WO1991009873A1 - Nouvelles proteines - Google Patents

Nouvelles proteines

Info

Publication number
WO1991009873A1
WO1991009873A1 PCT/EP1990/002164 EP9002164W WO9109873A1 WO 1991009873 A1 WO1991009873 A1 WO 1991009873A1 EP 9002164 W EP9002164 W EP 9002164W WO 9109873 A1 WO9109873 A1 WO 9109873A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
proteins
antibodies
cell
antibody
Prior art date
Application number
PCT/EP1990/002164
Other languages
German (de)
English (en)
Inventor
Anni Barbara Endler-Jobst
Stefan Meuer
Burkhard Schraven
Original Assignee
Basf Aktiengesellschaft
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 Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1991009873A1 publication Critical patent/WO1991009873A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70507—CD2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70589—CD45
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2806—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD2
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex

Definitions

  • T cells mediate the cellular cytotoxicity and represent the regulatory elements for other effector cells of the immune system, such as antibody-producing B cells or phagocytes of the myelo-monocytic cell series.
  • T-CD 3 T cell receptor for antigens
  • CD2 CD2 molecule
  • CD45 CD45 molecule
  • the invention relates to two new proteins, characterized in that they
  • the molecular weight of the new proteins was determined electrophoretically by comparison with standard substances.
  • T lymphocytes The existence of these new proteins could be demonstrated by external radioactive labeling of T lymphocytes and subsequent isolation of T cell surface proteins from cell lysates.
  • T cells which can be isolated from human blood (Eur. J. Immunol. 19, 337 (1989)).
  • the cells are then lysed.
  • the cell nuclei are removed and some of the non-specifically binding proteins are removed by binding to Protein A-Sepharose ® .
  • it is immunoprecipitated with CD2- or CD45-specific antibodies and the precipitate is electrophoretically separated.
  • the invention furthermore relates to antibodies, preferably monoclonal antibodies, which bind to the proteins according to claim 1.
  • the antibodies can be produced in a known manner (Monoclonal Antibodies, Kennet et al., Plenum Press 1980, pp. 363-419).
  • BALB / c mice are immunized by repeated injection of a small amount of the naturally purified protein or recombinantly produced protein. Immunization was also performed on human peripheral T cells. As soon as sufficient antibodies can be detected in the serum, the spleen cells of the animals are fused with myeloma cells and the hybridoma cells are cultivated.
  • the individual cultures are examined for their content of antibodies which are specifically directed against the new proteins using a screening test.
  • Colonies that are derived from single cells are isolated from suitable cultures using the "limiting dilution cloning" method.
  • the propagation of these hybrid cells can be carried out both in vitro and in vivo.
  • the higher multiplication rate in vivo makes this culture method particularly favorable.
  • cells pretreated with Pristan ® BALB / c mice are injected intraperitoneally from the individual hybridoma cultures.
  • the ascitic tumor that forms is harvested after about 8 to 10 days.
  • the purification of the monoclonal antibodies takes place either by purification from supernatants of the in vitro cultivated hybridoma cells or from ascetic fluid.
  • the cleaning is based on the method of Bruch et al. (3rd Immunol. Methods 1982, Vol. 53, 313-319).
  • the dysregulation of T cell activation plays a central role in a group of diseases that is at least equal to cancer, autoimmune aggression diseases such as multiple sclerosis, uvenile diabetes mellitus, lupus erythematosus, sarcoidosis, chronic hepatitis and rheumatoid arthritis.
  • autoimmune aggression diseases such as multiple sclerosis, uvenile diabetes mellitus, lupus erythematosus, sarcoidosis, chronic hepatitis and rheumatoid arthritis.
  • the proteins themselves, especially if they are in soluble form or by binding antibodies, for example, to the new proteins or their natural ligands, offer the possibility of intervening in the activation of the T lymphocytes.
  • the suppression of the immune system is in the foreground in the above-mentioned clinical pictures. The same applies to prophylactic and therapeutic intervention to prevent rejection after organ transplants. For example
  • the 125 iodine-labeled T cells were resuspended in PBS, pH 8.3 with a density of 5 ⁇ 10 6 cells / ml.
  • DSP dissolved in DMSO, was added to the T cells to a final concentration of 12.5 ⁇ g / ml.
  • An incubation phase of 60 min followed, which was carried out at room temperature.
  • the T cells were then washed twice with PBS, pH 7.4 and then at 4 ° C.
  • RIPA buffer (0.01 M NaH 2 PO 4 , 10 mM EDTA, 10 mM EGTA and 10 mM NaF) , which additionally contained 1% Triton® X-100, 0.15 M NaCl, 0.5% Na deoxycholate, 1 mM PMSF, 20 mM iodoacetamide and 2 ⁇ g / ml trypsin inhibitor.
  • the nuclei were then (min 10, 15000g) by a centrifugation and the separated postnucleäre cell lysate with 50 .mu.l of protein A-Sepharose ® beads incubated to non-specific binding proteins to separate.
  • the immunoprecipitates were washed four times with the following buffers: Buffer 1: RIPA buffer with 0.5% NaDOC, 0.5 NaCl, 1% Triton ® X-100; Buffer 2 and 3: RIPA with 0.05% SDS, 1% Triton X-100, 0.5% NaDOC, buffer 4: RIPA with 0.15 M NaCl, 1% Triton® X-100.
  • Buffer 1 RIPA buffer with 0.5% NaDOC, 0.5 NaCl, 1% Triton ® X-100
  • Buffer 2 and 3 RIPA with 0.05% SDS, 1% Triton X-100, 0.5% NaDOC
  • buffer 4 RIPA with 0.15 M NaCl, 1% Triton® X-100.
  • the radioactive immune complexes were then applied to a 5-15% SDS gradient flat gel and separated according to their molecular weight in electrophoresis under reducing conditions. The molecular weight of the precipitated proteins was determined from the dried gels using the autoradiography
  • the labeled T cells were immunoprecipitated with CD3-specific antibodies (monoclonal antibody OKT3 from Ortho Diagnostics) in order to show the specificity of the coprecipitation of the new proteins with CD2 and CD45.
  • CD3-specific antibodies monoclonal antibody OKT3 from Ortho Diagnostics
  • the figure shows immunoprecipitates of transmembrane molecules that are expressed on the surface of resting human T lymphocytes.
  • Lanes A, C and D of the figure show the immunoprecipitates that were obtained using mock crosslinking (DMSO was added to the T lymphocytes that were not to be crosslinked in order to avoid artifacts during immunoprecipitation) with the help of monoclonal antibodies that were specific to CD2 (track A), CD3 (track C) and CD45 (track D) react.
