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WO1995035500A1 - Procedes de production de lymphocytes t cytolytiques specifiques contre un complexe de peptide et de molecule de mhc - Google Patents

Procedes de production de lymphocytes t cytolytiques specifiques contre un complexe de peptide et de molecule de mhc Download PDF

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Publication number
WO1995035500A1
WO1995035500A1 PCT/US1995/007559 US9507559W WO9535500A1 WO 1995035500 A1 WO1995035500 A1 WO 1995035500A1 US 9507559 W US9507559 W US 9507559W WO 9535500 A1 WO9535500 A1 WO 9535500A1
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WIPO (PCT)
Prior art keywords
cells
peptide
cd8β
blood mononuclear
mononuclear cells
Prior art date
Application number
PCT/US1995/007559
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English (en)
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WO1995035500A9 (fr
Inventor
Pierre Coulie
Pierre Van Der Bruggen
Thierry Boon-Falleur
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Ludwig Institute For Cancer Research
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 Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to EP95923868A priority Critical patent/EP0767911A1/fr
Priority to AU28289/95A priority patent/AU690291B2/en
Priority to JP8502451A priority patent/JPH10501971A/ja
Publication of WO1995035500A1 publication Critical patent/WO1995035500A1/fr
Publication of WO1995035500A9 publication Critical patent/WO1995035500A9/fr
Priority to NO965347A priority patent/NO965347L/no
Priority to FI965038A priority patent/FI965038A0/fi

