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WO2003062272A1 - Nouvelle strategie de modulation de l'activation des lymphocytes t basee sur la regulation de l'interaction cd3ε- nck. - Google Patents

Nouvelle strategie de modulation de l'activation des lymphocytes t basee sur la regulation de l'interaction cd3ε- nck. Download PDF

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Publication number
WO2003062272A1
WO2003062272A1 PCT/ES2003/000039 ES0300039W WO03062272A1 WO 2003062272 A1 WO2003062272 A1 WO 2003062272A1 ES 0300039 W ES0300039 W ES 0300039W WO 03062272 A1 WO03062272 A1 WO 03062272A1
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nck
cd3ε
activation
interaction
protein
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Spanish (es)
Inventor
Balbino ALARCÓN SÁNCHEZ
Diana GIL PAGÉS
Wolfgang Schamel
Francisco SÁNCHEZ MADRID
María MONTOYA SÁNCHEZ
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Autonoma de Madrid
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Autonoma de Madrid
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the immune system and more specifically to the development of regulatory treatments for the T lymphocyte-mediated immune response in pathological processes.
  • the development of these methods is based on the possibility of identifying new regulatory agents for T lymphocyte antigen receptor (TCR) function and on the use of new gene therapy strategies modulating intracellular signal transmission from said TCR.
  • TCR T lymphocyte antigen receptor
  • the cells respond to extracellular stimuli by means of receptors that cross the plasma membrane. Although some receptors are channels that allow ions to pass into the cytoplasm, most receptors do not transmit material through the membrane and depend on interaction with intracellular effector molecules to activate signal transmission pathways. In these cases, after ligand binding, surface receptors initiate signaling cascades within cells either after undergoing a conformational change or after forming aggregates (Bachmann and Ohashi, 1999; Cochran et al., 2001; Jiang and Hunter , 1999; Reth, 2001; Stoddard et al., 1992; Weiss and Schlessinger, 1998).
  • T lymphocyte antigen The receptor for T lymphocyte antigen (TCR) is responsible for the recognition of specific antigens bound to the major histocompatibility complex (MHC) present in so-called antigen presenting cells (APC).
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • IPC immune synapse
  • the TCR is concentrated in the IS forming a Supramolecular activation aggregate (SMAC) surrounded by a ring of adhesion molecules (Monks et al, 1998).
  • SMAC Supramolecular activation aggregate
  • TCR is composed of a TCR ⁇ / ⁇ heterodimer (or TCR ⁇ / ⁇ in T ⁇ lymphocytes) bound to a set of four dimer-organized polypeptides: the CD3 ⁇ -CD3 ⁇ and CD3 ⁇ -CD3 ⁇ heterodimers. and the CD3 ⁇ -CD3 ⁇ homodimer (Clevers et al., 1988).
  • TCR ⁇ / ⁇ recognizes the antigen / MHC complex and somehow transmits this information to CD3 components.
  • TCR subunits are type I membrane proteins, but unlike the TCR ⁇ / ⁇ heterodimer, CD3 components have cytoplasmic stems that can interact with intracellular signal transmission molecules.
  • the CD3 subunits each have one (CD3 ⁇ , CD3 ⁇ and CD3 ⁇ ) or three (CD3 ⁇ ) tyrosine and leucine motifs, called tyrosine-based activation motifs present in immunoreceptors (ITAM) (Reth, 1989).
  • ITAM immunoreceptors
  • src type 2 (SH2) homology domains such as ZAP70 tyrosine kinase ( reviewed in (Kane et al., 2000; Lin and Weiss, 2001; Qian et al., 1997).
  • ITAM phosphorylation is believed to be the earliest activation event that occurs after ligand binding to TCR.
  • the entire cytoplasmic stem of the CD3 subunits has been conserved throughout evolution, to date no role in activation has been described for sequences located outside ITAMs.
  • T lymphocytes play a critical role in regulating the immune response
  • drugs that block the activation of T lymphocytes has become desirable for the prevention of organ allograft rejection and for the control of diseases of etiology.
  • autoimmune rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes, myasthenia gravis, to name a few, and allergic type.
  • it is also convenient to control the expansion of T lymphocytes in the case of T-cell lymphomas.
  • T lymphocytes For all these reasons, it is necessary to discover new agents that allow a selective inhibition of the activation of T lymphocytes and that therefore do not interfere with the functions of other tissues.
  • the specific activation pathways of T lymphocytes can be perfect targets for new and selective immunosuppressive agents and therapies.
  • current therapies for stimulating T lymphocytes in infections and tumors) are often insufficient to induce an adequate immune response, so that new strategies would allow expanding the efficacy of said treatments.
