WO1999026980A1 - Reactifs et procedes d'utilisation de proteines de la famille sap, nouveaux regulateurs de transduction de signaux - Google Patents

Reactifs et procedes d'utilisation de proteines de la famille sap, nouveaux regulateurs de transduction de signaux Download PDF

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Publication number
WO1999026980A1
WO1999026980A1 PCT/US1998/024976 US9824976W WO9926980A1 WO 1999026980 A1 WO1999026980 A1 WO 1999026980A1 US 9824976 W US9824976 W US 9824976W WO 9926980 A1 WO9926980 A1 WO 9926980A1
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Prior art keywords
sap
polypeptide
protein
family member
domain
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PCT/US1998/024976
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English (en)
Inventor
Cornelis P. Terhorst
Joan Sayos-Ortega
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Beth Israel Deaconess Medical Center
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Priority to AU15992/99A priority Critical patent/AU1599299A/en
Publication of WO1999026980A1 publication Critical patent/WO1999026980A1/fr

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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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to modulation of SH2 domain-containing protein- mediated signal transduction generally, and in particular antigen-specific T cell activation.
  • the signal transduction cascade emanating from a cell surface-localized receptor into the nucleus of the cell where activation-specific genes are upregulated involves a very large number of molecules, including kinases, phosphatases, and adaptor molecules.
  • the molecules involved in signal transduction events in both B and T lymphocytes, as well as in other non-lymphoid cells, both hematopoietic and non-hematopoietic often bear an internal domain with homology to a domain in the src p60 tyrosine kinase found in platelets and neuronal tissues. This domain, termed an SH2 domain, has been found to bind to phosphorylated tyrosine residues, thus mediating the interaction of molecules involved in the signal transduction cascade in a variety of cells.
  • the Signaling Lymphocyte Activation Molecule (SLAM) (CDwl50), a 70kD glycosylated type I transmembrane protein present on the surface of B and T cells, is a high-affinity self- ligand. Since triggering of SLAM co-activates T or B lymphocyte responses, it is considered to play a major role in bi-directional T ⁇ -B cell stimulation (Cocks et al, Nature 376: 260-263, 1995; Aversa et al, Immunol. Cell Biol. 75: 202- 205, 1997; Aversa et al, J. Immunol. 158: 4036-4044, 1997; Carballido et al, J. Immunol.
  • SAP SLAM-Associated Protein
  • the invention provides a substantially pure nucleic acid encoding a SAP polypeptide.
  • the SAP polypeptide is a fragment of the full length naturally-occurring SAP polypeptide.
  • the nucleic acid is operably linked to a second nucleic acid.
  • the second nucleic acid sequence may be a coding sequence and the nucleic acid operably linked to said second nucleic acid produces a fusion protein that includes the SAP polypeptide.
  • the second nucleic acid sequence may also be a gene promoter.
  • nucleic acid comprises a mutation that results in an amino acid alteration in the SAP polypeptide.
  • the nucleic acid includes a nucleic acid sequence that is substantially identical to SEQ ID NO: 3 or SEQ ID NO: 5.
  • the SAP polypeptide binds a phosphorylated tyrosine residue, or binds a non-phosphorylated tyrosine residue.
  • the nucleic acid includes a nucleic acid sequence encoding a naturally-occurring SH2 domain.
  • the nucleic acid encodes a SAP polypeptide that has SAP biological activity.
  • the SAP polypeptide modulates SH2 domain-containing protein-mediated signal transduction, such as antigen-specific T cell activation, which may be mediated by Thl cells.
  • the invention provides a substantially pure SAP polypeptide.
  • the polypeptide is a fragment of the full length naturally-occurring SAP polypeptide.
  • the polypeptide is part of a fusion protein.
  • the polypeptide includes an amino acid sequence that has a mutation as compared to the naturally-occurring amino acid sequence of the polypeptide.
  • the SAP polypeptide includes an amino acid sequence that is substantially identical to SEQ ID NO: 4 or SEQ ID NO: 6.
  • the SAP polypeptide binds a phosphorylated tyrosine residue, or binds a non-phosphorylated tyrosine residue.
  • the polypeptide includes a naturally- occurring SH2 domain.
  • the SAP polypeptide has SAP biological activity.
  • the polypeptide modulates SH2 domain-containing protein-mediated signal transduction, such as antigen-specific T cell activation which may be mediated by Thl cells.
