EP0539547A1 - Procede de preparation de lymphocytes b et t specifiques d'antigenes et anticorps monoclonaux ainsi obtenus - Google Patents

Procede de preparation de lymphocytes b et t specifiques d'antigenes et anticorps monoclonaux ainsi obtenus

Info

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
Authority
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.)
Withdrawn
Application number
EP92909718A
Other languages
German (de)
English (en)
Inventor
Johann Hinrich Peters
Susanne Lenzner
Uwe Scholtes
Carl Watzek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IDT AG fur IN VIVO DIAGNOSTIK und THERAPIE
Original Assignee
IDT AG fur IN VIVO DIAGNOSTIK und THERAPIE
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by IDT AG fur IN VIVO DIAGNOSTIK und THERAPIE filed Critical IDT AG fur IN VIVO DIAGNOSTIK und THERAPIE
Publication of EP0539547A1 publication Critical patent/EP0539547A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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.

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.
EP92909718A 1991-05-16 1992-05-18 Procede de preparation de lymphocytes b et t specifiques d'antigenes et anticorps monoclonaux ainsi obtenus Withdrawn EP0539547A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE4116078 1991-05-16
DE4116078 1991-05-16
DE4216355A DE4216355A1 (de) 1991-05-16 1992-05-18 Verfahren zur bereitstellung von antigen-spezifischen b- und t-lymphozyten sowie davon erhaltene monoklonale antikoerper
DE4216355 1992-05-18

Publications (1)

Publication Number Publication Date
EP0539547A1 true EP0539547A1 (fr) 1993-05-05

Family

ID=25903683

Family Applications (1)

Application Number Title Priority Date Filing Date
EP92909718A Withdrawn EP0539547A1 (fr) 1991-05-16 1992-05-18 Procede de preparation de lymphocytes b et t specifiques d'antigenes et anticorps monoclonaux ainsi obtenus

Country Status (5)

Country Link
EP (1) EP0539547A1 (fr)
JP (1) JPH06500023A (fr)
CA (1) CA2087157A1 (fr)
DE (1) DE4216355A1 (fr)
WO (1) WO1992020783A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100896540B1 (ko) * 2000-05-16 2009-05-07 길 아르노 너트

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4422020A1 (de) * 1993-07-29 1995-02-02 Bernhard Heising Pharmazeutische Zusammensetzung
CA2188165C (fr) 1994-04-28 2007-08-28 Marilyn Kehry Methode de multiplication et differenciation des cellules b et leur utilisation
JPH08333262A (ja) * 1995-04-07 1996-12-17 Chugai Pharmaceut Co Ltd 免疫抑制剤
JP6546219B2 (ja) * 2017-06-30 2019-07-17 テルモ株式会社 シート状細胞培養物の製造方法
JP7002497B2 (ja) * 2019-06-20 2022-01-20 テルモ株式会社 シート状細胞培養物の製造方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988007077A1 (fr) * 1987-03-11 1988-09-22 The Children's Hospital, Incorporated Procede de production de lignees de lymphocytes t a specificite antigenique et utilisation therapeutique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9220783A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100896540B1 (ko) * 2000-05-16 2009-05-07 길 아르노 너트

Also Published As

Publication number Publication date
CA2087157A1 (fr) 1992-11-17
DE4216355A1 (de) 1992-11-26
WO1992020783A1 (fr) 1992-11-26
JPH06500023A (ja) 1994-01-06

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