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WO1996036881A2 - Evaluation preliminaire d'inhibiteurs potentiels des interactions entre les recepteurs des lymphocytes t (rlt) et le complexe majeur d'histocompatibilite (cmh) - Google Patents

Evaluation preliminaire d'inhibiteurs potentiels des interactions entre les recepteurs des lymphocytes t (rlt) et le complexe majeur d'histocompatibilite (cmh) Download PDF

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Publication number
WO1996036881A2
WO1996036881A2 PCT/GB1996/001165 GB9601165W WO9636881A2 WO 1996036881 A2 WO1996036881 A2 WO 1996036881A2 GB 9601165 W GB9601165 W GB 9601165W WO 9636881 A2 WO9636881 A2 WO 9636881A2
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WIPO (PCT)
Prior art keywords
mhc
tcr
peptide
peptide ligand
cell
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Application number
PCT/GB1996/001165
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English (en)
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WO1996036881A3 (fr
Inventor
Dan Hung Barouch
Stephen John Rupert Vessey
Bent Karsten Jakobsen
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Cancer Research Campaign Technology Limited
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Application filed by Cancer Research Campaign Technology Limited filed Critical Cancer Research Campaign Technology Limited
Priority to AU57690/96A priority Critical patent/AU5769096A/en
Publication of WO1996036881A2 publication Critical patent/WO1996036881A2/fr
Publication of WO1996036881A3 publication Critical patent/WO1996036881A3/fr