  • the CD2 antibody precipitates the CD2 protein with a molecular weight of 50 kilodaltons (kd) under mock crosslinking conditions (lane A).
  • the CD3 antibody (lane C) recognizes three proteins with a molecular weight of approx.
  • the CD2 antibody precipitated the CD2 molecule (lane B, lower large arrow, the isoforms of the CD45 molecule (lane B, upper large arrow), and the three additional proteins with molecular weights of 150, 65 and 43 kDa (lane B, Small arrows) It is striking that the protein pattern is qualitatively similar to that which was precipitated from the CD45 antibody under crosslinking conditions
  • the upper large arrow in lane F marks the isoforms of the CD45 molecule, the lower large arrow the 50 kDa CD2 Molecule and the small arrows the three additional proteins
  • the CD3 antibody precipitated under crosslinking conditions in addition to the CD3 molecules two other proteins with molecular weights of approximately 45-55 kd (lane E) Make proteins the two chains (alpha and beta) of the T cell receptor, which is non-covalently associated with the CD3 molecules.
  • mice Female BALB / c mice were immunized intraperitoneally (ip) with 30 ⁇ g of the purified proteins in 0.5 ml of Freund's complete adjuvant or with 10 7 human T cells isolated from peripheral blood in 0.5 ml of PBS. 14 days later, the animals again received 30 ⁇ g of the antigen or 10 7 cells in 0.5 ml PBS intraperitoneally in incomplete Freund's adjuvant. Two more
  • Immunizations were carried out intraperitoneally at 14-day intervals with 30 ⁇ g antigen or with the cells. The spleens were removed from two animals three days after the last antigen administration.
  • the spleens were ground into a single cell suspension by pressing the organs through a stainless steel sieve (pore size 100 ⁇ m).
  • the cells were transferred to RPMI 1640, supplemented with 10% FCS, 1% penicillin-streptomycin (10,000 iu / ml penicillin, 10,000 ⁇ g / ml streptomycin) and 2% L-glutamine (200 nmol / l) (RPMI plus) , washed three times with the same medium and resuspended therein.
  • RPMI 1640 supplemented with 10% FCS, 1% penicillin-streptomycin (10,000 iu / ml penicillin, 10,000 ⁇ g / ml streptomycin) and 2% L-glutamine (200 nmol / l) (RPMI plus)
  • Myeloma cells from the Ag8.653 line (ATCC CRL 1580) were used as fusion partners.
  • the cells are resistant to 20 ⁇ g / ml 8-azaguanine and can no longer grow in RPMI 1640 plus, which contains hypoxanthine, aminopterine and thymidine (HAT). They were cultured in RPMI 1640 plus with 10% fetal calf serum. The cells were used in the logarithmic growth phase for the fusion.
  • the fusion was carried out according to a standard protocol (Monoclonal Antibodies J.H. Peters, H. Baumgarten, M. Schulze Springer Contract (1985), page 32).
  • the spleen cell suspension (5 x 10 7 cells) was mixed with the myeloma cells (107 cells) and washed with serum-free medium RPMI 1640. The cells were then resuspended in 30 ml of RPMI 1640 serum-free medium and centrifuged in a 50 ml conical polypropylene tube at 800 rpm for 5 minutes. The supernatant was completely aspirated down to 200 ⁇ l and 1.5 ml of a solution of polyethylene glycol (PEG) with the molecular weight 1500 were carefully added to the cell pellet within 3 min at 37 ° C. The cell pellet was thereby constantly light
  • PEG polyethylene glycol
  • the cells were cultivated in HAT medium at 37 ° C. with 7% CO 2 in a moist atmosphere. The cultures were fed twice a week by replacing half the media volume with fresh HAT medium. After 1 to 2 weeks, the hybridoma cell cultures were supernatant for the presence of specific mAb against the new proteins was examined. Such hybridomas, the supernatants of which gave a positive result, were cloned using the "limiting-dilution cloning" technique. An average of 0.3 cells per well of a 96 well culture plate were sown. 105 mouse spleen cells were added as "feeder cells". The antibody-producing cells selected according to this cloning method were grown, 107 cells were frozen in fetal calf serum with 10% dimethyl sulfoxide and stored in liquid nitrogen.
  • the monoclonal antibodies were characterized in more detail in an ELISA system.
  • the corresponding protein (5 ⁇ g / ml) was coated on the wells of a microtiter plate. Free binding sites were blocked with 1% BSA in PBS for 30 minutes at room temperature. After incubation for 1 hour at room temperature of this prepared plate with purified monoclonal antibodies from cell culture, a second incubation step (2 hours at room temperature) with rabbit anti
  • a peroxidase-labeled goat anti-rabbit (Miles) immunoglobul in antiserum was added. After incubation for one hour, the enzyme reaction was started by adding the color substrate tetramethylbenzidine (Miles) and hydrogen peroxide.
  • mice were given to condition the peritoneum
  • Ascitis from Balb / c mice injected with hybridoma cells was diluted 1: 3 in binding buffer (1.5 M sodium chloride, 0.75 M glycine, pH 8.9) and filtered (filter with 0.22 ⁇ m pore size).
  • binding buffer 1.5 M sodium chloride, 0.75 M glycine, pH 8.9
  • a 4 ml protein-A-Sepharose ® CL 4 column was equilibrated 20 min with binding buffer.
  • the diluted ascitis was applied to the column (6 ml of diluted ascitis was the maximum), running speed 1 ml / min.
  • the columns were rinsed again with binding buffer for 15 min.
  • the mAK was eluted with 0.1 M citrate buffer with different pH values. Elution was carried out first with citrate buffer pH 6.0 (elution from IgG1) for 20 min, then with buffer pH 4.5 (elution from IgG2a) and finally with pH 3.0 buffer (elution from IgG2b).
  • the optical density of the eluted fractions was determined at 280 nm using a photometer.
  • the fractions containing the antibody were pooled, the pH was adjusted to 7.0 with 1N sodium hydroxide solution and concentrated in vacuo using collodium tubes (size excl. 12 KD), dialyzed against PBS at 4 ° C. overnight and finally at -70 ° C frozen.
  • collodium tubes size excl. 12 KD
  • Purified protein was diluted to 3 ⁇ g / ml in PBS (phosphate buffered saline consisting of 0.8% NaCl and 0.02 molar sodium phosphate, adjusted to pH 7.4 with HCl or NaOH).
  • PBS phosphate buffered saline consisting of 0.8% NaCl and 0.02 molar sodium phosphate, adjusted to pH 7.4 with HCl or NaOH.
  • Wells of microtiter plates ® were loaded with 0.1 ml of this solution. After two hours at room temperature, the supernatant was suctioned off and the cells were treated with 0.3 ml of a 1% bovine serum albumin solution (Sigma, RIA grade) for at least 30 minutes.
  • a 1% bovine serum albumin solution Sigma, RIA grade

