EP0539547A1 - Process for preparing antigen-specific b and t lymphocytes and monoclonal antibodies obtained therefrom - Google Patents

Process for preparing antigen-specific b and t lymphocytes and monoclonal antibodies obtained therefrom

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Publication number
EP0539547A1
EP0539547A1 EP92909718A EP92909718A EP0539547A1 EP 0539547 A1 EP0539547 A1 EP 0539547A1 EP 92909718 A EP92909718 A EP 92909718A EP 92909718 A EP92909718 A EP 92909718A EP 0539547 A1 EP0539547 A1 EP 0539547A1
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EP
European Patent Office
Prior art keywords
antigen
cells
lymphocytes
adherent
presenting cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP92909718A
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German (de)
French (fr)
Inventor
Johann Hinrich Peters
Susanne Lenzner
Uwe Scholtes
Carl Watzek
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IDT AG fur IN VIVO DIAGNOSTIK und THERAPIE
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IDT AG fur IN VIVO DIAGNOSTIK und THERAPIE
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Publication of EP0539547A1 publication Critical patent/EP0539547A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin

Definitions

  • the present invention relates to a method for providing antigen-specific B and T lymphocytes, 5 in which body fluids and / or tissues from
  • Mammals isolated antigen-presenting cells and lymphocytes are incubated together for a specific adhesion and cultured according to conventional methods.
  • the present invention is therefore based on the object of providing a method with which this can be avoided.
  • antigen-presenting cells and lymphocytes are isolated from various body fluids and / or tissues from mammals.
  • Blood, lymph, pleural, peritoneal and synovial fluid and cerebrospinal fluid are to be mentioned here in particular as body fluids.
  • the body fluids can be taken from both healthy and sick mammals. The same applies to
  • Tissues of which the spleen, lymph nodes, bone marrow, skin and tumors are to be specified.
  • mammals 5 are meant in particular humans, domestic and laboratory animals, whereby domestic animals are also to be understood as domestic animals.
  • body fluids and / or tissues can originate from one or more mammals. It is not imperative that, when using several mammals, they all belong to one of the groups mentioned above.
  • the method according to the invention is just as limited to
  • Mammals rather it includes all living things from the animal kingdom, from which the above cells can be isolated.
  • Ficoll-Hypaque centrifugation method can be used to isolate the antigen-presenting cells and the lymphocytes.
  • body fluids are used directly, while solid tissues and tumors are centrifuged only after individual preparation by comminution and digestion with conventional enzymes, such as collagenase dispase or trypsin, under conditions such as 1 h at 37 ° C.
  • the antigen-presenting cells and the lymphocytes are obtained in the interphase and then washed several times with a physiological buffer, for example PBS, pH 7.2.
  • the antigen-presenting cells are separated from the lymphocytes.
  • Known methods can be used for this.
  • the cells are in one
  • Culture vessel incubated with serum-free medium, for example RPMI 1640 or CG medium, under customary conditions, such as 37 ° C. and 5% CO.
  • the culture vessel has a coating with plasma or serum, which comes from the same mammal (autologous) or from the same species (allogeneic) from which the antigen-presenting cells and the lymphocytes have been isolated.
  • the antigen-presenting cells spontaneously adhere to the coating, whereas the lymphocytes remain in suspension. After an hour's incubation, the lymphocytes are pipetted off, while the antigen-presenting cells remain.
  • the lymphocytes are freed from suppressive cells, for which purpose they are treated, for example, with leucine methyl ester (1.25 mM) for 30 minutes at room temperature and then several times in the above-mentioned, serum-free cells
  • the antigen-presenting cells are activated, whereby they are treated differently according to their maturity.
  • Antigen-presenting cells made of solid tissue are mature in many cases and therefore highly active. In contrast, they are often not mature from body fluids and are therefore incubated for about 24 hours in the culture vessels specified above, e.g. CG medium specified above is used.
  • Factors for induction such as interleukin-1, interleukin-6, adenosine or dibutyryl-cAMP, are advantageously added.
  • antigen-presenting cells from bone marrow are usually still present as precursor cells and are therefore incubated in one of the media specified above for about 1 week.
  • Factors such as linoleic acid (4.ug/ml), vitamin D3 (2.5 M), vitamin E (0.1%), multi-CSF (2 U / ml) and M-CSF (3 U / ml ) added.
  • Added antigen is, for example, in the range from 1 ⁇ g to 10 ⁇ g / ml culture medium. It is added from a few minutes to 24 hours.
  • Whole or parts of cells, pathogens and / or biomolecules can be mentioned as antigen. Under cells there are both normal and to understand virus-modified and pathologically degenerate.