  • Nck and CD3 ⁇ proteins as used in the present invention, indicate to all those proteins that, with respect to these and regardless of their origin, have a homology of at least 30%, preferably at least 85%, or more preferably at least 95%.
  • Nck the NcK protein as used as a general reference in the present invention comes to indicate both the NcK ⁇ protein and the NcK ⁇ protein.
  • T lymphocyte activation that occurs in inflammatory or immune reactions following activation from antigen / MHC binding to the TCR antigen receptor is provided in the present invention, demonstrating the key role of the specific interaction between certain domains of CD3 ⁇ and Nck proteins in the modulation of signal transmission that takes place in said activation of lymphocytes.
  • the present invention provides methods for the modulation of inflammatory or immune T lymphocyte activation dependent on the CD3 ⁇ and Nck interaction by methods that allow the alteration of the protein-protein interaction of CD3 ⁇ and Nck.
  • a specific object of the present invention is the modulation of said CD3 ⁇ -Nck interaction through the use of chemical substances that alter said interaction. If that such chemicals block the CD3 ⁇ -Nck interaction will be identified as substances that inhibit (antagonize) the immune or inflammatory response, whereas if they activate (agonists) said CD3 ⁇ -Nck interaction, substances that activate these immune or inflammatory responses will be identified.
  • a specific object of the present invention is the use of said substances that antagonize T lymphocyte activation in treatment protocols for diseases that cause pathological activation of T lymphocytes such as, among others, autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes and myasthenia gravis; allergic-type diseases; in the prevention and treatment of transplant rejection and T lymphocyte lymphomas.
  • a specific object of the present invention is the use of said T lymphocyte activation agonist substances in protocols for the treatment of diseases that have an inhibition of T lymphocytes such as, among others, infectious, bacterial and viral diseases, among others, and tumors.
  • Another object of the present invention is a method for the identification of said agonist and antagonist substances of the cellular function of the protein-protein CD3 ⁇ -Nck interaction that can occur in physiological and pathological processes, among others, inflammatory, autoimmune, allergic, infectious and tumorous.
  • a specific object of the present invention is a method for the identification of agonists and antagonists of the activation of T lymphocytes mediated by the interaction of CD3 ⁇ -Nck proteins, which comprises the following steps: a) generating a biological preparation of the protein interaction -protein that mimics the CD3 ⁇ -Nck interaction, b) adding the candidate chemical compound agonist or antagonist, and c) determining the blockade or activation of the protein-protein CD3 ⁇ -Nck interaction, identifying in the case of blocking an antagonist of the CD3 ⁇ interaction- Nck and in case of activation an agonist of the interaction
  • Another object of the present invention is the previous method of identifying agonists and antagonists of the CD3 ⁇ -Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3 ⁇ -Nck is carried out using the human CD3 ⁇ and Nck proteins, their homologous forms. or peptides
  • Another specific object of the present invention is a peptide that mimics the biological activity of CD3 ⁇ -binding NcK characterized by the amino acid sequence of the SH3.1 domain of NcK ⁇ (SEQ ID NO2).
  • Another specific object of the present invention is a peptide that mimics the biological activity of NcK binding to CD3 ⁇ characterized by the amino acid sequence of the SH3.1 domain of NcK ⁇ (SEQ ID NO3) as an element of said biological preparation.
  • Another specific object of the present invention is a peptide that mimics the biological activity of Nck-binding CD3 ⁇ characterized by the amino acid sequence of the proline rich region (PRS) of CD3 ⁇ described in the present invention (SEQ ID NO1) as an element of said biological preparation.
  • PRS proline rich region
  • Another object of the present invention is a fusion protein that comprises, in addition to the amino acid sequence of human CD3 ⁇ and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms, other amino acid sequences that facilitate the subsequent determination of the alteration of the CD3 ⁇ -Nck interaction as an element of said biological preparation (among others and as an example: GST-SH3.1, SH3.1-EGFP, Nck-EGFP and HA-Nck) .
  • Another object of the present invention is the previous method of identifying agonists and antagonists of the CD3 ⁇ -Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3 ⁇ -Nck is carried out using the human CD3 ⁇ and Nck proteins, their homologous forms. or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms derived from used cells or from preparations of purified or semi-purified proteins.
  • Another specific object of the present invention is a method of identifying agonists and antagonists of the CD3 ⁇ -Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3 ⁇ -Nck is carried out in a cell-free environment (among other possibilities, the immunoprecipitation assays carried out in example 1 and the stimulation assay of the purified TCR with the anti-CD3 antibody UCHT1 and subsequent blocking with the APAl / 1 antibody, see example 4).
  • the protein-protein complexes that mimic the CD3 ⁇ -Nck interaction and that are part of the procedure for identifying agonists and antagonists of the CD3 ⁇ -Nck interaction can be identified and quantified using a wide range of protein labeling techniques, among others, by immunological techniques, as performed in the present invention (anti-Flag and anti HA).
