EP0996639A2 - Human antibody against a fusion (poly) peptide or protein presenting a part having at least six histidines - Google Patents

Human antibody against a fusion (poly) peptide or protein presenting a part having at least six histidines

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Publication number
EP0996639A2
EP0996639A2 EP98943648A EP98943648A EP0996639A2 EP 0996639 A2 EP0996639 A2 EP 0996639A2 EP 98943648 A EP98943648 A EP 98943648A EP 98943648 A EP98943648 A EP 98943648A EP 0996639 A2 EP0996639 A2 EP 0996639A2
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EP
European Patent Office
Prior art keywords
dna
fusion
peptide
poly
antibody
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EP98943648A
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German (de)
French (fr)
Inventor
Melvyn Little
Martin Welschof
Sergey Kipriyanov
Timo KÜRSCHNER
Michael Braunagel
Heinz DÖRSAM
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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Publication of EP0996639A2 publication Critical patent/EP0996639A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • the present invention relates to human antibodies against a fusion (poly) peptide or protein which has at least six successive histidine residues, processes for their preparation and their use.
  • a polypeptide in the form of a histidine fusion polypeptide In such a histidine content of e.g. 5-1 8 successive histidine residues, mostly fused at the C or N terminus of the polypeptide.
  • polyclonal or monoclonal antibodies produced in mice or rabbits are now provided (German Patent DE-C-1 9 507 1 66).
  • the detection of the histidine fusion polypeptides is carried out using the antibodies in immune reactions, e.g. ELISAs using human serum as the standard. This has proven to be problematic since ELISA kits that offer human serum as standard are used in different countries, e.g. USA, can not be distributed or only with difficulty. In addition, it is often difficult to prepare a suitable serum sample in sufficient quantity and durability.
  • the present invention is therefore based on the object of providing an agent which, instead of human serum, can be used as a standard in immune reactions which are based on histidine fusion proteins.
  • this is achieved by a human antibody against a fusion (poly) peptide or protein that is at least six consecutive Histidine residues is directed.
  • Antibodies according to the invention are obtained from an antibody library which has been produced, for example, from human lymphocyte cDNA. They are isolated using the "phage display” technique (Welschof et al., Proc. Natl. Acad. Sei USA 94 (1997), pp. 1 902-1 907). Using this technique, the inventors have now for the first time succeeded in producing a human anti-histidine antibody. There has long been a need for a human antibody, but the immunization technique used in animals by injection of the histidine antigen and booster according to a certain schedule has of course never been possible in humans for ethical and medical reasons, so that it has not been possible to date to provide human antibodies to histidine fusion proteins.
  • the following steps can be used, for example, to obtain a human antibody according to the invention: 1) screening an IgM library with a hexahistidine fragment which is bound to a support (e.g. BSA); 2) selection of specific individual clones using phage ELISA; 3) Characterization of selected individual clones by phage ELISA and restriction duration; 4) recloning into an expression vector (e.g. E. coli); 5) Sequencing and sequence analysis of clones.
  • a support e.g. BSA
  • phage ELISA hexahistidine fragment which is bound to a support
  • Characterization of selected individual clones by phage ELISA and restriction duration e.g. E. coli
  • sequence analysis of clones e.g. E. coli
  • the antibody of the invention is preferably single chain, i.e. it consists only of the variable domain linked to a peptide linker.
  • fusion (poly) peptide or protein encompasses a peptide or protein of any type and length.
  • Such a (poly) peptide can be expressed by any cells, for example bacteria, yeast, insect, plant and animal cells, and organisms, for example transgenic animals.
  • An above histidine portion comprises, for example, 6-1 8, preferably 6-10, successive histidine residues and is fused to the N and / or C-terminus of the polypeptide or is in a cluster in the middle of the peptide.
  • a preferred antibody contains the amino acid sequence shown in FIG. 4 or a sequence differing therefrom by one or more amino acids.
  • the invention further relates to DNA coding for the antibody according to the invention. This DNA includes
  • hybridizing DNA indicates a DNA which hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA.
  • the DNA of FIG. 4 was deposited with the DSMZ (German Collection of Microorganisms and Cell Cultures) as clone A6 under DSM 1 1 595 on June 10, 1997.
  • Antibodies obtained above can be used to produce variations of the antibodies according to the invention (synthetic antibodies). For this purpose it is advisable to analyze the antigen binding regions of the antibodies and to identify the parts that are necessary and not necessary for the above recognition. The necessary parts can then be modified and the unnecessary parts can be completely or partially eliminated or replaced with parts that give the antibodies further favorable properties. Parts outside the binding regions of the antibodies can also be modified, eliminated or replaced.
  • DNA recombination technology is particularly suitable for the above measures. He is very familiar with this. Techniques are also known to the person skilled in the art to complete a single-chain antibody according to the invention by attaching light and heavy chains.
  • An advantage of the antibodies according to the invention is that they can be used universally in immunological detection methods instead of human serum.
  • Antibodies according to the invention are also distinguished by the fact that, like the anti-His antibodies produced in animals, they recognize any fusion (poly) peptides which have a histidine content.
  • the detection is based on the presence of the sequence of at least six histidines and is independent of the peptide content, i.e. the antibody according to the invention specifically recognizes the histidine portion of a histidine fusion (poly) peptide or protein.
  • the antibodies are therefore suitable for the rapid detection of the expression of such fusion polypeptides.
  • the detection is carried out in particular by Western blot, ELISA, immunoprecipitation or immunofluorescence.
  • the antibodies according to the invention can, if appropriate, be labeled or be used in combination with labeled antibodies directed against them.