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

  • This invention relates to methods of obtaining cytolytic T cell subpopulations specific for a peptide/MHC complex.
  • Lymphocytes are cells responsible for recognizing and differentiating the various antigens which the immune system encounters.
  • Each lymphocyte carries a surface receptor which is capable of recognizing a particular antigen. Although each lymphocyte carries only one type of receptor and therefore can only recognize one type of antigen, different lymphocytes have different receptors, so that the lymphocyte population as a whole can recognize a wide range of antigens.
  • Lymphocytes are of two main types; namely B cells which develop in the bone marrow or fetal liver and can differentiate into antibody-producing plasma cells, and
  • T cells which differentiate in the thymus and serve a number of different functions.
  • B and T cells The antigen receptors used by B and T cells are different.
  • B cells can recognize an unmodified antigen molecule, either free in solution or on the surface of other cells, whereas T cells can recognize antigen only when it is presented to them in association with molecules encoded by the major histocompatibility complex
  • MHC antigen presenting cells
  • APCs antigen presenting cells
  • HLAs human leukocyte antigens
  • Antigen presenting cells take up, sequester in intracellular compartments, and process the antigen, and then present the processed antigen at the cell surface membrane in an energy-requiring, dynamic process that requires cell viability. There is considerable circumstantial evidence that for most soluble protein antigens, the re- expressed surface antigen that is recognized by T cells is not in native form.
  • Agents that raise lysosomal pH such as ammonia and chloroquine inhibit the development of effective antigen presentation when given immediately after antigen binding to presenting cells but not at later times.
  • cytolytic T cells Prior methods of generating cytolytic T cells (CTLs) to specific antigen/MHC complexes have exposed a mixed lymphocyte culture to an antigen presenting cell that has already processed the antigen of interest intracellularly.
  • the antigen presenting cell initiates a response from cytolytic T cell precursors having T cell receptors that recognize the antigen/MHC complex, whereby those T cells are activated and begin cell division and proliferation.
  • This method of generating cytolytic T cell clones specific to a certain antigen/MHC complex suffers from a number of drawbacks. In particular, it relies upon the ability of the processing cell to generate the peptide of interest, as well as a source (e.g., a larger antigen) of the peptide presented. Thus, there exists a need for improved methods of generating cytolytic T cells to specific antigen/MHC complexes.
  • the inventors have surprisingly discovered that exposing blood mononuclear cells to peptides will bind the peptide directly to MHC molecules present on the surface of the blood mononuclear cells and that the exposed cells are capable of stimulating cytolytic T-cells specific to the complex of the peptide and MHC molecule.
  • the blood mononuclear cells do not process the peptide intracellularly.
  • the method of the invention provides for a method to obtain cytolytic T- cells specific to a particular peptide/MHC complex as well as a method to identify antigenic peptides.
  • a method of obtaining a cytolytic T cell subpopulation specific for a peptide/MHC complex comprising contacting a sample of blood mononuclear cells with said peptide to bind said peptide directly to MHC molecules present on the surface of said blood mononuclear cells, and contacting said sample to a population of CD8 ⁇ + cells under conditions favoring stimulation of any CD8 ⁇ + cells specific to complexes of said peptide and said MHC molecule to which it binds.
  • the present invention relates to an improved method of obtaining a cytolytic T cell subpopulation specific for a peptide/MHC complex by contacting a sample of blood mononuclear cells with said peptide to bind said peptide directly to MHC molecules present on the surface of said blood mononuclear cells, and contacting said sample to a population of CD8 ⁇ + cells, under conditions favoring stimulation of any
  • the peptide bound to the MHC molecule can be any peptide capable of eliciting a cytolytic T-cell response.
  • Certain MHC molecules are known to prefer certain amino acids at certain positions in peptides. See Barinaga, Matsumura et al., and Latron, supra.
  • the peptide is a MAGE derived peptide.
  • MAGE-1 peptide MZ2-E or a peptide derived from MAGE-3 and presented by HLA-A2 molecules, depicted herein as SEQ ID NO: 1
  • the cytolytic T cells raised in vitro according to the present invention can be administered to a patient utilizing known techniques to treat tumor-cell related conditions.
  • the cytolytic T-cells lyse tumor cells, thereby achieving the desired therapeutic goal.
  • the cytolytic T cells are also useful in a diagnostic context. For example, once one has obtained CTLs specific to a peptide/MHC complex, assays can be developed for the presence of that particular peptide/MHC complex, using well known techniques such as chromium release assays, and TNF release assays.
  • the present invention also can be used to identify antigenic peptides.
  • the peptide of interest is contacted to blood mononuclear cells to bind the peptide to the MHC molecules on the surface of the cell.
  • the cells are then contacted to a population of CD8 ⁇ + cells under conditions favoring stimulation of any CD8 ⁇ + cells specific to complexes of the peptide/MHC complex.
  • the peptides are preferably 9-20 amino acids in length and, more preferably, 9-12 amino acids in length. Most preferably, they are nonamers, i.e., 9 amino acids in length.
  • the medium used in the examples is "Iscove's medium” (GIBCO, Grand Island, NY), supplemented with 10% pooled ABO decomplemented human serum, L-arginine (0.55 mM), L-asparagine (0.24 mM), L-glutamine (1.5 mM) and 2- mercaptoethanol (5 x 10 "5 M).
  • the MZ2-E peptide consists of Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr (SEQ ID NO: 1
  • the anti-CD8 ⁇ antibody is monoclonal antibody 1 A3.3 coupled to fluorescein. However, any anti-CD8 ⁇ antibody may be used.
  • Interleukin-2(IL-2) was used as an agent to stimulate the proliferation of
  • CD8 ⁇ + cells One unit/ml is the concentration that allows half-maximal proliferation of the IL-2 dependent cell line CTLL-2.
  • Other agents that stimulate the proliferation of CD8 ⁇ + can also be used, such as other interleukins or cytokines, T cell growth factors, mitogens, and other substances well known to the art.
  • Clone C1R-Al.cl3 was generated by cotransfecting plasmid pcDSR ⁇ having the HLA-Al gene cloned therein and plasmid pSVtkneo ⁇ conferring resistance to geneticin into EBV-transformed B cell line C1R (HLA-A " , HLA-B ,HLA-Cw4 + ) using well known techniques not reiterated here. Clones were isolated from the genticin- resistant transfectants. Clone C1R-Al.cl3 expressed HLA-Al, as established by labelling with an anti-HLA-Al monoclonal antibody. R Isolation of CD8 ⁇ + Cells