  • Another object of the present invention is a method of identifying agonists and antagonists of the CD3 ⁇ -Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3 ⁇ -Nck is carried out in a cellular environment, among other possibilities: a) assay functional quantification of cell extension and deployment, actin cytoskeleton polymerization, (see example 5), b) functional assay for quantification of cytokine secretion, IL-2 and TNF ⁇ (see example 5), c) functional assay for quantification of cell proliferation (see example 5), d) functional assay to quantify the formation of conjugates between lymphocytes and APC cells (see example 6), and e) functional assay to quantify IS maturation (see example 6).
  • Another object of the present invention is a method of identifying agonists and antagonists of the CD3 ⁇ -Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3 ⁇ -Nck is carried out in a cellular environment, consisting of a two-hybrid assay (trap assay) (for a more detailed description of this type of assay as well as other examples of assays aimed at identifying compounds modulating protein interactions see: United States Patent 6,037,136 Beach, et al., March 14, 2000 Interactions between RaF proto-oncogenes and CDC25 phosphatases, and uses related thereto and United States Patent 5,723,436 Huang, et al, March 3, 1998 Calcineurin interacting protein compositions and methods).
  • trap assay for a more detailed description of this type of assay as well as other examples of assays aimed at identifying compounds modulating protein interactions see: United States Patent 6,037,136 Beach, et al., March 14, 2000 Interactions between RaF proto-onc
  • Another object of the present invention is the previous method of identifying agonists and antagonists of the CD3 ⁇ -Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3 ⁇ -Nck and the determination of the blocking or activation of said interaction after the addition of an agonist or antagonist candidate is performed in a basal or control situation, of stimulation (anti-CD3 and APC cells loaded with superantigen or antigen, among others) or blocking (overexpression of SH3.1 of Nck and transduction of APA1 antibody / 1, among others) of said interaction according to the interest in identifying one or the other agent.
  • a specific test to identify a possible antagonist of the Nck-CD3 ⁇ interaction could consist of the tests carried out in example 5 or 6 of the present invention, where an antagonist would produce a block of the activation obtained after stimulation of the TCR complex either with the anti CD3 antibody UCHT1 or with APCs loaded with SEE both in the control situation (EGFP) and in the case of cells transfected with Nck-EGFP.
  • Chemical compounds that are candidates for testing as modulators of the CD3 ⁇ -Nck interaction can be produced, for example, by bacteria, yeasts, and other microorganisms (natural products), chemically produced (for example, small molecules including peptidomimetics), or produced by genetic engineering.
  • Another object of the present invention are methods for modulating the CD3 ⁇ and Nck interaction by means of gene manipulation that allow blocking of the protein-protein interaction of CD3 ⁇ and Nck and therefore the implantation of new gene therapy treatments for diseases in which it is necessary to block the activation of T lymphocytes such as, among others, autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes and myasthenia gravis, allergic-type diseases, in the treatment and prevention of transplant rejection and treatment of T lymphocyte lymphomas.
  • autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes and myasthenia gravis, allergic-type diseases, in the treatment and prevention of transplant rejection and treatment of T lymphocyte lymphomas.
  • a specific object of the present invention is a treatment of gene therapy of diseases that occur with an activation of T lymphocytes characterized in that the blocking of the CD3 ⁇ -Nck interaction is carried out, among others, by overexpression of the SH3.1 domain of Nck or by transduction with a specific antibody ⁇ fico (APA1 / 1).
  • a nucleotide encoding the SH3.1 domain of Nck, the Nck protein, its homologous forms, or any other peptide (fragment) that mimics said functional activity of interaction with the PRS domain of CD3 ⁇ is inserted into a suitable vector, by example a plasmid.
  • Nucleic acid can be genomic DNA, cDNA or RNA.
  • DNA or RNA can be isolated and integrated into a vector by standard methods known in the state of the art. Such methods are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory 1989).
  • Another specific object of the present invention are the nucleotides (DNA, RNA or cDNA) that encode human CD3 ⁇ and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms and that they form part of any of the above methods of modulating the CD3 ⁇ and Nck interaction.
  • those vectors that comprise the above nucleotides as well as all those host cells that contain said vectors are part of the present invention.
  • Nck is recruited to CD3 ⁇ after binding of ligands to TCR.
  • A Nck binds to CD3 ⁇ but not to the other CD3 subunits. COS cells transfected with the indicated constructs were used and precipitation (pd) was performed with the GST-Nck fusion protein followed by immunoblotting with anti-Flag or anti-CD3 ⁇ antibodies. A fraction of the total lysate from each transfectant was hybridized with the corresponding antibody to demonstrate the presence of the protein.
