WO1992020783A1 - Verfahren zur bereitstellung von antigen-spezifischen b- und t-lymphozyten sowie davon erhaltene monoklonale antikörper - Google Patents

Verfahren zur bereitstellung von antigen-spezifischen b- und t-lymphozyten sowie davon erhaltene monoklonale antikörper Download PDF

Info

Publication number
WO1992020783A1
WO1992020783A1 PCT/EP1992/001089 EP9201089W WO9220783A1 WO 1992020783 A1 WO1992020783 A1 WO 1992020783A1 EP 9201089 W EP9201089 W EP 9201089W WO 9220783 A1 WO9220783 A1 WO 9220783A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
cells
lymphocytes
specific
mammals
Prior art date
Application number
PCT/EP1992/001089
Other languages
German (de)
English (en)
French (fr)
Inventor
Johann Hinrich Peters
Susanne Lenzner
Uwe Scholtes
Carl Watzek
Original Assignee
Idt Ag Für 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 Für In Vivo Diagnostik Und Therapie filed Critical Idt Ag Für In Vivo Diagnostik Und Therapie
Publication of WO1992020783A1 publication Critical patent/WO1992020783A1/de

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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/EP1992/001089 1991-05-16 1992-05-18 Verfahren zur bereitstellung von antigen-spezifischen b- und t-lymphozyten sowie davon erhaltene monoklonale antikörper WO1992020783A1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE4116078 1991-05-16
DEP4116078.9 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
DEP4216355.2 1992-05-18

Publications (1)

Publication Number Publication Date
WO1992020783A1 true WO1992020783A1 (de) 1992-11-26

Family

ID=25903683

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1992/001089 WO1992020783A1 (de) 1991-05-16 1992-05-18 Verfahren zur bereitstellung von antigen-spezifischen b- und t-lymphozyten sowie davon erhaltene monoklonale antikörper

Country Status (5)

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

Cited By (2)

* 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
US6297052B1 (en) 1994-04-28 2001-10-02 Boehringer Ingelheim Pharmaceuticals, Inc. B cell culture system comprising high density membrane bound CD40 ligand

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08333262A (ja) * 1995-04-07 1996-12-17 Chugai Pharmaceut Co Ltd 免疫抑制剤
DE10023675C2 (de) * 2000-05-16 2003-04-17 Arno Giehl Mutter
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

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988007077A1 (en) * 1987-03-11 1988-09-22 The Children's Hospital, Incorporated Method for the generation of antigen-specific t cell lines and therapeutic use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988007077A1 (en) * 1987-03-11 1988-09-22 The Children's Hospital, Incorporated Method for the generation of antigen-specific t cell lines and therapeutic use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THE EMBO JOURNAL, Bd. 8, Nr. 13, Dezember 1989, EYNSHAM, OXFORD GB Seiten 4049 - 4052; E. MOZES ET AL.: 'Direct binding of a myasthenia gravis related epitope to MHC class II molecules on living murine antigen-presenting cells.' *

Cited By (2)

* 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
US6297052B1 (en) 1994-04-28 2001-10-02 Boehringer Ingelheim Pharmaceuticals, Inc. B cell culture system comprising high density membrane bound CD40 ligand

Also Published As

Publication number Publication date
CA2087157A1 (en) 1992-11-17
JPH06500023A (ja) 1994-01-06
EP0539547A1 (de) 1993-05-05
DE4216355A1 (de) 1992-11-26

Similar Documents

Publication Publication Date Title
US5849589A (en) Culturing monocytes with IL-4, TNF-α and GM-CSF TO induce differentiation to dendric cells
DE60218302T2 (de) Verfahren zur gewinnung antigenspezifischer tr1-regualtorischer lymphozyten
Inaba et al. Isolation of dendritic cells
RU2202615C2 (ru) Моноклональное антитело, способное распознавать антиген, вызывающий апоптоз миелоидных клеток костного мозга, и обладающее свойством вызывать апоптоз миеломных клеток, его фрагмент и гибридома
DE60210623T2 (de) Ex-vivo isolierte cd25+cd4+ t zellen mit immunsuppressiver aktivität und deren anwendungen
KR100223395B1 (ko) 사람수지상세포의시험관내생성방법및이의용도
DE69534595T2 (de) Methode zur proliferation und differenzierung von b-zellen und verwendungen dafür
US8603815B2 (en) CD4+ CD25− T cells and Tr1-like regulatory T cells
JPS59169489A (ja) ヒト培養株化細胞
EP0093436B1 (de) Verfahren zur Herstellung permanenter tierischer und humaner Zellinien und deren Verwendung
EP2495312B1 (en) Method for producing antigen-specific b cell population
CH683526A5 (de) Zellinie für die Herstellung von Antikörper bildenden Hybridomen, Verfahren zur Herstellung einer menschlichen Zellinie und Antikörper produzierendes Hybridom.
WO1992020783A1 (de) Verfahren zur bereitstellung von antigen-spezifischen b- und t-lymphozyten sowie davon erhaltene monoklonale antikörper
Nozawa et al. Inhibiton by glucocorticoids and choleragen of the conditional growth of poorly adherent mononuclear phagocytes of newborn hamster liver and lung (Hormonal control of macrophage growth)
DE60117167T2 (de) Verfahren zur herstellung von submikropartikel-suspensionen pharmazeutischer substanzen
Baghestanian et al. Thrombin augments vascular cell-dependent migration of human mast cells: role of MGF
DE69012105T2 (de) Proliferation von cd4-lymfocyten.
EP0431125A1 (de) Herstellungsverfahren fur humane monoklonale antikörper
Goodell et al. Canine dendritic cells from peripheral blood and lymph nodes
EP2025747A1 (de) Verfahren zur in-vitro-Amplifikation von regulatorischen T-Zellen
KR101913353B1 (ko) 면역 억제 능력을 가지는 수지상세포-유사 세포 및 그 제조방법
EP0910624B1 (de) Verfahren zur herstellung und vermehrung von lymphozyten
WO2008056734A1 (fr) Procédé de production de cellules dendritiques à partir de cellules souches d'embryons humains
Pegrum et al. The mitogenic effects of isologous nuclear material
EP1391504A1 (en) CD4+ CD25- T cells and Tr1-like regulatory T cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

WWE Wipo information: entry into national phase

Ref document number: 1992909718

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2087157

Country of ref document: CA

WWP Wipo information: published in national office

Ref document number: 1992909718

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1992909718

Country of ref document: EP