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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
    • 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
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • This invention relates to interactions between T-cell receptors and their specific MHC/peptide ligand and the potential therapeutic applications of inhibitors of this interaction.
  • TCRs T-cell receptors
  • MHC- peptide ligands MHC- peptide ligands
  • the responder cell expressing the TCR preferably has the following properties: (i) it should be easy to culture; (ii) the correctly folded TCR should be stably expressed at high levels on the cell surface; (iii) the signal generated by the TCR-MHC interaction should be easily detectable by conventional means and simulates the functioning of the TCR in its native T- cell environment.
  • An example of such a responder cell is a mast cell transfected with recombinant DNA encoding the TCR protein and which undergoes degranulation in response to the TCR-MHC interaction on the cell surface.
  • Stimulatory MHC-peptide complexes may be created by immobilising soluble recombinant MHC on a solid support, such as "Dynabeads" (a form of plastic beads).
  • the MHC is typically obtained by recombinant DNA expression, and is a single specific MHC molecule or fragment thereof.
  • the present invention is based upon the following experimental data.
  • TCR T cell receptor
  • Figure la shows the cloning and expression of TCR genes encoding V ⁇ 2.2 and VjSl.l.
  • Figure lb shows the FACS analysis of stably transfected RBLs expressing the TCZR.
  • Figure lc shows, in the preparation and purification of MHC-peptide complexes, a sample HPLC gel filtration profile of purified, soluble, recombinant MHC protein.
  • Figure 2a-d shows specific response of RBL/TCZR transfectants to peptide-pulsed target cells and purified MHC proteins. The results are means from triplicate assays. A typical experiment is shown in each case.
  • Figure 3a-f shows competition with monomeric MHC-peptide complexes and controls.
  • Figure 4 shows diagrammatically a model for TCZR signalling.
  • TCR genes were amplified by PCR from cDNA prepared from a CTL clone specific for HLA A2.1 restricted HIV pol peptide.
  • Primer sequences were: V ⁇ 2.2 forward (5' CAC CGC TCG AGC CGC CAT CAT GAT GAA ATC CTT GAG A 3'), Nc.2.2 back (5' CTG GTA CAC GGC CGG GTC AGG GTT CTG GAT ATT 3')
  • VjSl.l forward (5'ATG CAA GTC AGT CGA CCC GCC ACC ATG GGC TTC AGG CTC CTC T 3'), and VjSl.
  • Stable transfectants were selected by the addition of 700 g/ml G418 (Sigma), 15 ⁇ g/ml hypoxanthine, lO ⁇ g/ml aminopterin, 2 ⁇ g/ml thymidine, 250 ⁇ g/ml xanthine and 5 ⁇ g/ml mycophenolic acid.
  • Protein concentrations were determined by a combination of spectrophotometry, scanning densitometry on SDS-PAGE gels, and HPLC peak quantitation. The concentrations of active protein in each preparation were confirmed by an ELISA detecting immobilized MHC protein with a panel of monoclonal antibodies (data not shown).
  • peptides complexed with the A2 proteins were: pol (ILKEPVHGV), gag (SLYNTVATL), a random library of A2-binding peptides, pol-8E (ILKEPVHEV), a self peptide TLW (TLWVDPYEV), and null (SLAAAAAAL).
  • RBLs were cultured in flat bottomed 96 well plates (Falcon) at a density of 5x10"* cells/well for 12 hours at 37°C in DMEM 10%FCS (Sigma) with 3 H-hydroxytryptamine creatinine sulphate (DuPont) at a final concentration of 0.2 / xCi/ml.
  • the RBLs were washed 3 times in warm DMEM 10%FCS, incubated or 20 mins and then washed once more.
  • C1R cells were resuspended at 5xl0 6 /100 ⁇ l and pulsed with various concentrations of peptide for 2hrs at 37°C. After one wash these APCs were added to the RBLs and coincubated for 60mins at 37° C. Supernatants were collected and responses were measured as corrected counts per minute (ccpm) by liquid scintillation counting.
  • Figure 2a shows the response to HA-A2.1 C1R cells pulsed with a range of concentrations of the HIV pol peptide.
  • Figure 2b shows the response to HA-A2.1 or HA-B7 C1R cells pulsed with individual peptides.
  • Degranulation assays were performed as above except either monomeric MHC protein or 10 million MHC-saturated dynabeads were added to the wells instead of peptide-pulsed cells.
  • sheep anti-mouse dynabeads were first saturated with an affinity-purified monoclonal coupling antibody according to manufacturer's protocols (Dynal). Coupling antibodies used were W6/32 (monomorphic anti- MHC) and BB7.2 (anti-A2, directed against the alpha-2 helix of the MHC). Following three washes, the beads were saturated with MHC-peptide complexes for 2 hours at 4° and then washed again three times before addition to the assay.
  • Biotinylated A2-peptide complexes were prepared by surface biotinylation of refolded ⁇ 2M protein for 1 hour at room temperature using a 3-fold molar excess of NHS- SS-biotin (Pierce) gel filtration purification to remove free biotin, and seeding a folding reaction essentially as before (Fig lc) using folded biotinylated ⁇ 2M, denatured heavy chain, and a synthetic peptide.