  • B Nck is recruited to the TCR after stimulation of intact T lymphocytes. Jurkat cells were stimulated for 5 min (time 5) with 10 ⁇ g / ml of the anti-CD3 antibody UCHT1 or incubated without antibody (time 0).
  • the TCR was immunoprecipitated with the anti-CD3 ⁇ SP34 antibody and immunoblotting was performed with an anti-Nck antibody.
  • a fraction of the total lysate (TL) from unstimulated cells was run in parallel to indicate the position of Nck.
  • C Transfected Nck binds to endogenous TCR upon activation. Jurkat cells were transfected, or not transfected, with a vector expressing Nck ⁇ labeled with an HA epitope. Immunoprecipitation with UCHT1 and immunoblotting with anti-HA was performed. Proteins running below the HA-Nck ⁇ position (arrow) probably correspond to fragments of the antibodies used for stimulation and immunoprecipitation.
  • D Nck constitutively binds to immobilized CD3 ⁇ .
  • Nck ⁇ and Nck ⁇ can be associated with TCR. Sepharose bead-coupled GST-Nck ⁇ and GST-Nck ⁇ were used to precipitate the TCR of Used from Jurkat cells not stimulated or stimulated with UCHT1.
  • D Identification of the TCR binding site in Nck. The indicated GST fusion proteins were used to precipitate the TCR, revealed by immunoblot with anti-CD3 ⁇ antibody. Nck ⁇ trn is a Nck mutant lacking the SH2 domain.
  • TCR to Nck Binding of TCR to Nck is independent of tyrosine kinase activity.
  • GST-SH3.1 was used to precipitate the TCR from Jurkat cells stimulated for 5 min with UCHTl, stimulated with pervanadate (PV) or not stimulated.
  • An immunoblot was performed with the anti-CD3 ⁇ antibody (upper left panel).
  • immunoprecipitation and immunoblotting with the anti-phosphotyrosine antibody 4G10 was performed from the same Usages (lower left panel).
  • the purified TCR was subsequently incubated with 50 ng / ml of the UCHT1 stimulating antibody, and subsequently with 1 ⁇ g / ml of the anti-CD3 ⁇ APA1 / 1 antibody or with 1 ⁇ g / ml of the control anti-CD3 ⁇ APA1 / 2 antibody before precipitation of the TCR with GST-SH3.1.
  • a monovalent Fab fragment of the anti-TCR stimulating antibody induces the conformational change in the TCR that promotes Nck binding.
  • TCR purified from 31.13 scV ⁇ 3 cells was incubated with 50 ng / ml UCHtl or 50 ng / ml of its Fab fragment before precipitation with GST-SH3.1.
  • C Model of T cell activation by the TCR. The binding of the antigen / MHC to the variable regions of the TCR ⁇ / ⁇ heterodimer promotes both aggregation and a conformational change in the TCR.
  • TCR aggregation would be necessary for activation of TCR-associated Lk and Fyn kinases from the src family, which subsequently phosphorylate ITAMs.
  • a conformational change occurs in the cytoplasmic stems of the CD3 subunits resulting in exposure of the proline-rich sequence of CD3 ⁇ (indicated as a P in the Figure) and in Nck recruitment.
  • Located at a second level would be the recruitment of ZAP70 tyrosine kinase and other signaling molecules to phosphorylated ITAMs and the recruitment of WASP, WD ?, SLP76 and Pak to Nck.
  • NcK ⁇ an adapter protein involved in the regulation of the actin cytoskeleton promoted by receptor binding (Buday, 1999; Li and She, 2000; Myung et al, 2000), interacts with the cytoplasmic stem of CD3 ⁇ specifically and that this interaction induces a modification in the TCR receptor that allows the binding of the entire TCR complex to Nck and, therefore, the activation of T lymphocytes.
  • this interaction also occurs in different types of T lymphocytes after being stimulated: Jurkat human T lymphocyte cell line,
  • the regions involved in both proteins, NcK and CD3 ⁇ have been identified at the molecular level in said interaction.
  • a proline-rich sequence (PRS) of the cytoplasmic domain of CD3 ⁇ has been identified, which is absent in the other CD3 subunits, and which has been identified in the present invention as being present within the region of CD3 ⁇ deleted in the mutants 9 and 10 ( Figure 2C) (SEQ ID NO 1) as directly related to the interaction with NcK ⁇ (Example 2).
  • the amino-terminal SH3 domain (SH3-1) of NcK ⁇ SEQ ID NO 2 has been identified as the region of this protein involved in interaction with CD3 ⁇ (Example 2). It should be noted that two human genes corresponding to NcK have been described that share 67% homology (Buday, 1997).