  • antibodies according to the invention represent an alternative method for the purification of recombinant proteins with a sequence of at least 6 histidines by affinity chromatography. They also make it easier to identify the proteins when they are purified by chromatography, e.g. using metal chelate chromatography.
  • Another advantageous property of the antibodies according to the invention is that they have not been produced in any animal and therefore do not comprise any elements which would cause an immune reaction in humans. They are therefore ideal for in-vivo use, which can be both diagnostic and therapeutic. It is also possible to produce a vaccine with this antibody.
  • Figure 1 shows the cloning scheme for generating an antibody expression library 2 shows the enrichment of the antigen-specific phagemids
  • 6-histidine-BSA single clones The plates were coated with 6-histidine-BSA or BSA as a negative control. The OD 655 nm values of the individual clones are shown. If a clone has a low value for the detection of BSA and a high value for the detection of 6-histidine-BSA, this is an indication of the production of antigen-specific phagemid particles.
  • RNA from human peripheral lymphocytes was used to produce an IgM phage surface expression library.
  • IgM phage surface expression library (Dowel, Meth. Mol. Cell Biol. 3 (1,992), pp. 47-52).
  • Messenger RNA was generated using the Optiprep. 2 kits manufactured by Biometra (Göttingen) and rewritten in cDNA using a corresponding kit (Amersham, Braunschweig) in compliance with the manufacturer's instructions.
  • the cDNA was converted into a library by means of PCR (conditions according to Welschof et al., J. Immunol. Methods 179 (1 995), pp. 203-414) with a primer set which was developed on the basis of amino acid consensus sequences constructed.
  • This PCR primer set contained 1 2 primers for the amplification of the variable regions of the immunoglobulin chains, and one primer each for the amplification of the constant regions of the heavy and the two light chains (Welschof et al., See above).
  • the light chain repertoire was created in the phagemid surface expression vector pSEX 81 (Welschof et al., Proc. Natl. Acad. Be USA 94 (1 997), p. 1 902-1 907). After combining with the v H DNA repertoire (see FIG. 1), the entire vector was transformed into the expression system E. coli XL1 blue (Stratagene).
  • the bacteria transformed with the vector were infected with the helper phage M1 3KO7, whereby phage particles with single-chain Fv expressing on the surface were formed. After phage isolation from the culture supernatant, the phage library, which contained 4 ⁇ 10 7 independent clones, could be used for screening.
  • the phagemids obtained from the colonies were infected with helper phages (M 1 3KO7) with an MOI of 20 and packaged as phages.
  • the titer was determined from the resulting new infectious phages.
  • the above procedure of preincubation and subsequent administration in coated "Maxisorb Immunotubes" and infection of E. coli XL1 blue bacteria and packaging with helper phages M 1 3KO7 was repeated two to three times to enrich specifically binding phages. With increasing accumulation, the number of phagemid particles eluted should increase as more and more antigen-specific phagemid particles are eluted from round to round. An increasing elution titer is therefore a good measure of enrichment. This enrichment effect is shown in FIG. 2.
  • the eluted phagemids were then infected with E. coli XL1 blue and plated on LB Amp agar plates. 95 individual clones were picked from this plate, taken up in LB medium and infected with helper phage M 1 3KO7 with an MOI of 20. After culturing overnight at 37 ° C., 100 ⁇ l of culture supernatant were removed from each individual clone, which was each in a well of an ELISA plate which had been coated with a hexahistidine fragment (6-His-BSA; 1 ⁇ g / ml) coupled to BSA. was introduced. After overnight incubation at 4 ° C, 3 short washing steps with PBS followed.
  • EXAMPLE 2 ELISA detection of His fusion polypeptides by antibodies according to the invention
  • the phages were incubated with 1: 5000 diluted mouse monoclonal antibody directed against the phage main code protein pVIII and with peroxidase-coupled goat anti-mouse antibody (dilution according to the manufacturer). After 30 minutes of incubation at 37 ° C., 8 washing steps followed and then the peroxidase detection reaction with developer solution (50 mM sodium acetate, 0.4 mM 3.3 ', 5.5'-tetramethylbenzidine dihydrochloride, 4.4 mM H 2 O 2 ) at room temperature. The OD at 655nm was determined.
  • developer solution 50 mM sodium acetate, 0.4 mM 3.3 ', 5.5'-tetramethylbenzidine dihydrochloride, 4.4 mM H 2 O 2
  • the phage expresses the antibody according to the invention on the surface and specifically recognizes a histidine-BSA fusion polypeptide, but not BSA without a histidine component.

Abstract

The present invention relates to human antibodies against a fusion (poly) peptide presenting at least six consecutive histidines, a method for their production and their use.

Description

Humaner Antikörper gegen ein Fusions(poly)peptid bzw. -protein, das einen Anteil von mindestens sechs Histidinen aufweistHuman antibody against a fusion (poly) peptide or protein which contains at least six histidines
Die vorliegende Erfindung betrifft humane Antikörper gegen ein Fusions(po- ly)peptid bzw. -protein, das mindestens sechs aufeinanderfolgende Histidinreste aufweist, Verfahren zu ihrer Herstellung und ihre Verwendung.The present invention relates to human antibodies against a fusion (poly) peptide or protein which has at least six successive histidine residues, processes for their preparation and their use.