  • Blood mononuclear cells from patient MZ2 were isolated by density gradient centrifugation and stored at -80°C in medium containing 10% DMSO. These were thawed and labelled with mAb 1A3.3 coupled to fluorescein.
  • the CD8 ⁇ + lymphocytes (20% of cells in this case) were sorted on an ATC 3000 flow cytometer, and seeded at 800 cells/well in 96 N-bottom microwells in 25 ml of Iscove's medium.
  • blood mononuclear cells were thawed and resuspended at 5xl0 6 cells/ml in serum-free medium X-NIVO 10 containing 5 mg/ml of human ⁇ 2- microglobulin and lOOmM of the MZ2-E peptide referred to supra. After incubating for 2 hours at 37°C, blood mononuclear cells were irradiated with 3,000 rads from a cesium source, and adjusted to 4x10 s cells/ml with medium. IL-2 was added (20 U/ml) and 25 ml of the suspension (corresponding to 10,000 stimulator cells) were added to the microwells containing the CD8 ⁇ + lymphocytes obtained from step (B).
  • microcultures were incubated at 37°C in 8% CO 2 . On day 3, the microcultures were restimulated by the addition of 25 ml of medium containing 3 mM of the MZ2-E peptide and 10 U/ml of IL-2.
  • the stimulator cells were transferred into flat-bottom microwells ( ⁇ unc), and 160 ml of medium containing 10 U/ml of IL-2 was added.
  • microcultures were restimulated by E peptide as follows: cells were resuspended at 1-5X10 5 cells/ml in medium containing 600 nM of peptide, and incubated at 37°C for 30 minutes. Control cells were incubated in medium without peptide. Cells were centnfuged, resuspended in medium, counted, and added (1000 target cells/microwell) to the effector cells.
  • Lytic activities of microcultures of MZ2 CD8B + lymphocytes stimulated with autologous irradiated blood mononuclear cells contacted with the MAGE-1 MZ2-E nonapeptide Percentages of specific lysis are shown for two sets of 36 microcultures assayed on days 14, 18 and 23 of the stimulation.
  • Target cells are the EBN- transformed B cell line C1R-Al.cl3, that expresses HLA-Al but not the MZ2-E antigen, and the same cells contacted with the MZ2-E peptide.
  • Lytic activities of microcultures that show specificity for the peptide are boxed. Twenty-three microcultures that showed specificity for the MZ2-E peptide were amplified in vitro using well known techniques.
  • Cytolytic T cell clones were transferred into 2 ml wells and restimulated on days 28, 37 and 44 by the addition of medium supplemented with MZ2-E peptide (200 nM), IL-2 (30 U/ml) and irradiated (10,000 rads) allogeneic EBN-B cells (lOVwell) as feeder cells.
  • Lytic activities of anti-MZ2 nonapeptide microcultures and of the stable anti-MZ2-MEL CTL clones derived from these microcultures Only microcultures that were specific for the MZ2-E peptide at day 23 are shown. Values of lysis observed at day 23 are indicated. These microcultures were then amplified and their lytic activities were assayed on days 48 and 68, with defined Effector/target ratios and with additional targets: the autologous MZ2-MEL melanoma cell line and its E- antigen-loss variant. As a control, lytic activity of anti-MZ2-E CTL clone 82/30 is shown. This CTL clone is described in
  • the medium used in the examples is "Iscove's medium” supplemented with 10% pooled ABO decomplemented human serum, L-arginine (0.55 mM), L- asparagine (0.24 mM), L-glutamine (1.5 mM) and 2-mercaptoethanol (5 x 10 "5 M).
  • MAGE-3 peptides Phe-Leu-Trp-Gly-Pro-Arg-Ala-Leu-Nal (SEQ ID NO: 2) (codons 271-279; hereinafter "LB-373-1"), Ala-Leu-Ser-Arg-Lys-Val-Ala-Glu-Leu-
  • A2-I- and express MAGE-3 were isolated by density gradient centrifugation and stored at -80°C in medium containing 10% DMSO. Blood mononuclear cells were thawed and labelled with mAb 1A3.3 coupled to fluorescein.
  • the CD8B + lymphocytes were sorted on an ATC 3000 flow cytometer, and seeded at 1000 cells/well in 96 N-bottom microwells in 25 ml of medium. The final volume included 100 ⁇ l Iscove's medium supplemented with 10% human serum, asparagine-arginine- glutamine, 10 U/ml of r-hu-IL-2 and 10 ng/ml of r-hu-IL-7.
  • T2 cells which present HLA-A2, but have an antigen processing defect which results in an increased capacity to present exogenous peptides (Cerundolo et al., Nature 345:449 (1990)), were resuspended in Iscove's medium without serum + 1/3 (v/v) anti human class I mab W6/32 culture supernatant, incubated at 4°C for 30 minutes, and irradiated with 100 Gy from a cesium source.
  • the cells were then centrifuged and the pellet was resuspended in X-Vivo medium with 2.5 ⁇ g/ml human B2-microglobulin and lOO g/ml of either LB-373-1, LB-373-2, or LB-373-3 peptide. Cells were incubated for at least 1 hour at 37°C, then centrifuged, and resuspended in Iscove's medium supplemented with 10% human serum.
  • microcultures were restimulated by addition of 100 ⁇ l of fresh medium containing 10 U/ml of r-hu-IL-2 and 10 ng/ml of r-hu-IL-7, 6,000 LB373- PBLs preincubated with LB-373-1, LB-373-2, or LB-373-3 peptide. These were prepared, as described, supra.
  • the CD8 ⁇ + cells were transferred into flat-bottom microwells and restimulated by the addition of 100 ⁇ l of fresh medium containing 10 U/ml of r-hu- IL-2 and 10 ng/ml of r-hu-IL-7, 6,000 LB373-PBLs preincubated with LB-373-1, LB- 373-2, or LB-373-3 peptide. These were prepared, as described, supra. On day 16, because of strong proliferation, microcultures were divided in two (2/3 and 1/3).
  • lymphocytes were tested for their lytic activity on HLA-A2 + target cells preincubated with or without one of the Mage-3 peptides.
  • microcultures (1/3) were restimulated by the addition of 100 ⁇ l of fresh medium containing 10 U/ml of r-hu-IL2 and 10 ng/ml of r-hu-IL7, 25,000 LB373-PBL preincubated with LB-373-1 peptide.
  • lymphocytes from the different microcultures were tested for their lytic activity on HLA-A2 target cells preincubated with or without one of the MAGE-3 peptides.
  • the lymphocytes had higher lytic activity on a HLA-A2 + target preincubated with Phe-Leu-Tip-Gly-Pro-Arg-Ala-Leu-Nal than on the same target without peptides.
  • "Positive" microcultures were then restimulated weekly with different HLA-A2 + stimulator cells. The following table shows the lytic activity of some of the "CTL clones".
  • the order of steps is not critical. For example, while it may be preferred to combine the APCs with peptide prior to contact to the CD8 ⁇ + cells, it is also possible to combine the two cell types prior to adding the peptide. Similarly, one may add proliferation stimulating agents to the cells before or after they are combined. Further, the culture conditions may vary. While the examples show the use of serum free medium for culturing BMCs, any appropriate medium can be used. Other possible variations on the specific examples given herein will be apparent to the skilled artisan, and need not be elaborated upon here.