  • PRS-SH3 made it difficult to explain how Nck recruitment to TCR could be inducible. Since the PRS in CD3 ⁇ is located near ITAM, one possible explanation was that tyrosine phosphorylation in some way influenced the accessibility of PRS to Nck. However, in the present invention it could be concluded that the change in TCR after binding of Nck to TCR is independent of PTK activity and precedes ITAM phosphorylation in tyrosine (Example 3).
  • the characterization of the NcK-CD3 ⁇ interaction described for the first time in the present invention identifies NcK as an immediate effector of the TCR framed in the following model:
  • the binding of the antigen / MHC to the variable regions of the TCR ⁇ / ⁇ heterodimer promotes at the same time the aggregation and a conformational change in the TCR.
  • TCR aggregation would be necessary for activation of TCR-associated Lk and Fyn kinases from the src family, which subsequently phosphorylate ITAMs.
  • a conformational change occurs in the stems cytoplasmic subunits of CD3 resulting in exposure of the proline-rich sequence of CD3 ⁇ (indicated as a P in Figure 4C) and in Nck recruitment.
  • Located at a second level would be the recruitment of ZAP70 tyrosine kinase and other signaling molecules to the phosphorylated ITAMs.
  • Also at this level would be the recruitment to Nck of other proteins through domains other than SH3.1. These proteins are: WASP (third domain SH3), WD? (second SH3 domain), SLP76 (SH2 domain) and Pakl (second SH3 domain) (Buday, 1999; Li and She, 2000). Therefore, Nck has the potential to simultaneously bind to TCR and other important proteins involved in actin cytoskeleton polymerization and signal transduction, and thus can become a modulating site for T cell activation.
  • Example 1 Nck binds to CD3 ⁇ after the interaction of the TCR with its ligands.
  • yeast two-hybrid system based on recruitment of
  • Nck ⁇ interacts with the cytoplasmic stem of CD3 ⁇ .
  • Nck ⁇ specifically bound to a bait construct consisting of two tandem copies of the cytoplasmic stem of CD3 ⁇ , and also to a single copy (Table 1).
  • a GST-Nck ⁇ fusion protein to precipitate from Used COS cells transfected with the different CD3 subunits.
  • GST-Nck ⁇ bound CD3 ⁇ but not CD3 ⁇ , nor CD3 ⁇ , nor CD3 ⁇ , indicating that CD3 ⁇ is the only subunit of the CD3 complex that interacts with Nck ⁇ ( Figure 1A).
  • Nck and CD3 ⁇ interact in T lymphocytes
  • immunoprecipitation with anti-CD3 ⁇ was performed from the human T cell line Jurkat, followed by immunoblotting with anti-Nck (Figure IB).
  • Nck was co-precipitated together with CD3 ⁇ from Jurkat cells stimulated with the anti-CD3 antibody UCHTl for 5 min, but not from unstimulated cells.
  • the protein band detected by blot represents Nck
  • Jurkat cells were transiently transfected with Nck ⁇ labeled with an HA epitope (Figure 1C). Immunoblotting with anti-HA confirmed the presence of Nck ⁇ in CD3 ⁇ immunoprecipitates only when starting from cells transfected with HA-Nck ⁇ ( Figure 1C).
  • CD3 ⁇ of TCR stimulation a series of precipitation experiments were performed to identify the responsible regions in both CD3 ⁇ and Nck.
  • a set of deletion mutants comprising the entire cytoplasmic region of CD3 ⁇ ( Figure 2A) was transfected into COS cells. After metabolic labeling with 35 S-methionine, cells were used and GST-Nck ⁇ was used for precipitation assays.
  • Nck ⁇ interacts with wild-type CD3 ⁇ and also with mutants 7, 8, 11, and 12 but not with mutants 9 and 10 ( Figure 2B, upper panel).
  • a proline-rich sequence (PRS) that is absent in the other CD3 subunits, is contained within the CD3 ⁇ region deleted in mutants 9 and 10 ( Figure 2A). Deletion of this sequence prevents binding to Nck.
  • Nck Two human genes corresponding to Nck have been described that share 67% homology (Buday, 1999). These have an identical organization in three SH3 domains and one C-terminal SH2 domain.
  • Nck ⁇ the form we initially identified in the two-hybrid assay was Nck ⁇
  • the two forms bind comparably to TCR as demonstrated in precipitation experiments with GST-Nck ⁇ and GST-Nck ⁇ ( Figure 2C). Consequently, constructs of all or part of Nck ⁇ and Nck ⁇ were used interchangeably for the following experiments. Since Nck contains three SH3 domains, it seemed feasible that the Nck-CD3 ⁇ interaction was mediated by the binding of one or more of these SH3 domains to the CD3 ⁇ PRS.
  • Example 3. The change in the TCR that promotes its binding to Nck occurs before and independently of protein phosphorylation in tyrosine.
