursprungs codierendes Nucleinsäuresegment umfaßt, das ein Gensegment einschließt, das einen Bereich der schweren Kette eines menschlichen Immunglobulins codiert, wobei das Gensegment nicht in vollständig rearrangierter Form vorliegt, wobei die DNA in dem Tier rearrangiert wird, so daß sie eine Anzahl von Immunglobulinen mit einem Teil oder Teilen, die von der inserierten DNA stammen, codiert, und in den Zellen oder der Körperflüssigkeit des Tieres exprimiert wird; und die Gewinnung von Immunglobulin aus Zellen oder geeigneter Körperflüssigkeit des Tieres.
2. Verfahren zur Herstellung eines Immunglobulins gegen ein bestimmtes Antigen, umfassend die Produktion eines transgenen nicht-menschlichen Tieres durch Insertion von DNA fremden Ursprungs in die Keimbahn des Tieres, die mindestens einen Teil eines Immunglobulins menschlichen Ursprungs codiert, das ein einen Bereich der schweren Kette eines menschlichen Immunglobulins codierendes Gensegment einschließt, wobei die DNA fremden Ursprungs einem Rearrangement oder einer Mutation im Lymphoidgewebe des transgenen Tieres unterliegt, so daß eine Vielzahl von rearrangierten Genen, die Immunglobuline, Immunglobulinfragmente oder chimere Immunglobuline codieren, produziert werden, so daß, nach Exposition des transgenen Tieres mit einem bestimmten Antigen, das Immunglobulin gegen das Antigen, das durch die rearrangierte oder mutierte DNA codiert wird, in Zellen oder Körperflüssigkeit des Tieres exprimiert wird; und Gewinnung von Immunglobulin aus Zellen oder geeigneter Körperflüssigkeit des Tieres.
3. Verfahren nach Anspruch 1 oder 2, wobei das Immunglobulin umfassendes polyclonales Antiserum von dem Tier erhalten wird.
4. Verfahren nach Anspruch 1 oder 2, wobei das Immunglobulin umfassende monoclonale Antikörper unter Verwendung von von dem Tier erhaltenen Zellen produziert werden.
5. Verfahren nach Anspruch 1 oder 2, wobei das Immunglobulin in einer Körperflüssigkeit oder einem Sekret des Tieres produziert wird.
6. Verfahren nach Anspruch 1 oder 2, wobei das Immunglobulin in vitro aus vom Tier erhaltenen Zellen produziert wird.
7. Verfahren nach einem der vorangehenden Ansprüche, wobei die inserierte DNA im wesentlichen die gesamten Speziesspezifischen Bereiche eines Immunglobulins codiert.
8. Verfahren nach einem der vorangehenden Ansprüche, wobei das transgene Tier eine Maus ist.
9. Verfahren nach einem der vorangehenden Ansprüche, wobei die inserierte DNA ein Plasmid oder Cosmid, multiple Plasmide oder Cosmide, ein künstliches Hefechromosom oder Vertebratenchromosom oder DNA-Fragmente umfaßt.
10. Verfahren nach einem der vorangehenden Ansprüche, wobei die DNA durch Injektion oder andere Verfahren in befruchtete hier oder embryonale Stammzellen inseriert wird.
11. Verfahren nach einem der vorangehenden Ansprüche, wobei das Tier zunächst kein genetisches Material trägt, das konstante Bereiche von Immunglobulin codiert.
12. Immunglobulin fremden Ursprungs, erhältlich durch das Verfahren nach einem der vorangehenden Ansprüche.
13. Transgenes nicht-menschliches Tier, in dessen Keimbahn DNA inseriert ist, die ein mindestens einen Teil eines Immunglobulin menschlichen Ursprungs codierendes Nucleinsäuresegment umfaßt, das ein Gensegment einschließt, welches einen Bereich der schweren Kette eines menschlichen Immunglobulins codiert, wobei das Gensegment nicht in vollständig rearrangierter Form vorliegt, wobei die DNA in dem Tier rearrangiert wird, so daß sie eine Anzahl von Immunglobulinen mit einem Teil oder Teilen, die von der inserierten DNA stammen, codiert, und in Zellen oder geeigneter Körperflüssigkeit des Tieres exprimiert wird.
DE68926508T 1988-10-12 1989-10-12 Erzeugung von antikörpern aus transgenen tieren Expired - Lifetime DE68926508T2 (de)

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ATE138104T1 (de) 1996-06-15
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AU4417389A (en) 1990-05-01
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US5545807A (en) 1996-08-13
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JP2003192699A (ja) 2003-07-09
EP0438474B1 (de) 1996-05-15
WO1990004036A1 (en) 1990-04-19
JPH04500911A (ja) 1992-02-20

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