  • the pathogens include viruses, bacteria and allergens, while the biomolecules include antibodies, mediators, enzymes and neurotransmitters.
  • the antigens can be used as such alone or in combination with conventional carrier molecules, both of which are also used in mixtures with common adjuvants.
  • the antigen-presenting cells and the lymphocytes are incubated together for specific adhesion.
  • the cells are introduced into the culture vessels specified above, the lymphocytes being in a 10 to 1000-fold excess, and cultured with one of the media mentioned above.
  • the cultivation time is 15 minutes to 2 days at 37 ° C or a maximum of 24 hours at 4 ° C.
  • the non-adherent cells are separated from the adherent ones. To do this, wash the coated ones carefully by rinsing them carefully
  • the adherent cells are further cultivated by customary methods. They are incubated in the culture vessels specified above.
  • As the medium an above-mentioned one is used, to which factors that require proliferation, such as interleukin-2, -4 or -6, can be added.
  • a conditioned medium which is obtained, for example, from permanent cell cultures, can also be added.
  • the incubation leads to the proliferation of antigen-specific B and T lymphocytes. Unspecific lymphocytes are only formed in an extremely small amount. After 4 days of incubation, antibodies are obtained in the supernatants, the amount of which can be increased by further incubation. It is also advantageous to add fresh medium with proliferation-promoting factors every week. If necessary, antigen-presenting cells can also be added, but these should correspond to the originally used cells.
  • the antibodies obtained according to the invention are highly specific against the antigens used, in particular after cloning. It is then a monoclonal idiotype
  • Antibodies also anti-idiotype antibodies if an antibody has been used as the antigen.
  • the antigen-specific B and T lymphocytes obtained according to the invention are extremely homogeneous in themselves. After cloning, they are particularly suitable for the isolation of specific nucleic acid precursors, for example mRNA, which can then be used for gene cloning purposes and for the amplification of specific nucleic acid sequences using known techniques, for example PCR methods. Furthermore, the B- and T lymphocytes for adoptive immunotherapy. Furthermore, they can also be immortalized by conventional methods, such as fusion with hybridoma cells. The B lymphocytes obtained according to the invention can furthermore be cultured and expanded further by adding anti-CD40 antibodies.
  • Ficoll-Hypaque centrifugation method isolated and separated from each other in culture vessels with autologous plasma coating.
  • the lymphocytes were freed from suppressive cells by treatment with leucine methyl ester, while the antigen-presenting cells were incubated with 0, 1 and 10 ng tetanus toxoid / ml medium for 24 h as described above. The antigen was then washed out.
  • the antigen-presenting cells and the lymphocytes were incubated together with CG medium in the culture vessels described above.
  • the lymphocytes were in 200-fold excess. After 24 hours of incubation, the non- adherent cells removed by careful pipetting. Fresh medium supplemented with conditioned medium was added to the adherent cells. After 6 days of incubation, cell count, total antibody amount and specific
  • Antibody amount determined.
  • the specific antibody synthesis was determined in an ELISA using a tetanus toxoid bound to plastic as the antigen.
  • the non-specific antibody synthesis was also determined in an ELISA using a mouse antibody directed against human Ig.
  • antigen-specific B and T lymphocytes are preferably provided in the methods according to the invention, whereas non-antigen-specific ones are largely diluted.

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Abstract

Selon un procédé de préparation de lymphocytes B et T spécifiques d'antigènes, on isole des fluides et/ou des tissus organiques de mammifères des cellules et des lymphocytes présentant des antigènes, on les incube ensemble afin d'obtenir une adhérence spécifique et on les cultive par un procédé usuel. Ce procédé se caractérise par le fait que l'on sépare les cellules adhérentes des cellules non adhérentes et que l'on ne cultive pas les cellules non adhérentes, mais uniquement les cellules adhérentes.A method for preparing antigen-specific B and T lymphocytes involves isolating antigen-presenting cells and lymphocytes from mammalian body fluids and/or tissues, incubating them together to achieve specific adhesion, and cultivated by a usual process. This process is characterized by the fact that the adherent cells are separated from the non-adherent cells and that the non-adherent cells are not cultured, but only the adherent cells.

Description

Verfahren zur Bereitstellung von Antigen-spezifischen B- und T- ymphozyten sowie davon erhaltene monoklonale Anti¬ körperYmphozyten method for providing antigen-specific B and T and obtained therefrom monoclonal anti ¬ body
Die vorliegende Erfindung betrifft ein Verfahren zur Bereitstellung Antigen-spezifischer B- und T-Lymphozyten, 5 bei dem aus Körperflüssigkeiten und/oder Geweben vonThe present invention relates to a method for providing antigen-specific B and T lymphocytes, 5 in which body fluids and / or tissues from
Säugetieren antigenpräsentierende Zellen und Lymphozyten isoliert, diese zu einer spezifischen Adhäsion gemeinsam inkubiert und nach üblichen Verfahren kultiviert werden.Mammals isolated antigen-presenting cells and lymphocytes, these are incubated together for a specific adhesion and cultured according to conventional methods.