  • Nck-TCR binding is mediated by a SH3 domain suggests that binding of a ligand (antibody or antigen) to TCR promotes a conformational change that results in CD3 ⁇ PRS exposure.
  • a cell-free assay was performed from extracts of Jurkat cells transfected with the Fv (single inoglobulin chain) fragment attached to the N-terminus of TCR ⁇ . The Fv fragment that we used recognizes the NP and NIP haptens.
  • Example 5 The recruitment of Nck to the TCR is required for the activation of T lymphocytes.
  • Clones expressing EGFP alone or a fusion protein consisting of complete Nck bound to EGFP were also selected. It has been previously described that stimulation of Jurkat cells on anti-CD3 coated coverslips results in their extension and deployment in a few minutes, forming an F-actin ring that borders the periphery of the cells (Borroto et al., 2000; Bunnell et al., 2001). Since Nck is postulated to have a role in the rearrangement of the actin cytoskeleton (Buday, 1999), we performed a deployment test to test the effect of SH3.1 overexpression. Cells stably transfected with SH3.1-EGFP were less deployed and their actin cytoskeleton less polymerized than in cells expressing EGFP ( Figure 5A). It is notable that although the level of expression of Nck-EGFP was lower than that of EGFP and SH3.1-EGFP (data not shown), there was a clear enhancement of cell deployment.
  • the second approach consisted of transduction of T lymphocytes with the APA1 / 1 antibody, capable of blocking the Nck-CD3 ⁇ interaction by binding to PRS in CD3 ⁇ (Borroto et al, 1998).
  • Peripheral blood mononuclear cells (PBMC) from healthy human donors were transduced with fluorescein-labeled APA 1/1. While anti-CD3 stimulation induced TCR to Nck association in non-transduced PBMC, the interaction was inhibited in APA1 / 1 transduced cells, suggesting that APA1 / 1 was blocking CD3 ⁇ PRS (Figure 5F).
  • Example 6 The overexpression of the Nck SH3.1 domain inhibits the formation of conjugates between T lymphocytes and APC and the maturation of the immune synapse.
  • T lymphocytes we analyzed the formation of T: APC conjugates.
  • TCR-induced signals increase integrin-mediated adhesion in a process dependent on the polymerization of the actin cytoskeleton (Dustin and Cooper, 2000). Since overexpression of the Nck SH3.1 domain inhibits TCR-stimulated induced actin polymerization, we predicted that conjugate formation would also be affected. This was confirmed in an experiment where Jurkat cells were stimulated with Raji APC loaded with SEE superantigen. Incubation with SEE produced a considerable increase in conjugate formation between Raji cells and the EGFP transfected Jurkat cell clone but not with the SH3.1 -EGFP transfected clone ( Figure 6A).
  • the human cell line of origin T named Jurkat was maintained in RPMI culture medium supplemented with fetal bovine serum (FBS, Sigma), 10%.
  • the COS7 monkey cell line was maintained in DMEM medium supplemented with 5% FBS.
  • the Jurkat line lacking the TCR ⁇ 31.13 chain was kindly provided by Dr. A. Alcover (Institut Pasteur, Paris).
  • the flag-CD3 ⁇ clone was obtained by stable transfection of the expression plasmid pSR ⁇ -FlagCD3 ⁇ in the Jurkat line.
  • the clones named EGFP, Nck-EGFP and SH3.1-EGFP were obtained by stable transfection of the corresponding expression vectors in a Jurkat line named J77clon20 (Niedergang et al., 1997).
  • the 31.13 scV ⁇ 3 clone was obtained by transfection of the expression vector for fusion of the V ⁇ 3 chain fused with the single chain Fv.
  • the Fv chain used is a derivative of the antibody against the hapten 3-nitro-4hydroxyphenylacetate Bl-8 (Reth et al., 1978).
  • Thymus and spleen cells were isolated from transgenic mice with an AND-type TCR (Rag2 - / -) (Kaye et al., 1992) or from BL6 mice. As presenting cells they they used the DCEKs (Kuhlmam et al., 1991).
  • the cd25h yeast strain was provided by Stratagene.
  • Plasmids.- The SOS ⁇ fusion protein was obtained by inserting the coding fragment by the cytoplasmic domain of human CD3 ⁇ into the yeast expression vector pSOS (Stratagene).
  • the vector pSOS ⁇ tandem encodes the fusion of the SOS protein with two copies of the human tail-directed cytoplasmic domain of human CD3 ⁇ .
  • Vectors encoding the control fusion proteins p SOS-MafB, pMyr-MafB, and pMyr-LamC were provided by Stratagene.
  • PCR fragments corresponding to the complete molecule or to a deletion of the amino terminal SH2 domain were obtained starting from the human Nck ⁇ gene.