Es ist bekannt, ein Polypeptid in Form eines Histidin-Fusionspolypeptids zu ex- primieren. In einem solchen liegt ein Histidin-Anteil von z.B. 5-1 8 aufeinander folgenden Histidinresten, meist fusioniert am C- oder N-Terminus des Polypep- tids, vor. Zum Nachweis solcher Histidin-Fusionspolypeptide werden inzwischen in Mäusen oder Kaninchen erzeugte polyklonale oder monoklonale Antikörper bereitgestellt (Deutsches Patent DE-C-1 9 507 1 66). Der Nachweis der Histidin- Fusionspolypeptide erfolgt dabei unter Anwendung der Antikörper in Immunreaktionen, wie z.B. ELISAs, wobei Humanserum als Standard verwendet wird. Dies hat sich als problematisch erwiesen, da ELISA-Kits, die Humanserum als Standard anbieten, in verschiedenen Staaten, z.B. USA, nicht oder nur erschwert vertrieben werden können. Außerdem ist es häufig schwer, eine geeignete Serum-Probe in genügender Menge und ausreichender Haltbarkeit herzustellen.It is known to express a polypeptide in the form of a histidine fusion polypeptide. In such a histidine content of e.g. 5-1 8 successive histidine residues, mostly fused at the C or N terminus of the polypeptide. For the detection of such histidine fusion polypeptides, polyclonal or monoclonal antibodies produced in mice or rabbits are now provided (German Patent DE-C-1 9 507 1 66). The detection of the histidine fusion polypeptides is carried out using the antibodies in immune reactions, e.g. ELISAs using human serum as the standard. This has proven to be problematic since ELISA kits that offer human serum as standard are used in different countries, e.g. USA, can not be distributed or only with difficulty. In addition, it is often difficult to prepare a suitable serum sample in sufficient quantity and durability.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzustellen, das anstelle von Humanserum in Immunreaktionen, die auf Histidin-Fu- sionsproteinen aufgebaut sind, als Standard verwendet werden kann.The present invention is therefore based on the object of providing an agent which, instead of human serum, can be used as a standard in immune reactions which are based on histidine fusion proteins.
Diese Aufgabe wird durch die Gegenstände der Patentansprüche gelöst.This object is solved by the subject matter of the claims.
Insbesondere wird dies durch einen humanen Antikörper erreicht, der gegen ein Fusions(poly)peptid bzw. -protein, das mindestens sechs aufeinanderfolgende Histidinreste aufweist, gerichtet ist.In particular, this is achieved by a human antibody against a fusion (poly) peptide or protein that is at least six consecutive Histidine residues is directed.
Erfindungsgemäße Antikörper werden aus einer Antikörper-Bibliothek erhalten, die beispielsweise aus menschlicher Lymphozyten-cDNA hergestellt wurde. Sie werden mit Hilfe der "Phage Display" Technik (Welschof et al., Proc. Natl. Acad. Sei USA 94 (1997), S. 1 902-1 907) isoliert. Unter Anwendung dieser Technik ist es den Erfindern jetzt erstmals gelungen, einen humanen anti-Histidin Antikörper herzustellen. Der Bedarf nach einem humanen Antikörper bestand schon lange, aber die bei Tieren angewendete Immunisierungstechnik durch Injektion des Histidin-Antigens und Booster nach einem bestimmten Zeitschema konnte beim Menschen aus ethischen und medizinischen Gründen natürlich nie durchgeführt werden, so daß es bisher nicht möglich war, einen humanen Antikörper gegen Histidin-Fusionsproteine bereitzustellen.Antibodies according to the invention are obtained from an antibody library which has been produced, for example, from human lymphocyte cDNA. They are isolated using the "phage display" technique (Welschof et al., Proc. Natl. Acad. Sei USA 94 (1997), pp. 1 902-1 907). Using this technique, the inventors have now for the first time succeeded in producing a human anti-histidine antibody. There has long been a need for a human antibody, but the immunization technique used in animals by injection of the histidine antigen and booster according to a certain schedule has of course never been possible in humans for ethical and medical reasons, so that it has not been possible to date to provide human antibodies to histidine fusion proteins.
Zum Erhalt eines erfindungsgemäßen humanen Antikörpers können beispielsweise die folgenden Schritte angewendet werden: 1 ) Screening einer IgM-Biblio- thek mit einem Hexahistidinfragment, das an einen Träger (z.B. BSA) gebunden ist; 2) Auswahl von spezifischen Einzelklonen mittels Phagen-ELISA; 3) Charakterisierung ausgewählter Einzelklone durch Phagen-ELISA und Restriktionsver- dau; 4) Umklonierung in einen Expressionsvektor (z.B. E. coli); 5) Sequenzierung und Sequenzanalyse von Klonen. Die Einzelschritte zum Erhalt eines erfindungsgemäßen Antikörpers sind nachfolgend in den Beispielen ausführlicher beschrieben.The following steps can be used, for example, to obtain a human antibody according to the invention: 1) screening an IgM library with a hexahistidine fragment which is bound to a support (e.g. BSA); 2) selection of specific individual clones using phage ELISA; 3) Characterization of selected individual clones by phage ELISA and restriction duration; 4) recloning into an expression vector (e.g. E. coli); 5) Sequencing and sequence analysis of clones. The individual steps for obtaining an antibody according to the invention are described in more detail below in the examples.
Der erfindungsgemäße Antikörper ist vorzugsweise einzelkettig, d.h. er besteht nur aus der mit einem Peptidlinker verbundenen variablen Domäne.The antibody of the invention is preferably single chain, i.e. it consists only of the variable domain linked to a peptide linker.