IQ Ein solches Verfahren ist aus Maeda, T. et al., Hybridoma,I Q Such a method is from Maeda, T. et al., Hybridoma,
Band 5, Nr. 1 (1986), 33-41 bekannt.Volume 5, No. 1 (1986), 33-41.
Es hat sich nun gezeigt, daß dieses Verfahren in großem Maße zu unspezifischen, nicht-Antigen-spezifischen B- und jg T-Lymphozyten führt.It has now been shown that this method leads to a large extent to non-specific, non-antigen-specific B and j g T lymphocytes.
Der vorliegenden Erfindung liegt daher die Aufgäbe zugrunde, ein Verfahren bereitzustellen, mit dem dies vermieden werden kann. 0The present invention is therefore based on the object of providing a method with which this can be avoided. 0
Erfindungsgemäß wird dies dadurch erreicht, daß in obigem Verfahren die adhärenten Zellen von den nicht-adhärenten abgetrennt und nur die adhärenten Zellen, nicht aber die nicht-adhärenten kultiviert werden. 5This is achieved according to the invention in that in the above process the adherent cells are separated from the non-adherent cells and only the adherent cells, but not the non-adherent ones, are cultivated. 5
Erfindungsgemäß werden antigenpräsentierende Zellen und Lymphozyten aus verschiedenen Körperflüssigkeiten und/oder Geweben von Säugetieren isoliert. Als Körperflüssigkeit sind hier insbesondere Blut, Lymphe, Pleura-, Peritoneal- und 0 Gelenkflüssigkeit sowie Liquor cerebrospinalis zu nennen. Die Körperflüssigkeiten können sowohl gesunden wie auch kranken Säugetieren entnommen werden. Gleiches gilt auch fürAccording to the invention, antigen-presenting cells and lymphocytes are isolated from various body fluids and / or tissues from mammals. Blood, lymph, pleural, peritoneal and synovial fluid and cerebrospinal fluid are to be mentioned here in particular as body fluids. The body fluids can be taken from both healthy and sick mammals. The same applies to
Gewebe, von denen insbesondere Milz, Lymphknoten, Knochenmark, Haut und Tumore anzugeben sind. Als Säugetiere 5 sind insbesondere Mensch, Haus- und Labortier gemeint, wobei unter Haustier auch Nutztier zu verstehen ist.Tissues, of which the spleen, lymph nodes, bone marrow, skin and tumors are to be specified. As mammals 5 are meant in particular humans, domestic and laboratory animals, whereby domestic animals are also to be understood as domestic animals.
Für das er indungsgemäße Verfahren können Körper- flüssigkeiten und/oder Gewebe von ein oder mehreren Säugetieren stammen. Es ist nicht zwingend, daß bei Verwendung mehrerer Säugetiere diese alle einer der vorstehend genannten Gruppen angehören. Genausowenig beschränkt sich das erfindungsgemäße Verfahren nur aufFor the method according to the invention, body fluids and / or tissues can originate from one or more mammals. It is not imperative that, when using several mammals, they all belong to one of the groups mentioned above. The method according to the invention is just as limited to
Säugetiere, vielmehr umfaßt es alle Lebewesen des Tierreichs, aus denen obige Zellen isoliert werden können.Mammals, rather it includes all living things from the animal kingdom, from which the above cells can be isolated.
Zur Isolierung der antigenprasentierenden Zellen und der Lymphozyten können bekannte Verfahren, beispielsweise das Ficoll-Hypaque-Zentrifugationsverfahren, angewandt werden. Hierzu werden Körperflüssigkeiten direkt eingesetzt, während solide Gewebe und Tumore erst nach Einzelpräparation durch Zerkleinerung und Verdauung mit üblichen Enzymen, wie Kollagenase-Dispase oder Trypsin, unter Bedingungen, wie 1 h bei 37°C, zentrifugiert werden. Die antigenprasentierenden Zellen und die Lymphozyten werden in der Interphase erhalten und dann mehrmals einer Waschung mit einem physiologischen Puffer, beispielsweise PBS, pH 7.2, unterzogen.Known methods, for example the Ficoll-Hypaque centrifugation method, can be used to isolate the antigen-presenting cells and the lymphocytes. For this purpose, body fluids are used directly, while solid tissues and tumors are centrifuged only after individual preparation by comminution and digestion with conventional enzymes, such as collagenase dispase or trypsin, under conditions such as 1 h at 37 ° C. The antigen-presenting cells and the lymphocytes are obtained in the interphase and then washed several times with a physiological buffer, for example PBS, pH 7.2.