  • the Ig- ⁇ immunoglobulin leader sequence including the signal for peptidase cleavage and the Flag epitope, was amplified by PCR from the vector pEVmb-INneo (Schamel and Reth, 2000). The resulting fragment replaced the human leader sequence in the human CD3 chains in obtaining the expression vectors pSR ⁇ Flag-CD3 ⁇ , -CD3 ⁇ and -CD3 ⁇ . Cytoplasmic deletions of the human CD3 ⁇ chain were generated by a PCR-based mutagenesis method as previously described (Mallabiabarrena et al., 1992).
  • the PCR products encoding the complete human Nck ⁇ protein and its first SH3 domain were cloned into the mammalian expression vector pEFGPNl (Clontech), to obtain the Nck-EGFP and SH3.1-EGFP fusion proteins.
  • Antibodies and Reagents. The mouse antibodies APA1 / 1, SP34 and APA1 / 2, which recognize the cytoplasmic portion of human CD3 ⁇ , the extracellular portion of human CD3 ⁇ and the cytoplasmic tail of CD3 ⁇ respectively have been previously described (Alarcón et al.
  • the rabbit anti-Nck antibody which recognizes both Nck ⁇ and Nck ⁇ , was obtained from the Pharmingen house, the mouse anti-phosphotyrosine monoclonal antibody, 4G10, from Upstate Biotechnology, the mouse anti-HA monoclonal antibody, 12CA5, from Boehringer Manriheim and Sigma's anti-Flag mouse monoclonal antibody, M2.
  • Staphylococcus aureus A and E enterotoxins, SEE and SEA were purchased from Toxin Technologies.
  • the Src family tyrosine kinase inhibitor, PP2 was purchased from Calbiochem.
  • the TCR AND specific antigenic peptide, PCC corresponding to amino acids 88-100 of pigeon cytochrome C, was synthesized by the method of N - (- 9-fluorenyl) methoxycarbonyl, (Fmoc), and purified by HPLC. All the culture media, amino acids and sugars necessary for the growth and transformation of the yeasts were purchased from Sigma and Merck.
  • Precipitation was then carried out under the same conditions for at least 4 hours or at most overnight.
  • the precipitates were washed 3 to 5 times with the cell lysis buffer (Brij 96 0.3%, 150mM NaCl, 20mM Tris-HCl, pH 7.8, lOmM Iodoacetamide, lmM phenylmethylsulfonylfluoride, 1 ⁇ g / ml aproptinin, 1 ⁇ g / ml leupeptin, 1mM sodium orthovanadate and 20mM sodium fluoride).
  • the precipitates were subjected to SDS-PAGE and transfer to nitrocellulose filters (BioRad).
  • Treatment with the Src family tyrosine kinase inhibitor carried out during some stimulations consisted of pre-incubating the cells before stimulation for 30 minutes with the drug PP2 at a concentration of 20 ⁇ M, which was maintained afterwards during stimulation. Stimulations of less than 1 minute in duration were performed differently.
  • the cell suspension was concentrated by reducing its volume to half ml and was added already tempered at 37 ° C on an eppendorf with the 10 ⁇ g of stimulating antibody also tempered.
  • the lysis buffer was prepared at double the final concentration of use and kept on ice in aliquots of half ml during the stimulation to immediately add the stimulated cells. After lysis, postnuclear fractions were obtained that were subjected to previously described immunoprecipitation protocols (Alarcón et al., 1991).
  • TCR-CD3 complex The purification of the modified TCR-CD3 complex from the stable clone 31.13 was v ⁇ 3 with NP-sepharose and elution with NIP buffer was carried out as previously described (Schamel and Reth, 2000). Antibody transduction.- In order to introduce the APA1 / 1- Fitc and HP26-Fitc antibodies into living cells, the Gene Therapy Systems system called Bioporter Reagent was used. In-house specifications were followed, using the antibodies at a concentration of 250 ⁇ g / ml. The cells used were PBMCs isolated from healthy donor with Ficoll Hypaque (Rafer) and 2 ⁇ l of the reagent was used for every 2 x 10 6 cells.
  • Human Thl / Th2 Cytokine Cytometric Bead Assay kit Human Thl / Th2 Cytokine Cytometric Bead Assay kit.
  • Jurkat cells were stimulated on wells of p96 culture plates, treated with the anti-CD3 antibody UCHTl for 24 hours, and the supernatants were analyzed with the kit.
  • the cells were stimulated with Raji presenter cells loaded 30 minutes previously with SEE in different doses, in a 1: 1 ratio.
  • PBMCs (1 x 10 5 per dot) were seeded in p96 wells, treated with the anti-CD3 antibody UCHTl, at different doses, for 24 hours . Then 1 ⁇ Ci of [ 3 H] thymidine (Amersham) per well was added and the incorporation of this metabolite was measured 24 hours later.