Der Ausdruck "Fusions(poly)peptid bzw. -protein" umfaßt ein Peptid bzw. Protein jeglicher Art und Länge. Ein solches (Poly)peptid kann von jeglichen Zellen, z.B. Bakterien, Hefen, Insekten-, Pflanzen- und tierischen Zellen, sowie Organismen, z.B. transgenen Tieren, exprimiert sein. Ein vorstehender Histidin- Anteil umfaßt z.B. 6-1 8, vorzugsweise 6-10, aufeinander folgende Histidinreste und liegt fusioniert am N und/oder C-Terminus des Polypeptids vor oder aber befindet sich in einer Anhäufung in der Mitte des Peptids.The term "fusion (poly) peptide or protein" encompasses a peptide or protein of any type and length. Such a (poly) peptide can be expressed by any cells, for example bacteria, yeast, insect, plant and animal cells, and organisms, for example transgenic animals. An above histidine portion comprises, for example, 6-1 8, preferably 6-10, successive histidine residues and is fused to the N and / or C-terminus of the polypeptide or is in a cluster in the middle of the peptide.
Ein bevorzugter Antikörper enthält die in Fig. 4 angegebene Aminosäuresequenz oder eine sich hiervon durch eine oder mehrere Aminosäuren unterscheidende Sequenz. Weiter betrifft die Erfindung DNA kodierend für den erfindungsgemäßen Antikörper. Diese DNA umfaßtA preferred antibody contains the amino acid sequence shown in FIG. 4 or a sequence differing therefrom by one or more amino acids. The invention further relates to DNA coding for the antibody according to the invention. This DNA includes
(a) die DNA von Fig. 4 oder einen Teil davon,(a) the DNA of Fig. 4 or part thereof,
(b) eine mit der DNA von (a) hybridisierende DNA oder(b) a DNA hybridizing with the DNA of (a) or
(c) eine mit der DNA von (a) oder (b) über den degenerierten genetischen Code verwandte DNA.(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.
Der Ausdruck "hybridisierende DNA" weist auf eine DNA hin, die unter üblichen Bedingungen, insbesondere bei 20°C unter dem Schmelzpunkt der DNA, mit einer DNA von (a) hybridisiert.The term "hybridizing DNA" indicates a DNA which hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA.
Die DNA von Fig. 4 wurde bei der DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) als Klon A6 unter DSM 1 1 595 am 10. Juni 1997 hinterlegt.The DNA of FIG. 4 was deposited with the DSMZ (German Collection of Microorganisms and Cell Cultures) as clone A6 under DSM 1 1 595 on June 10, 1997.
Zur Herstellung von Variationen der erfindungsgemäßen Antikörper (synthetische Antikörper) kann von vorstehend erhaltenen Antikörpern ausgegangen werden. Hierzu bietet sich an, die Antigen-Bindungsregionen der Antikörper zu analysieren und die für vorstehende Erkennung notwendigen und nicht notwendigen Teile zu identifizieren. Die notwendigen Teile können dann modifiziert und die nicht notwendigen ganz oder teilweise eliminiert bzw. durch Teile ersetzt werden, die den Antikörpern weitere günstige Eigenschaften verleihen. Auch können Teile außerhalb der Bindungsregionen der Antikörper modifiziert, eliminiert oder ersetzt werden. Der Fachmann weiß, daß sich für vorstehende Maßnahmen insbesondere die DNA-Rekombinationstechnologie eignet. Diese ist ihm bestens vertraut. Dem Fachmann sind auch Techniken bekannt, einen erfindungsgemäßen einzelkettigen Antikörper durch Anhängen von leichter und schwerer Kette zu vervollständigen. Ein Vorteil der erfindungsgemäßen Antikörper ist, daß sie anstelle von Humanserum universell in immunologischen Nachweisverfahren einsetzbar sind.Antibodies obtained above can be used to produce variations of the antibodies according to the invention (synthetic antibodies). For this purpose it is advisable to analyze the antigen binding regions of the antibodies and to identify the parts that are necessary and not necessary for the above recognition. The necessary parts can then be modified and the unnecessary parts can be completely or partially eliminated or replaced with parts that give the antibodies further favorable properties. Parts outside the binding regions of the antibodies can also be modified, eliminated or replaced. The person skilled in the art knows that DNA recombination technology is particularly suitable for the above measures. He is very familiar with this. Techniques are also known to the person skilled in the art to complete a single-chain antibody according to the invention by attaching light and heavy chains. An advantage of the antibodies according to the invention is that they can be used universally in immunological detection methods instead of human serum.
Erfindungsgemäße Antikörper zeichnen sich auch dadurch aus, daß sie ebenso wie die im Tier erzeugten anti-His-Antikörper beliebige Fusions(poly)peptide erkennen, die einen Histidin-Anteil aufweisen. Die Erkennung basiert auf dem Vorhandensein der Abfolge von mindestens sechs Histidinen und ist unabhängig vom Peptidanteil, d.h. der erfindungsgemäße Antikörper erkennt spezifisch den Histidin-Anteil eines Histidin-Fusions(poly)peptids bzw. -proteins. Die Antikörper eignen sich daher zum schnellen Nachweis der Expression solcher Fusionspo- lypeptide. Der Nachweis erfolgt insbesondere durch Western Blot, ELISA, Im- munpräzipitation oder Immunfluoreszenz. Hierzu können die erfindungsgemäßen Antikörper, wenn es angebracht ist, markiert sein oder in Kombination mit markierten gegen sie gerichteten Antikörpern eingesetzt werden.Antibodies according to the invention are also distinguished by the fact that, like the anti-His antibodies produced in animals, they recognize any fusion (poly) peptides which have a histidine content. The detection is based on the presence of the sequence of at least six histidines and is independent of the peptide content, i.e. the antibody according to the invention specifically recognizes the histidine portion of a histidine fusion (poly) peptide or protein. The antibodies are therefore suitable for the rapid detection of the expression of such fusion polypeptides. The detection is carried out in particular by Western blot, ELISA, immunoprecipitation or immunofluorescence. For this purpose, the antibodies according to the invention can, if appropriate, be labeled or be used in combination with labeled antibodies directed against them.