Erfindungsgemäß werden die antigenprasentierenden Zellen von den Lymphozyten getrennt. Hierfür können bekannte Verfahren verwendet werden. Vorzugsweise werden die Zellen in einemAccording to the invention, the antigen-presenting cells are separated from the lymphocytes. Known methods can be used for this. Preferably the cells are in one
Kulturgefäß mit serumfreiem Medium, beispielsweise RPMI 1640 oder CG-Medium, unter üblichen Bedingungen, wie 37°C und 5 % C0_ inkubiert. Das Kulturgefäß weist eine Beschichtung mit Plasma oder Serum auf, wobei dieses aus dem gleichen Säuge- tier (autolog) oder einem der gleichen Spezies (allogen) stammt, aus dem die antigenprasentierenden Zellen und die Lymphozyten isoliert worden sind. Die antigenprasentierenden Zellen adhärieren spontan an die Beschichtung, wohingegen die Lymphozyten in Suspension bleiben. Nach einstündiger Inkubation werden die Lymphozyten abpipettiert, während die antigenprasentierenden Zellen zurückbleiben.Culture vessel incubated with serum-free medium, for example RPMI 1640 or CG medium, under customary conditions, such as 37 ° C. and 5% CO. The culture vessel has a coating with plasma or serum, which comes from the same mammal (autologous) or from the same species (allogeneic) from which the antigen-presenting cells and the lymphocytes have been isolated. The antigen-presenting cells spontaneously adhere to the coating, whereas the lymphocytes remain in suspension. After an hour's incubation, the lymphocytes are pipetted off, while the antigen-presenting cells remain.
Erfindungsgemäß werden die Lymphozyten von supprimierenden Zellen befreit, wozu sie beispielsweise mit Leucin- methylester (1,25 mM) , 30 min bei Raumtemperatur behandelt und dann mehrfach in vorstehend angegebenem, serumfreienAccording to the invention, the lymphocytes are freed from suppressive cells, for which purpose they are treated, for example, with leucine methyl ester (1.25 mM) for 30 minutes at room temperature and then several times in the above-mentioned, serum-free cells
Medium gewaschen werden.Medium can be washed.
Die antigenprasentierenden Zellen werden dagegen aktiviert, wobei sie entsprechend ihres Reifezustands unterschiedlich behandelt werden. Antigenpräsentierende Zellen aus solidem Gewebe sind vielfach ausgereift und somit hochaktiv. Im Gegensatz dazu sind sie aus Körperflüssigkeiten oftmals nicht ausgereift und werden daher etwa 24 h in vorstehend angegebenen Kulturgefäßen inkubiert, wobei z.B. vorstehend angegebenes CG-Medium verwendet wird. Vorteilhafterweise werden hierbei Faktoren zur Induktion, wie Interleukin-1, Interleukin-6, Adenosin oder Dibutyryl-cAMP, zugegeben. Desweiteren liegen antigenpräsentierende Zellen aus Knochenmark zumeist noch als Vorläuferzellen vor und werden daher in einem der vorstehend angegebenen Medien etwa 1 Woche inkubiert. Diesem werden Faktoren, wie Linolsäure (4.ug/ml), Vitamin D3 (2,5 M) , Vitamin E (0,1 %) , Multi-CSF (2 U/ml) und M-CSF (3 U/ml) zugegeben.The antigen-presenting cells, on the other hand, are activated, whereby they are treated differently according to their maturity. Antigen-presenting cells made of solid tissue are mature in many cases and therefore highly active. In contrast, they are often not mature from body fluids and are therefore incubated for about 24 hours in the culture vessels specified above, e.g. CG medium specified above is used. Factors for induction, such as interleukin-1, interleukin-6, adenosine or dibutyryl-cAMP, are advantageously added. Furthermore, antigen-presenting cells from bone marrow are usually still present as precursor cells and are therefore incubated in one of the media specified above for about 1 week. Factors such as linoleic acid (4.ug/ml), vitamin D3 (2.5 M), vitamin E (0.1%), multi-CSF (2 U / ml) and M-CSF (3 U / ml ) added.