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On décrit deux nouvelles protéines d'un poids moléculaire de 65 et 150 kd, qui sont présentes à la surface des cellules T, qui influencent l'activation des cellules T et qui coprécipitent avec CD2 ou CD45.
PCT/EP1990/002164 1989-12-22 1990-12-13 Nouvelles proteines WO1991009873A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19893942579 DE3942579A1 (de) 1989-12-22 1989-12-22 Neue proteine
DEP3942579.7 1989-12-22

Publications (1)

Publication Number Publication Date
WO1991009873A1 true WO1991009873A1 (fr) 1991-07-11

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ID=6396207

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1990/002164 WO1991009873A1 (fr) 1989-12-22 1990-12-13 Nouvelles proteines

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DE (1) DE3942579A1 (fr)
WO (1) WO1991009873A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2686087A1 (fr) * 1992-01-13 1993-07-16 Inst Nat Sante Rech Med Nouvel antigene lymphocytaire, anticorps correspondant et leurs applications.
DE10251458B4 (de) 2001-11-21 2007-12-13 Siemens Ag Kryostat

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
INTERNATIONAL IMMUNOLOGY vol. 2, no. 4, 1990, pages 353 - 360; P. ALTEVOGT et al.: "Association of CD2 and T200 CD45 in mouse T lymphocytes." *
NATURE. vol. 256, 7 August 1975, LONDON GB pages 495 - 497; G. KöHLER et al.: "Continuous cultures of fused cells secreting antibody to predefined specificity." see the whole document *
NATURE. vol. 345, 3 May 1990, LONDON GB pages 71 - 74; B. SCHRAVEN et al.: "Association of CD2 and CD45 on human T lymphocytes" see page 74, lines 42 - 48; figure 2 *

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DE3942579A1 (de) 1991-06-27

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