Weitere Vorteile der erfindungsgemäßen Antikörper sind, daß sie eine alternative Methode für die Reinigung von rekombinanten Proteinen mit einer Abfolge von mindestens 6 Histidinen durch Affinitätschromatografie darstellen. Außerdem erleichtern sie die Identifikation der Proteine, wenn diese chromatografisch gereinigt werden, z.B. mittels Metallchelatchromatografie.Further advantages of the antibodies according to the invention are that they represent an alternative method for the purification of recombinant proteins with a sequence of at least 6 histidines by affinity chromatography. They also make it easier to identify the proteins when they are purified by chromatography, e.g. using metal chelate chromatography.
Eine weitere vorteilhafte Eigenschaft der erfindungsgemäßen Antikörper ist, daß sie in keinem Tier erzeugt worden sind und daher auch keine Elemente umfassen, die im Menschen eine Immunreaktion bedingen würden. Sie eignen sich damit bestens für den in-vivo Einsatz, der sowohl diagnostischer als auch therapeutischer Art sein kann. Weiter ist es möglich mit diesem Antikörper eine Vakzine herzustellen.Another advantageous property of the antibodies according to the invention is that they have not been produced in any animal and therefore do not comprise any elements which would cause an immune reaction in humans. They are therefore ideal for in-vivo use, which can be both diagnostic and therapeutic. It is also possible to produce a vaccine with this antibody.
Die vorliegende Erfindung wird durch die folgenden Figuren weiter erläutert:The present invention is further illustrated by the following figures:
Fig. 1 zeigt das Klonierungsschema zum Erzeugen einer Antikörper-Expressionsbibliothek Fig. 2 zeigt die Anreicherung der Antigen-spezifischen PhagemideFigure 1 shows the cloning scheme for generating an antibody expression library 2 shows the enrichment of the antigen-specific phagemids
Fig. 3 zeigt einen Phagen-ELISA zum Nachweis spezifischer Bindung der3 shows a phage ELISA for the detection of specific binding of the
6-Histidin-BSA Einzelklone. Die Platten wurden mit 6-Histidin-BSA bzw. BSA als Negativkontrolle beschichtet. Dargestellt sind die OD 655 nm-Werte der Einzelklone. Wenn ein Klon einen niedrigen Wert für die Erkennung von BSA und einen hohen Wert für die Erkennung von 6-Histidin-BSA besitzt, ist dies ein Indiz für die Produktion Antigen-spezifischer Phagemidpartikel.6-histidine-BSA single clones. The plates were coated with 6-histidine-BSA or BSA as a negative control. The OD 655 nm values of the individual clones are shown. If a clone has a low value for the detection of BSA and a high value for the detection of 6-histidine-BSA, this is an indication of the production of antigen-specific phagemid particles.
Fig. 4 zeigt die Sequenzanalyse des Klons A6, der für einen erfindungsgemäßen Antikörper kodiert.4 shows the sequence analysis of clone A6, which codes for an antibody according to the invention.
Die vorliegende Erfindung wird weiter durch die nachstehenden Beispiele erläutert.The present invention is further illustrated by the following examples.
BEISPIEL 1 : Herstellung erfindungsgemäßer AntikörperEXAMPLE 1: Preparation of Antibodies According to the Invention
Zur Herstellung einer IgM Phagen-Oberflächen-Expressionsbibliothek wurde Ge- samt-RNA aus menschlichen peripheren Lymphozyten (erhalten aus Spenderblut) eingesetzt. (Dübel, Meth. Mol. Cell Biol. 3 ( 1 992), S. 47-52). Messenger-RNA wurde unter Verwendung des Optiprep. 2-Kits der Firma Biometra (Göttingen) hergestellt und in cDNA unter Verwendung eines entsprechenden Kits (Amers- ham, Braunschweig) unter Beachtung der Herstellerhinweise umgeschrieben. Aus der cDNA wurde mittels PCR (Bedingungen gemäß Welschof et al., J. Immunol. Methods 179 (1 995), S. 203-414) mit einem Primersatz, der auf der Grundlage von Aminosäuren-Konsensus-Sequenzen entwickelt wurde, eine Bibliothek konstruiert. Dieser PCR-Primersatz beinhaltete 1 2 Primer zur Am- plifikation der variablen Bereiche der Immunglobulinketten, sowie je einen Primer für die Amplifikation der konstanten Bereiche der schweren und der beiden leichten Ketten (Welschof et al., s. oben) . Das Leichtketten-Repertoire wurde in den Phagemid-Oberflächenexpressionsvektor pSEX 81 (Welschof et al., Proc. Natl. Acad. Sei USA 94 (1 997), S. 1 902-1 907) kloniert. Nach Kombinieren mit dem vH DNA-Repertoire (s. Fig. 1 ) wurde der gesamte Vektor in das Expressionssystem E. coli XL1 blue (Stratagene) transformiert. Die mit dem Vektor transformierten Bakterien wurden mit dem Helferphagen M1 3KO7 infiziert, wodurch Phagen-Partikel mit auf der Oberfläche exprimierenden einzelkettigen Fv entstanden. Nach der Phagenisolierung aus dem Kulturüberstand ließ sich die Phagenbibliothek, die 4 x 1 07 unabhängige Klone enthielt, zum Screening benutzen.Total RNA from human peripheral lymphocytes (obtained from donor blood) was used to produce an IgM phage surface expression library. (Dowel, Meth. Mol. Cell Biol. 3 (1,992), pp. 47-52). Messenger RNA was generated using the Optiprep. 2 kits manufactured by Biometra (Göttingen) and rewritten in cDNA using a corresponding kit (Amersham, Braunschweig) in compliance with the manufacturer's instructions. The cDNA was converted into a library by means of PCR (conditions according to Welschof et al., J. Immunol. Methods 179 (1 995), pp. 203-414) with a primer set which was developed on the basis of amino acid consensus sequences constructed. This PCR primer set contained 1 2 primers for the amplification of the variable regions of the immunoglobulin chains, and one primer each for the amplification of the constant regions of the heavy and the two light chains (Welschof et al., See above). The light chain repertoire was created in the phagemid surface expression vector pSEX 81 (Welschof et al., Proc. Natl. Acad. Be USA 94 (1 997), p. 1 902-1 907). After combining with the v H DNA repertoire (see FIG. 1), the entire vector was transformed into the expression system E. coli XL1 blue (Stratagene). The bacteria transformed with the vector were infected with the helper phage M1 3KO7, whereby phage particles with single-chain Fv expressing on the surface were formed. After phage isolation from the culture supernatant, the phage library, which contained 4 × 10 7 independent clones, could be used for screening.