  • Results shown are means of 3 to 8 independent experiments each performed in duplicate and are reported as a percent of the uncompeted response. Standard error of the mean is shown in Fig 3a.
  • Degranulation assays were performed (as in Fig 2) using 10 million A2-pol coated streptavidin dynabeads (as in Fig 2d) per well.
  • a sub-saturating concentration of MHC protein was used (l ⁇ g MHC per 10 million beads), and the response with no added competitor was approximately 1000 ccpm.
  • Soluble, monomeric, non-biotinylated MHC protein was checked by gel filtration to ensure that there was no detectable aggregation of the protein.
  • the protein was diluted in media and added to the assay together with the A2- pol coated dynabeads.
  • Figure 3c shows that the stimulatory capacity of streptavidin coated dynabeads bearing multivalent arrays of MHC-peptide is unaffected by preincubation with monomeric complexes.
  • Biotinylated A2-pol or A2-gag complexes were linked to streptavidin coated dynabeads as described above. Beads were then incubated for 60 mins at 37°C with either medium alone or 10 ⁇ M non-biotinylated monomeric complexes.
  • Multivalent A2-pol was incubated with monomeric A2-gag and vice versa. After washing the beads 3 times in warm DMEM 10% FCS to remove free monomeric complexes they were used in degranulation assays as described above.
  • Figure 3d shows that the response to multivalent A2-pol is unaffected by the presence of free gag peptide. Assays were performed as described above in the presence of ascending concentrations of uncomplexed gag peptide.
  • Figure 3f shows that RBL/TCZR degranulation is unaffected by preincubation of transfectants with monomeric MHC-peptide complexes.
  • RBL/TCZR transfectants were preincubated with monomeric MHC-peptide complexes for 40 mins at 37 °C and men washed with warm DMEM 10% FCS. Degranulation assays were then performed as in Fig 2 but with 10 million streptavidin dynabeads coated with A2-pol per well.
  • Figure 4 proposes a model for TCZR signalling based on the results presented here together with the findings in references 20-22 and possible similarities with the EGF receptor 23 .
  • Figure 4a shows TCZR engaged by a multivalent array of MHC-peptide complexes.
  • Figure 4b shows how specific ligand can induce conformational change allowing stable receptor aggregation. Non-specific ligand induces no conformational change.
  • Figure 4c shows TCZR engaged by soluble monomeric MHC-peptide complexes.
  • Figure 4d shows conformational change induced, but insufficient cross-linking of receptors takes place. Results and discussion
  • Eukaryotic expression vectors encoding and ⁇ TCZR chimeric receptor chains 4 were constructed using TCR genes from a CTL clone specific for an HLA -A2.1 restricted HIV pol peptide (residues 476-484; ILKEPVHGV) 5 (Fig la).
  • Stably transfected RBL 2H3 cells expressing the TCZR (Fig lb) showed peptide specific and MHC restricted degranulation using peptide pulsed target cells (Fig 2a, b).
  • the response to multivalent A2-pol was also inhibited by monomeric soluble A2 protein complexed with some other peptides, including a variant of the index pol peptide, a random library of A2 binding peptides 8 , and a supposedly irrelevant HIV gag peptide pl7 (residues 77-85) 9 (Fig 3b).
  • monomeric soluble A2 protein complexed with some other peptides including a variant of the index pol peptide, a random library of A2 binding peptides 8 , and a supposedly irrelevant HIV gag peptide pl7 (residues 77-85) 9 (Fig 3b).
  • the present invention thus allows a very concise assay to be set up for the screening of substances, eg peptides or mimetics, that inhibit the interaction between TCRs and MHC- peptide ligands in vitro.
  • substances eg peptides or mimetics
  • the use of a RBL (a type of mast cell) expressing TCRs as repsonder cells is useful since degranulation of these cells provides a convenient signal of effective TCR-MHC-peptide interactions.
  • the RBLs are also useful in that other surface proteins are not known to interact with MHC, such as would be the case with T-cells which normally express TCRs.
  • other responder cells could be used, for example employing expression of a suitable reporter molecule such as jS-galactosidase as the detectable signal.
  • inhibitors of their binding could typically be highly specific peptides, MHC-peptide complexes or MHC-peptide mimetics, antibodies, TCRs, or other immunospecific molecules, or fragments of any of the above.
  • Other inhibitors could be more general in their action and inhibit many or all TCR-MHC interactions. These inhibitors could act by direct competition, allosteric changes, or other means.
  • TCR-MHC-peptide interactions could be used in a variety of therapeutic applications, for example in blocking T-cells which give rise to autoimmune disease such as diabetes, rheumatoid arthritis, groves disease etc, organ transplant rejection, or other T-cell mediated conditions.