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Abstract

L'invention concerne des procédés d'obtention de sous-populations de lymphocytes T cytolytiques spécifiques contre un complexe peptide/MHC.
PCT/US1995/007559 1994-06-17 1995-06-14 Procedes de production de lymphocytes t cytolytiques specifiques contre un complexe de peptide et de molecule de mhc WO1995035500A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP95923868A EP0767911A1 (fr) 1994-06-17 1995-06-14 Procedes de production de lymphocytes t cytolytiques specifiques contre un complexe de peptide et de molecule de mhc
AU28289/95A AU690291B2 (en) 1994-06-17 1995-06-14 Method for producing cytolytic T cells specific for a complex of peptide and MHC-molecule
JP8502451A JPH10501971A (ja) 1994-06-17 1995-06-14 ペプチドとｍｈｃ−分子との複合体に対して特異的な細胞傷害性ｔ細胞の製造方法
NO965347A NO965347L (no) 1994-06-17 1996-12-13 Fremgangsmåte for fremstilling av cytolytiske T-celler som er spesifikke for et peptid/MHC-kompleks
FI965038A FI965038A0 (fi) 1994-06-17 1996-12-16 Menetelmä peptidin ja MHC-molekyylin kompleksille spesifisten sytolyyttisten T-solujen tuottamiseksi

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26154194A 1994-06-17 1994-06-17
US08/261,541 1994-06-17

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WO1995035500A1 true WO1995035500A1 (fr) 1995-12-28
WO1995035500A9 WO1995035500A9 (fr) 1996-02-01

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PCT/US1995/007559 WO1995035500A1 (fr) 1994-06-17 1995-06-14 Procedes de production de lymphocytes t cytolytiques specifiques contre un complexe de peptide et de molecule de mhc

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EP (1) EP0767911A1 (fr)
JP (1) JPH10501971A (fr)
CN (1) CN1171156A (fr)
AU (1) AU690291B2 (fr)
CA (1) CA2192562A1 (fr)
FI (1) FI965038A0 (fr)
NO (1) NO965347L (fr)
WO (1) WO1995035500A1 (fr)
ZA (1) ZA954986B (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044667A3 (fr) * 1996-05-21 1998-03-19 Pasteur Institut Methodes d'utilisation de complexes peptide/complexe majeur d'histocompatibilite pour obtenir ou purifier des cellules T antigene-specifiques et pour stimuler des cellules T
EP1132465A1 (fr) * 2000-03-11 2001-09-12 Forschungszentrum Jülich Gmbh Combinaison de nutriments pour la culture cellulaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 360, issued 03 December 1993, HILL et al., "Molecular analysis of the Association of HLA-B53 and Resistence to Severe Malaria", pages 434-439. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 91, issued March 1994, E. CELIS et al., "Induction of Anti-Tumor Cytotoxic T Lymphocytes in Normal Humans Using Primary Cultures and Synthetic Peptide Epitopes", pages 2105-2109. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044667A3 (fr) * 1996-05-21 1998-03-19 Pasteur Institut Methodes d'utilisation de complexes peptide/complexe majeur d'histocompatibilite pour obtenir ou purifier des cellules T antigene-specifiques et pour stimuler des cellules T
EP1132465A1 (fr) * 2000-03-11 2001-09-12 Forschungszentrum Jülich Gmbh Combinaison de nutriments pour la culture cellulaire

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AU2828995A (en) 1996-01-15
FI965038L (fi) 1996-12-16
CA2192562A1 (fr) 1995-12-28
NO965347L (no) 1996-12-17
ZA954986B (en) 1996-05-17
AU690291B2 (en) 1998-04-23
JPH10501971A (ja) 1998-02-24
FI965038A0 (fi) 1996-12-16
NO965347D0 (no) 1996-12-13
EP0767911A1 (fr) 1997-04-16
CN1171156A (zh) 1998-01-21

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