1011 bis 1012 rekombinante Phagen aus der obigen Bibliothek mit einer Komplexität von 4 x 1 07 wurden in PBS enthaltend 2% Milchpulver 2 Stunden präabsorbiert. Ein "Maxisorb lmmunotube"-Röhrchen (Nunc, Roskilde, Dänemark) wurde mit 1 25 μg Hexahistidinfragment konjugiert an BSA ( = Antigen) beschichtet. Ein zweites "Maxisorb lmmunotube"-Röhrchen wurde mit 1 25 μg BSA beschichtet. In diese Röhrchen wurden die präadsorbierten Phagen gefüllt und eine Stunde inkubiert. Die Röhrchen wurden 20 mal mit PBS-0, 1 % Tween und 20 mal mit PBS gewaschen. Zur Elution der Phagen wurde 1 ml 100 mmol Triethylamin in die Röhrchen hinzugefügt, 5 Min. inbubiert und mit 1 ml Tris-HCI neutralisiert. Die nun neutrale Phagenlösung wurden aus den Röhrchen entfernt und zu Bakterien (E. coli XL1 blue), die sich in der log-Phase befinden, gegeben. Nach einer Inkubationszeit von 1 Stunden wurden die Bakterien abzentrifugiert, die sedimentierten Bakterien in 1 ml LB-Kulturmedium aufgenommen und auf LB- Amp-Agarplatten ausplattiert. Die Platten wurden über Nacht bei 37 °C inkubiert. Am nächsten Morgen wurden die Kolonien geerntet. Die erhaltenen Phagemide aus den Kolonien wurden mit Helferphagen (M 1 3KO7) mit einer MOI von 20 infiziert und als Phagen verpackt. Von den entstandenen neuen infektiösen Phagen wurde der Titer bestimmt. Die obige Prozedur der Präinkubation und anschließende Gabe in beschichtete "Maxisorb Immunotubes" sowie Infektion von E. coli XL1 blue-Bakterien und Verpacken mit Helferphagen M 1 3KO7 wurde zur Anreicherung spezifisch bindender Phagen zwei- bis dreimal wiederholt. Mit zunehmender Anreicherung sollte die Anzahl an eluierten Phagemidpartikeln zunehmen, da von Runde zu Runde immer mehr Antigen-spezifische Phagemidp- artikel eluiert werden. Somit ist ein steigender Elutionstiter ein gutes Maß für eine Anreicherung. Dieser Anreicherungseffekt ist in Fig. 2 gezeigt.10 11 to 10 12 recombinant phages from the above library with a complexity of 4 × 10 7 were pre-absorbed in PBS containing 2% milk powder for 2 hours. A "Maxisorb immunotube" tube (Nunc, Roskilde, Denmark) was coated with 1 25 μg hexahistidine fragment conjugated to BSA (= antigen). A second "Maxisorb Immunotube" tube was coated with 1 25 µg BSA. The pre-adsorbed phages were filled into these tubes and incubated for one hour. The tubes were washed 20 times with PBS-0, 1% Tween and 20 times with PBS. To elute the phages, 1 ml of 100 mmol of triethylamine was added to the tubes, incubated for 5 minutes and neutralized with 1 ml of Tris-HCl. The now neutral phage solution was removed from the tubes and added to bacteria (E. coli XL1 blue) that are in the log phase. After an incubation period of 1 hour, the bacteria were centrifuged off, the sedimented bacteria were taken up in 1 ml LB culture medium and plated out on LB amp agar plates. The plates were incubated overnight at 37 ° C. The colonies were harvested the next morning. The phagemids obtained from the colonies were infected with helper phages (M 1 3KO7) with an MOI of 20 and packaged as phages. The titer was determined from the resulting new infectious phages. The above procedure of preincubation and subsequent administration in coated "Maxisorb Immunotubes" and infection of E. coli XL1 blue bacteria and packaging with helper phages M 1 3KO7 was repeated two to three times to enrich specifically binding phages. With increasing accumulation, the number of phagemid particles eluted should increase as more and more antigen-specific phagemid particles are eluted from round to round. An increasing elution titer is therefore a good measure of enrichment. This enrichment effect is shown in FIG. 2.