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Abstract

Cette invention concerne les interactions entre les RLT et leur ligand CMH-peptide spécifique. Un procédé d'évaluation préliminaire permet d'identifier les substances qui inhibent l'interaction spécifique entre le RLT et un ligand CMH-peptide: il consiste à incuber des cellules sensibles exprimant un RLT avec (i) un ligand CMH-peptide qui stimule ces cellules sensibles et (ii) une substance potentiellement inhibitrice; à mesurer le signal produit par les cellules sensibles quand le RLT entre en contact avec le ligand CMH-peptide; et à comparer le signal produit par ces cellules sensibles, lors du contact du RLT avec le ligand CMH-peptide, avec un signal de référence obtenu avec les mêmes cellules sensibles lorsqu'elles sont incubées avec (i) mais sans (ii). La diminution du signal associée à la présence de (ii) dans le milieu d'incubation représente l'efficacité de l'inhibiteur.
PCT/GB1996/001165 1995-05-16 1996-05-16 Evaluation preliminaire d'inhibiteurs potentiels des interactions entre les recepteurs des lymphocytes t (rlt) et le complexe majeur d'histocompatibilite (cmh) WO1996036881A2 (fr)

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AU57690/96A AU5769096A (en) 1995-05-16 1996-05-16 Screening for inhibitors of tcr-mhc interactions

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GBGB9509844.8A GB9509844D0 (en) 1995-05-16 1995-05-16 Screening for inhibitors of TCR-MHC interactions
GB9509844.8 1995-05-16

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Cited By (6)

* 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
WO1999038526A1 (fr) * 1998-01-29 1999-08-05 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Variants de ligands peptidiques induisant selectivement l'apoptose
US6309820B1 (en) 1995-04-07 2001-10-30 University Of North Carolina At Chapel Hill Polypeptides having a functional domain of interest and methods of identifying and using same
WO2001090747A3 (fr) * 2000-05-25 2002-07-11 Sunol Molecular Corp Modulation des interactions des recepteurs des cellules t
US6709821B2 (en) 1995-04-07 2004-03-23 University Of North Carolina At Chapel Hill Polypeptides having a functional domain of interest and methods of identifying and using same
WO2020028595A3 (fr) * 2018-07-31 2020-04-23 Janux Therapeutics, Inc. Peptides inhibiteurs de récepteur de lymphocyte t et procédés de découverte

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06507630A (ja) * 1991-03-22 1994-09-01 ザ　トラスティーズ　オブ　ザ　ユニバーシティ　オブ　ペンシルベニア 哺乳類ｔ細胞の応答を調節する方法
ES2318848T3 (es) * 1993-09-14 2009-05-01 Pharmexa Inc. Peptidos que se unen a pan dr para potenciar la respuesta inmunitaria.

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6309820B1 (en) 1995-04-07 2001-10-30 University Of North Carolina At Chapel Hill Polypeptides having a functional domain of interest and methods of identifying and using same
US6709821B2 (en) 1995-04-07 2004-03-23 University Of North Carolina At Chapel Hill Polypeptides having a functional domain of interest and methods of identifying and using same
EP0833941A4 (fr) * 1995-04-07 2004-12-08 Cytogen Corp Polypeptides presentant un domaine fonctionnel important, et leurs procedes d'identification et d'utilisation
US7223547B2 (en) 1995-04-07 2007-05-29 Cytogen Corporation Polypeptides having a functional domain of interest and methods of identifying and using same
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
WO1999038526A1 (fr) * 1998-01-29 1999-08-05 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Variants de ligands peptidiques induisant selectivement l'apoptose
WO2001090747A3 (fr) * 2000-05-25 2002-07-11 Sunol Molecular Corp Modulation des interactions des recepteurs des cellules t
WO2020028595A3 (fr) * 2018-07-31 2020-04-23 Janux Therapeutics, Inc. Peptides inhibiteurs de récepteur de lymphocyte t et procédés de découverte
US20210371849A1 (en) * 2018-07-31 2021-12-02 Janux Therapeutics, Inc. Inhibitory t cell receptor peptides and discovery methods

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WO1996036881A3 (fr) 1997-01-09
AU5769096A (en) 1996-11-29
GB9509844D0 (en) 1995-07-12

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