Erfindungsgemäß wird den antigenprasentierenden ZellenAccording to the antigen-presenting cells
Antigen zugegeben. Seine Menge liegt z.B. im Bereich von 1 pg - 10 .ug/ml Kulturmedium. Es wird wenige Minuten bis 24 Stunden zugegeben. Als Antigen sind ganze oder Teile von Zellen, Krankheitserregern und/oder Biomolekülen zu nennen. Unter Zellen sind dabei sowohl normale wie auch virusmodifizierte und krankhaft entartete zu verstehen. Die Krankheitserreger umfassen Viren, Bakterien und Allergene, während zu den Biomolekülen Antikörper, Mediatoren, Enzyme und Neurotransmitter zu rechnen sind.Added antigen. Its amount is, for example, in the range from 1 μg to 10 μg / ml culture medium. It is added from a few minutes to 24 hours. Whole or parts of cells, pathogens and / or biomolecules can be mentioned as antigen. Under cells there are both normal and to understand virus-modified and pathologically degenerate. The pathogens include viruses, bacteria and allergens, while the biomolecules include antibodies, mediators, enzymes and neurotransmitters.
Die Antigene können als solche allein oder gekoppelt mit üblichen Carriermolekülen eingesetzt werden, wobei beide auch in Gemischen mit gängigen Adjuvantien verwendet werden.The antigens can be used as such alone or in combination with conventional carrier molecules, both of which are also used in mixtures with common adjuvants.
In manchen Fällen kann es aber von Vorteil sein, auf die Zugabe von Antigen zu verzichten, insbesondere dann, wenn die antigenprasentierenden Zellen bereits im Organismus hinreichend mit Antigen in Kontakt gekommen sind. Dies ist insbesondere bei aus einem Tumor und aus rheumatischem Gewebe isolierten Zellen der Fall. Desweiteren kann es hier, wie auch in anderen Fällen, von Vorteil sein, ganz auf die vorstehend angegebene Trennung von antigenprasentierenden Zellen und Lymphozyten zu verzichten und die aus dem Ficoll-Hypaque-Zentrifugations- verfahren erhaltene Interphase direkt weiter zu verwenden.In some cases, however, it may be advantageous to dispense with the addition of antigen, especially if the antigen-presenting cells have already come into sufficient contact with antigen in the organism. This is particularly the case with cells isolated from a tumor and from rheumatic tissue. Furthermore, as in other cases, it can be advantageous to do without the separation of antigen-presenting cells and lymphocytes and to continue using the interphase obtained from the Ficoll-Hypaque centrifugation process.
Erfindungsgemäß werden die antigenprasentierenden Zellen und die Lymphozyten zu einer spezifischen Adhäsion gemeinsam inkubiert. Hierzu werden die Zellen in vorstehend angegebene Kulturgefäße eingebracht, wobei die Lymphozyten in 10- bis 1000-fachem Überschuß vorliegen, und mit einem der vorstehend genannten Medien kultiviert. Als Kultivierungszeit sind 15 min bis 2 Tage bei 37°C oder maximal 24 h bei 4°C anzugeben.According to the invention, the antigen-presenting cells and the lymphocytes are incubated together for specific adhesion. For this purpose, the cells are introduced into the culture vessels specified above, the lymphocytes being in a 10 to 1000-fold excess, and cultured with one of the media mentioned above. The cultivation time is 15 minutes to 2 days at 37 ° C or a maximum of 24 hours at 4 ° C.
Erfindungsgemäß werden die nicht-adhärenten Zellen von den adhärenten abgetrennt. Hierzu werden erstere durch vorsichtiges Spülen von den an den beschichtetenAccording to the invention, the non-adherent cells are separated from the adherent ones. To do this, wash the coated ones carefully by rinsing them carefully
Kulturgefäßen adhärenten Zellen abgezogen. Desweiteren werden, wenn schwimmende, aneinander adhärente Zellen vorliegen, die nicht-adhärenten durch eine wenige Minuten dauernde Geschwindigkeits-Sedimentation bei 1 bis 1500 g abgetrennt.Cells adhering to culture vessels are withdrawn. Furthermore, if there are floating, mutually adherent cells, the non-adherent cells will be replaced by a few minutes Velocity sedimentation separated at 1 to 1500 g.