Danach wurden die eluierten Phagemide mit E. coli XL1 blue infiziert und auf LBAmp-Agarplatten ausplattiert. Von diesem Platten wurden 95 Einzelklone gepickt, in LB-Medium aufgenommen und mit Helferphagen M 1 3KO7 mit einer MOI von 20 infiziert. Von jedem einzelnen Klon wurden nach Kultivieren über Nacht bei 37°C 100 μl Kulturüberstand abgenommen, welcher in jeweils eine Vertiefung einer ELISA-Platte, die mit an BSA gekoppeltem Hexahistidinfragment (6-His-BSA; 1 μg/ml) beschichtet worden war, eingebracht wurde. Nach Inkubation über Nacht bei 4°C schlössen sich 3 kurze Waschschritte mit PBS an. Anschließend erfolgte die Blockierung freier Bindungsstellen des polymeren Trägers durch einstündige Inkubation mit 2 % Milchpulver in PBS bei Raumtemperatur. Dazu wurde ein gegen das Phagen-Hauptcodeprotein pVIII gerichteter monoklonaler Antikörper aus Maus sowie ein Peroxidase-gekoppelte Ziege-antiMaus Antikörper (Verdünnung nach Angabe der Hersteller) zugegeben. Nach 30- minütiger Inkubation bei 37°C folgten 8 Waschschritte und anschließend die Peroxidase-Nachweisreaktion mit Entwicklerlösung (50 mM Natriumacetat, 0,4 mM 3,3', 5,5'-Tetramethyl-benzidin-dihydrochlorid, 4,4 mM H2O2) bei Raumtemperatur. Die OD bei 655nm wurde bestimmt. Zur Kontrolle wurden statt der mit BSA-Histidinfragment beschichteten ELISA-Platte eine nur mit BSA beschichtete ELISA-Platte eingesetzt. Das Ergebnis des Phagen-ELISA's ist in Fig. 3 gezeigt.The eluted phagemids were then infected with E. coli XL1 blue and plated on LB Amp agar plates. 95 individual clones were picked from this plate, taken up in LB medium and infected with helper phage M 1 3KO7 with an MOI of 20. After culturing overnight at 37 ° C., 100 μl of culture supernatant were removed from each individual clone, which was each in a well of an ELISA plate which had been coated with a hexahistidine fragment (6-His-BSA; 1 μg / ml) coupled to BSA. was introduced. After overnight incubation at 4 ° C, 3 short washing steps with PBS followed. The free binding sites of the polymeric carrier were then blocked by incubation for 1 hour with 2% milk powder in PBS at room temperature. For this purpose, a mouse monoclonal antibody directed against the phage main code protein pVIII and a peroxidase-coupled goat anti-mouse antibody (dilution according to the manufacturer's instructions) were added. After 30 minutes of incubation at 37 ° C., 8 washing steps followed and then the peroxidase detection reaction with developer solution (50 mM sodium acetate, 0.4 mM 3.3 ', 5.5'-tetramethylbenzidine dihydrochloride, 4.4 mM H) 2 O 2 ) at room temperature. The OD at 655nm was determined. As a control, an ELISA plate only coated with BSA was used instead of the ELISA plate coated with BSA-histidine fragment. The result of the phage ELISA is shown in Fig. 3.
Aus den 95 Einzelklonen wurden 10 Klone, die ein signifikantes Signal für das jeweilige Screening-Antigen (6-His-BSA) zeigten, ausgewählt und in einem zweiten Phagen-ELISA auf ihre Reaktivität gegenüber 6-His-BSA und andere 5- bzw. 6-His-getaggte Antikörper, die als Kontroll-Antigene eingesetzt wurden, getestet.From the 95 individual clones, 10 clones which showed a significant signal for the respective screening antigen (6-His-BSA) were selected and their reactivity towards 6-His-BSA and other 5- or Tested 6-His tagged antibodies used as control antigens.
Ein Restriktionsverdau und die Sequenzanlyse der 10 Einzelklone ergab, daß diese sich nicht voneinander unterscheiden, sondern von einem Urklon abstam- men. Die Sequenzanalyse erfolgte unter Verwendung der Didesoxy-Termina- tionsmethode mit dem Vektor pSEX-81 (Welschof et al., Proc. Natl. Acad. Sei. USA 94 (1 997), S. 1 902-1 907). Die Sequenzierung des Klons A6 ergab die in Figur 4 gezeigte Sequenz.Restriction digestion and sequence analysis of the 10 individual clones showed that they do not differ from one another, but are derived from an original clone. men. The sequence analysis was carried out using the dideoxy termination method with the vector pSEX-81 (Welschof et al., Proc. Natl. Acad. Sci. USA 94 (1 997), p. 1 902-1 907). The sequencing of clone A6 gave the sequence shown in FIG. 4.