  • CM-TMR loaded Raji cells (5mM for 20 min at 37 ° C) were added onto the chamber. Confocal images were acquired using a Leica TCS-SP confocal laser unit. Serial immunofluorescence and DIC images were obtained simultaneously at the indicated times. The most representative optical section of the green channel (EGFP, J77 cells), its corresponding DIC image (cell morphology) and the image of the red channel (CM-TMR, Raji cells) were superimposed to form a single image.
  • CD3 -gamma and CD3 -delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR / CD3 complexes.
  • T cell receptor / CD3 complex a dynamic protein ensemble.
  • Laczko I., Hollosi, M., Vass, E., Hegedus, Z., Monostori, E., and Toth, G. K. (1998).
  • the SH2 and SH3 adapter Nck a two-gene family and a linker between tyrosine kinases and multiple signaling networks. Histol Histopathol 75, 947-55.
  • Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling. J Exp Med 755, 1253-9.
  • Oligomeric antigen receptors a new view on signaling for the selection of lymphocytes. Trends Immunol 22, 356-60.
  • TCR / CD3 complex CD3 epsilon / delta and CD3 epsilon / gamma dimers associate indistinctly with both TCR alpha and TCR beta chains.

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Abstract

La présente invention concerne le mécanisme moléculaire d'activation des lymphocytes T se produisant dans les réactions inflammatoires ou immunitaires suite à l'activation à partir de l'union de l'antigène/MHC au récepteur de l'antigène de TCR, démontrant le rôle clé de l'interaction spécifique entre certains domaines des protéines CD3ε et Nck dans la modulation de la transmission des signaux se produisant au cours de ladite activation des lymphocytes. La présente invention concerne également des procédés de modulation de l'activation des lymphocytes T au moyen de procédés permettant d'altérer l'interaction protéine-protéine de CD3ε et Nck.
PCT/ES2003/000039 2002-01-24 2003-01-24 Nouvelle strategie de modulation de l'activation des lymphocytes t basee sur la regulation de l'interaction cd3ε- nck. Ceased WO2003062272A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102083809A (zh) * 2008-06-30 2011-06-01 西班牙高等科研理事会 基于阻断TCR-Nck相互作用的免疫抑制物
US10106518B2 (en) 2013-10-18 2018-10-23 Artax Biopharma Inc. Chromene derivatives as inhibitors of TCR-Nck interaction
US10131647B2 (en) 2013-10-18 2018-11-20 Artex Biopharma Inc. Chromene derivatives substituted by alkoxide as inhibitors of the TCR-Nck interaction
US11807633B2 (en) 2018-02-27 2023-11-07 Artax Biopharma Inc. Chromene derivatives as inhibitors of TCR-Nck interaction

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2001036443A1 (fr) * 1999-11-19 2001-05-25 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de nck-2

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WO2001036443A1 (fr) * 1999-11-19 2001-05-25 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de nck-2

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CLEVERS H.C. ET AL.: "Human CD3-epsilon gene contains three miniexons and is transcribed from a non-TATA promoter", PROC. NATL. ACAD. SCI. USA, vol. 85, November 1988 (1988-11-01), pages 8156 - 8160 *
GIL D. ET AL.: "Recruitment of NCK by CD3epsilon reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation", CELL, vol. 109, 28 June 2002 (2002-06-28), pages 901 - 912, XP002301957, DOI: doi:10.1016/S0092-8674(02)00799-7 *
LEHMANN J.M. ET AL.: "Nck, a melanoma cDNA encoding a cytoplasmatic protein consisting of the src homology units SH2 and SH3", NUCLEIC ACIDS RESEARCH, vol. 18, no. 4, 1990, pages 1048, XP002915759 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102083809A (zh) * 2008-06-30 2011-06-01 西班牙高等科研理事会 基于阻断TCR-Nck相互作用的免疫抑制物
EP2308860A4 (fr) * 2008-06-30 2012-01-18 Consejo Superior Investigacion Immunosuppresseur fondé sur l'interruption de l'interaction tcr-nck
US10106518B2 (en) 2013-10-18 2018-10-23 Artax Biopharma Inc. Chromene derivatives as inhibitors of TCR-Nck interaction
US10131647B2 (en) 2013-10-18 2018-11-20 Artex Biopharma Inc. Chromene derivatives substituted by alkoxide as inhibitors of the TCR-Nck interaction
US11807633B2 (en) 2018-02-27 2023-11-07 Artax Biopharma Inc. Chromene derivatives as inhibitors of TCR-Nck interaction
US12319675B2 (en) 2018-02-27 2025-06-03 Artax Biopharma Inc. Chromene derivatives as inhibitors of TCR-Nck interaction

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