Erfindungsgemäß werden die adhärenten Zellen weiter nach üblichen Verfahren kultiviert. Sie werden in vorstehend angegebenen Kulturgefäßen inkubiert. Als Medium wird ein vorstehend genanntes verwendet, dem proliferationsfordernde Faktoren, wie Interleukin-2, -4 oder -6 zugegeben werden können. Auch kann ein konditioniertes Medium, das beispielsweise von permanenten Zellkulturen gewonnen wird, zugesetzt werden. Die Inkubation führt zur Proliferation von antigenspezifischen B- und T-Lymphozyten. Unspezifische Lymphozyten werden nur in äußerst geringer Menge gebildet. Nach 4-tägiger Inkubation werden Antikörper in den Überständen erhalten, deren Menge durch weitere Inkubation gesteigert werden kann. Vorteilhaft ist es auch, jeweils nach einer Woche frisches Medium mit proliferationsfordernden Faktoren zuzugeben. Gegebenenfalls können auch antigenpräsentierende Zellen zugegeben werden, wobei diese jedoch den ursprünglich eingesetzten entsprechen sollen.According to the invention, the adherent cells are further cultivated by customary methods. They are incubated in the culture vessels specified above. As the medium, an above-mentioned one is used, to which factors that require proliferation, such as interleukin-2, -4 or -6, can be added. A conditioned medium, which is obtained, for example, from permanent cell cultures, can also be added. The incubation leads to the proliferation of antigen-specific B and T lymphocytes. Unspecific lymphocytes are only formed in an extremely small amount. After 4 days of incubation, antibodies are obtained in the supernatants, the amount of which can be increased by further incubation. It is also advantageous to add fresh medium with proliferation-promoting factors every week. If necessary, antigen-presenting cells can also be added, but these should correspond to the originally used cells.
Die erfindungsgemäß erhaltenen Antikörper sind insbesondere nach Klonierung hochspezifisch gegen die verwendeten Antigene. Es handelt sich dann um monoklonale Idiotyp-The antibodies obtained according to the invention are highly specific against the antigens used, in particular after cloning. It is then a monoclonal idiotype
Antikörper, ferner auch um Anti-Idiotyp-Antikörper, wenn als Antigen ein Antikörper eingesetzt worden ist.Antibodies, also anti-idiotype antibodies if an antibody has been used as the antigen.
Die erfindungsgemäß erhaltenen, antigenspezifischen B- und T-Lymphozyten sind in sich äußerst homogen. Sie eignen sich insbesondere nach Klonierung zur Isolierung von spezifischen Nukleinsäurevorstufen, beispielsweise mRNA, die dann für Genklonierungszwecke und zur Vermehrung spezifischer Nukleinsäuresequenzen über bekannte Techniken, beispielsweise PCR-Verfahren genutzt werden können. Ferner eignen sich die erfindungsgemäß hergestellten B- und T-Lymphozyten zur adoptiven Immuntherapie. Desweiteren können sie auch durch übliche Verfahren, wie Fusion mit Hybridomzellen, immortalisiert werden. Die erfindungsgemäß erhaltenen B-Lymphozyten können darüberhinaus durch Zugabe von Anti-CD40-Antikörpern weiter kultiviert und expandiert werden.The antigen-specific B and T lymphocytes obtained according to the invention are extremely homogeneous in themselves. After cloning, they are particularly suitable for the isolation of specific nucleic acid precursors, for example mRNA, which can then be used for gene cloning purposes and for the amplification of specific nucleic acid sequences using known techniques, for example PCR methods. Furthermore, the B- and T lymphocytes for adoptive immunotherapy. Furthermore, they can also be immortalized by conventional methods, such as fusion with hybridoma cells. The B lymphocytes obtained according to the invention can furthermore be cultured and expanded further by adding anti-CD40 antibodies.
Die nachfolgenden Beispiele erläutern die Erfindung.The following examples illustrate the invention.
Beispiel 1example 1
Isolierung, Trennung und Behandlung von antigen¬ prasentierenden Zellen und Lymphozyten aus BlutIsolation, separation and treatment of antigen-presenting cells and lymphocytes from blood
Aus gesunden, mit Tetanusantigen immunisierten Spendern wurden, wie vorstehend beschrieben, antigenpräsentierende Zellen und Lymphozyten über dasHealthy donor immunized with tetanus antigen, as described above, antigen presenting cells and lymphocytes over the
Ficoll-Hypaque-Zentrifugationsverfahren isoliert und in Kulturgefäßen mit autologer Plasmabeschichtung voneinander getrennt. Die Lymphozyten wurden durch Behandlung mit Leucin- methylester von supprimierenden Zellen befreit, während die antigenprasentierenden Zellen 24 h, wie vorstehend beschrieben, mit 0, 1 und 10 ng Tetanustoxoid/ml Medium inkubiert wurden. Das Antigen wurde anschließend ausgewaschen.Ficoll-Hypaque centrifugation method isolated and separated from each other in culture vessels with autologous plasma coating. The lymphocytes were freed from suppressive cells by treatment with leucine methyl ester, while the antigen-presenting cells were incubated with 0, 1 and 10 ng tetanus toxoid / ml medium for 24 h as described above. The antigen was then washed out.
Beispiel 2Example 2
Gemeinsame Inkubation der antigenprasentierenden Zellen und der Lymphozyten sowie anschließende Abtrennung der nicht- adhärenten Zellen.Joint incubation of the antigen-presenting cells and the lymphocytes and subsequent separation of the non-adherent cells.