BEISPIEL 2: ELISA-Nachweis von His-Fusionspolypeptiden durch erfindungsgemäße AntikörperEXAMPLE 2: ELISA detection of His fusion polypeptides by antibodies according to the invention
In eine 96-Loch-Platte wurden pro Loch je 1 00μl mit 1 μg Histidin-BSA-Fusions- polypeptid bzw. als Kontrolle BSA einpipettiert. Nach Inkubation über Nacht bei 4°C schlössen sich 3 kurze Waschschritte mit PBS an. Anschließend erfolgte die Blockierung freier Bindungsstellen des polymeren Trägers durch einstündige Inkubation mit 1 % BSA + 2% Milchpulver in PBS bei 37 °C. 109 Phagen in Phagenverdünnungspuffer d O mM Tris-HCI, pH 7,5; 20 mM NaCI; 2 mM EDTA) wurden hinzugefügt. Die Phagen wurden mit 1 :5000 verdünntem gegen das Phagen-Hauptcodeprotein pVIII gerichtetem monoklonalem Antikörper aus Maus sowie mit Peroxidase-gekoppeltem Ziege-anti-Maus Antikörper (Verdünnung nach Angabe der Hersteller) inkubiert. Nach 30-minütiger Inkubation bei 37°C folgten 8 Waschschritte und anschließend die Peroxidase-Nachweisreaktion mit Entwicklerlösung (50 mM Natriumacetat, 0,4 mM 3,3', 5,5'-Tetramethyl-benzi- din-dihydrochlorid, 4,4 mM H2O2) bei Raumtemperatur. Die OD bei 655nm wurde bestimmt.100 μl of 1 μg of histidine-BSA fusion polypeptide or, as a control, of BSA were pipetted into a 96-well plate. After overnight incubation at 4 ° C, 3 short washing steps with PBS followed. The free binding sites of the polymeric carrier were then blocked by incubation for 1 hour with 1% BSA + 2% milk powder in PBS at 37 ° C. 10 9 phages in phage dilution buffer dO mM Tris-HCl, pH 7.5; 20 mM NaCl; 2 mM EDTA) were added. The phages were incubated with 1: 5000 diluted mouse monoclonal antibody directed against the phage main code protein pVIII and with peroxidase-coupled goat anti-mouse antibody (dilution according to the manufacturer). After 30 minutes of incubation at 37 ° C., 8 washing steps followed and then the peroxidase detection reaction with developer solution (50 mM sodium acetate, 0.4 mM 3.3 ', 5.5'-tetramethylbenzidine dihydrochloride, 4.4 mM H 2 O 2 ) at room temperature. The OD at 655nm was determined.
Es zeigte sich, daß der Phage den erfindungsgemäßen Antikörper auf der Oberfläche exprimiert und spezifisch ein Histidin-BSA-Fusionspolypeptid, nicht aber BSA ohne Histidin-Anteil, erkannt wird. It was shown that the phage expresses the antibody according to the invention on the surface and specifically recognizes a histidine-BSA fusion polypeptide, but not BSA without a histidine component.

Claims

Patentansprüche claims
1 . Humaner Antikörper gegen ein Fusions(poly)peptid bzw. -protein, das einen Anteil von mindestens sechs aufeinanderfolgenden Histidinen aufweist.1 . Human antibody against a fusion (poly) peptide or protein which contains at least six successive histidines.
2. Antikörper nach Anspruch 1 , dadurch gekennzeichnet, daß er die in Fig. 4 gezeigte Aminosäuresequenz oder sich hiervon durch eine oder mehrere Aminosäuren unterscheidet.2. Antibody according to claim 1, characterized in that it differs from the amino acid sequence shown in FIG. 4 or differs therefrom by one or more amino acids.
3. DNA kodierend für einen Antikörper nach Anspruch 1 oder 2 umfaßend3. DNA encoding for an antibody according to claim 1 or 2 comprising
(a) die DNA von Fig. 4 oder einen Teil davon,(a) the DNA of Fig. 4 or part thereof,
(b) eine mit der DNA von (a) hybridisierende DNA oder(b) a DNA hybridizing with the DNA of (a) or
(c) eine mit der DNA von (a) oder (b) über den degenerierten genetischen Code verwandte DNA.(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.
4. DNA nach Anspruch 3, dadurch gekennzeichnet, daß sie bei der DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) unter DSM 1 1 595 am 10. Juni 1 997 hinterlegt worden ist.4. DNA according to claim 3, characterized in that it has been deposited with the DSMZ (German Collection of Microorganisms and Cell Cultures) under DSM 1 1 595 on June 10, 997.
5. Verfahren zur Herstellung eines Antikörpers nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß er aus einer IgM scFv-Phagen-Oberflächen- Expressionsbibliothek, die aus menschlichen peripheren Lymphozyten hergestellt worden ist, unter Anwendung von Screeningschritten gewonnen wird.5. A method for producing an antibody according to claim 1 or 2, characterized in that it is obtained from an IgM scFv phage surface expression library which has been prepared from human peripheral lymphocytes using screening steps.
6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß ein an BSA gebundenes Hexahistidin-Fragment zum Screening der Bibliothek eingesetzt wird. 6. The method according to claim 5, characterized in that a hexahistidine fragment bound to BSA is used for screening the library.
7. Verwendung eines Antikörpers nach Anspruch 1 oder 2 in einem Nachweisverfahren für ein Fusions(poly)peptid bzw. -protein, das einen Anteil von mindestens sechs aufeinanderfolgenden Histidinen aufweist.7. Use of an antibody according to claim 1 or 2 in a detection method for a fusion (poly) peptide or protein which has a proportion of at least six consecutive histidines.
8. Verwendung nach Anspruch 7, wobei das Nachweisverfahren ein We- stern-Blot, ein ELISA, eine Immunfluoreszenz oder eine Immunpräzipitation ist. 8. Use according to claim 7, wherein the detection method is a Western blot, an ELISA, an immunofluorescence or an immunoprecipitation.
EP98943648A 1997-07-04 1998-07-03 Human antibody against a fusion (poly) peptide or protein presenting a part having at least six histidines Withdrawn EP0996639A2 (en)

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