Die antigenprasentierenden Zellen und die Lymphozyten wurden in vorstehend beschriebenen Kulturgefäßen mit CG-Medium gemeinsam inkubiert. Die Lymphozyten lagen in 200-fachem Überschuß vor. Nach 24 Stunden Inkubation wurden die nicht- adhärenten Zellen durch vorsichtiges Pipettieren entfernt. Den adhärenten Zellen wurde frisches Medium, ergänzt mit konditioniertem Medium, zugegeben. Nach 6 Tagen Inkubation wurden Zellzahl, Gesamtantikörpermenge und spezifischeThe antigen-presenting cells and the lymphocytes were incubated together with CG medium in the culture vessels described above. The lymphocytes were in 200-fold excess. After 24 hours of incubation, the non- adherent cells removed by careful pipetting. Fresh medium supplemented with conditioned medium was added to the adherent cells. After 6 days of incubation, cell count, total antibody amount and specific
Antikörpermenge bestimmt. Die spezifische AntikörperSynthese wurde in einem ELISA mittels eines an Plastik gebundenen Tetanustoxoids als Antigen bestimmt. Die unspezifische Antikörpersynthese wurde ebenfalls in einem ELISA bestimmt, bei dem ein gegen humanes Ig gerichteter Maus-Antikörper verwendet wurde.Antibody amount determined. The specific antibody synthesis was determined in an ELISA using a tetanus toxoid bound to plastic as the antigen. The non-specific antibody synthesis was also determined in an ELISA using a mouse antibody directed against human Ig.
Die Ergebnisse in der Figur zeigen folgendes: In Abwesenheit von antigenprasentierenden Zellen (m-AC) findet sich in den gespülten Ansätzen keine signifikante spezifische Anti¬ körpersynthese, während diese in den Ansätzen in Anwesenheit von m-AC (schraffierte Säulen) vorliegt. Bei Zugabe von 10 ng/ l Antigen ist die Ausbeute am höchsten. Bei 1 ng/ml stark, und selbst ohne Zugabe des Antigens findet sich eine signifikante, spezifische Antikörperproduktion. DieThe results in the figure show the following: In the absence of antigen-presenting cells (m-AC), there is no significant specific antibody synthesis in the rinsed batches, while this is present in the batches in the presence of m-AC (hatched columns). The yield is highest when 10 ng / l antigen is added. Strong at 1 ng / ml, and even without adding the antigen, there is significant, specific antibody production. The
Gesamtantikörpermenge (rechte Ordinate) ist in diesen Ansätzen außerordentlich niedrig (A ) • In den nicht-gespülten Kontrollen, bei denen also adhärente und nicht-adhärente Zellen vorliegen, finden sich zwar signifikante Mengen von spezifischem Antikörper, jedoch liegt auch eine sehr hohe Menge an nicht-spezifischen Antikörpern vor. Gleichzeitig ist die Zellzahl in den nicht-gespülten Kontrollen etwa um den Faktor 10 höher als in den gespülten Ansätzen.Total antibody amount (right ordinate) is extremely low in these approaches (A) • In the non-rinsed controls, which contain adherent and non-adherent cells, there are significant amounts of specific antibody, but there is also a very high amount non-specific antibodies. At the same time, the number of cells in the non-rinsed controls is about a factor of 10 higher than in the rinsed batches.
Dies unterstreicht, daß in den erfindungsgemäßen Verfahren antigenspezifische B- und T-Lymphozyten bevorzugt bereitgestellt, nicht-antigenspezifische dagegen weitgehend ausverdünnt werden. This underlines that antigen-specific B and T lymphocytes are preferably provided in the methods according to the invention, whereas non-antigen-specific ones are largely diluted.

Claims

Patentansprüche Claims
1. Verfahren zur Bereitstellung Antigen-spezifischer B- und T-Lymphozyten, bei dem aus Körperflüssigkeiten und/oder Geweben von Säugetieren antigenpräsentierende Zellen und Lymphozyten isoliert, diese zu einer spezifischen Adhäsion gemeinsam inkubiert und nach üblichen Verfahren kultiviert werden, dadurch gekennzeichnet, daß die adhärenten Zellen von den nicht-adhärenten getrennt und nur die adhärenten Zellen, nicht aber die nicht- adhärenten kultiviert werden.1. A method for providing antigen-specific B and T lymphocytes, in which antigen-presenting cells and lymphocytes are isolated from body fluids and / or tissues of mammals, these are incubated together for a specific adhesion and cultured according to conventional methods, characterized in that the adherent cells are separated from the non-adherent and only the adherent cells, but not the non-adherent, are cultivated.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die Körperflüssigkeiten Blut, Lymphe, Pleura-, Peritoneal- und Gelenkflüssigkeit sowie Liquor cereprospinalis umfassen.2. The method according to claim 1, characterized in that the body fluids include blood, lymph, pleural, peritoneal and synovial fluid and cerebrospinal fluid.
3. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die Gewebe Milz, Lymphknoten, Knochenmark, Haut und Tumore umfassen.3. The method according to claim 1, characterized in that the tissues include spleen, lymph nodes, bone marrow, skin and tumors.
4. Verfahren nach einem der Ansprüche 1 - 3, dadurch gekennzeichnet, daß die Säugetiere Mensch, Haus- und Labortier umfassen.4. The method according to any one of claims 1-3, characterized in that the mammals include humans, domestic and laboratory animals.
5. Verfahren nach einem der Ansprüche l und 4, dadurch gekennzeichnet, daß die Körperflüssigkeiten und/oder5. The method according to any one of claims l and 4, characterized in that the body fluids and / or
Gewebe aus ein oder mehreren Säugetieren stammen.Tissues come from one or more mammals.
6. Verfahren nach einem der Ansprüche 1 - 5, dadurch gekennzeichnet, daß vor Inkubation zur Adhäsion die antigenprasentierenden Zellen aktiviert und die6. The method according to any one of claims 1-5, characterized in that prior to incubation for adhesion, the antigen-presenting cells are activated and the
Lymphozyten von supprimierenden Zellen befreit werden, Lymphocytes are freed from suppressive cells,
7. Verfahren nach Anspruch 6,. dadurch gekennzeichnet, daß die Aktivierung der antigenprasentierenden Zellen die Zugabe von Antigen umfaßt.7. The method according to claim 6,. characterized in that the activation of the antigen-presenting cells comprises the addition of antigen.
8. Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß als Antigen ganze oder Teile von Zellen, Krankheits¬ erregern und/oder Biomolekülen zugegeben werden.8. The method according to claim 7, characterized in that whole or parts of cells, pathogens and / or biomolecules are added as antigen.
9. Verfahren nach Anspruch 8, dadurch gekennzeichnet, daß die Zellen normale, virusinfizierte und krankhaft entartete, die Krankheitserreger Viren, Bakterien und Allergene und die Biomoleküle Antikörper, Mediatoren, Enzyme und Neurotransmitter umfassen.9. The method according to claim 8, characterized in that the cells include normal, virus-infected and pathologically degenerate, the pathogens viruses, bacteria and allergens and the biomolecules include antibodies, mediators, enzymes and neurotransmitters.
10. Verfahren nach einem der Ansprüche 1 - 9, dadurch gekennzeichnet, daß die Inkubation zur Adhäsion in mit Plasma oder Serum beschichteten Kulturgefäßen erfolgt, wobei das Plasma oder Serum zumindest aus einem der Säugetiere stammt, das zur Isolierung der antigen¬ prasentierenden Zellen, und Lymphozyten herangezogen worden ist.10. The method according to any one of claims 1-9, characterized in that the incubation for adhesion takes place in culture vessels coated with plasma or serum, the plasma or serum originating at least from one of the mammals which is used to isolate the antigen-presenting cells, and Lymphocytes have been used.
11. Verfahren nach einem der Ansprüche 1 -10, dadurch gekennzeichnet, daß die üblichen Kultivierungsverfahren die Klonierung und Fusion mit Myelomzellen umfassen.11. The method according to any one of claims 1-10, characterized in that the usual cultivation methods include cloning and fusion with myeloma cells.
12. Verfahren nach einem der Ansprüche 1 - 10, dadurch gekennzeichnet, daß die üblichen Kultivierungsverfahren für die B-Lymphozyten die Klonierung und Zugabe von Anti-CD 40 Antikörpern umfassen.12. The method according to any one of claims 1-10, characterized in that the usual cultivation methods for the B lymphocytes include the cloning and addition of anti-CD 40 antibodies.
13. Antikörper, erhalten nach dem Verfahren nach einem der Ansprüche 1 - 12. 13. Antibodies obtained by the method according to any one of claims 1-12.
EP92909718A 1991-05-16 1992-05-18 Process for preparing antigen-specific b and t lymphocytes and monoclonal antibodies obtained therefrom Withdrawn EP0539547A1 (en)

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JPH08333262A (en) * 1995-04-07 1996-12-17 Chugai Pharmaceut Co Ltd Immunosuppressive agent
JP6546219B2 (en) * 2017-06-30 2019-07-17 テルモ株式会社 Method for producing sheet-like cell culture
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