  • the invention provides a method for detecting a disease involving aberrant SH2 domain-containing protein-mediated signal transduction in a patient that includes: (a) isolating a cell from the patient and (b) measuring the level of expression of a SAP family member polypeptide in the cell, where an alteration in the level in the patient relative to the level in a cell from a healthy control indicates the presence of a disease involving aberrant SH2 domain-containing protein-mediated signal transduction in the patient.
  • the disease is X-linked proliferative disease.
  • the invention provides a method for detecting a disease involving aberrant SH2 domain-containing protein-mediated signal transduction in a patient that includes: (a) isolating a SAP family member polypeptide from the patient and (b) determining the amino acid sequence of the SAP family member polypeptide, where an alteration in the amino acid sequence in the patient relative to the amino acid sequence of a SAP family member polypeptide isolated from a healthy control indicates the presence of a disease involving aberrant SH2 domain-containing protein- mediated signal transduction in the patient.
  • the disease is X-linked proliferative disease.
  • the invention provides a method for treating a disease involving aberrant SH2 domain-containing protein-mediated signal transduction in a patient that includes administering to the patient a SAP family member polypeptide or a fragment, mutant, or fusion thereof.
  • the SAP family member polypeptide includes an amino acid sequence substantially identical to SEQ ID NO: 4 or SEQ ID NO: 6.
  • the disease is X-linked proliferative disease.
  • the SAP family member polypeptide is EAT-2.
  • the SAP polypeptide binds a phosphorylated tyrosine residue or non-phosphorylated tyrosine residue.
  • the SH2 domain-containing protein-mediated signal transduction is antigen-specific T cell activation, which may be mediated by Thl cells.
  • the invention provides a method for identifying a compound that modulates SH2 domain-containing protein-mediated signal transduction that includes: (a) providing a cell that includes a SAP family member-encoding gene; (b) contacting the cell with a candidate compound; and (c) monitoring expression of the SAP family member-encoding gene, where an alteration in the level of the expression of the gene in response to the candidate compound indicates the presence of a compound that modulates SH2 domain-containing protein-mediated signal transduction.
  • the SAP family member-encoding gene encodes a SAP polypeptide.
  • the SAP family member encoding gene encodes an EAT-2 polypeptide.
  • the invention provides a method for identifying a compound that modulates SH2 domain-containing protein-mediated signal transduction that includes: (a) providing a cell including a reporter gene operably linked to a promoter from a SAP family member encoding gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where an alteration in the level of the expression of the reporter gene in response to the candidate compound indicates the presence of a compound that modulates SH2 domain-containing protein-mediated signal transduction.
  • the SAP family member-encoding gene encodes a SAP polypeptide.
  • the SAP family member encoding gene encodes an EAT-2 polypeptide.
  • the invention provides a method for identifying a compound that modulates SH2 domain-containing protein-mediated signal transduction that includes: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein that includes a SAP family member polypeptide covalently bonded to a binding moiety that is capable of specifically binding to the DNA-binding-protein recognition site; and (iii) a second fusion gene capable of expressing a second fusion protein that includes a SLAM polypeptide covalently bonded to a gene activating moiety; (b) exposing the cell to a candidate compound; and (c) measuring reporter gene expression in the cell, where an alteration in the level of the expression of the reporter gene in response to the candidate compound indicates the presence of a compound that modulates SH2 domain-containing protein-mediated signal transduction.
  • the cell is a yeast cell.
  • the invention provides a method for identifying a compound that modulates SH2 domain-containing protein-mediated signal transduction that includes: (a) providing a cell having: (i) a reporter gene operably linked to a DNA- binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein that includes a SLAM polypeptide covalently bonded to a binding moiety that is capable of specifically binding to the DNA-binding-protein recognition site; and (iii) a second fusion gene capable of expressing a second fusion protein that includes a SAP family member polypeptide covalently bonded to a gene activating moiety; (b) exposing the cell to the compound; and (c) measuring reporter gene expression in the cell, where an alteration in the level of the expression of the reporter gene in response to the candidate compound indicates the presence of a compound that modulates SH2 domain-containing protein-mediated signal transduction.
  • the cell is a yeast cell.
  • the SH2 domain-containing protein-mediated signal transduction is antigen-specific T cell activation, which may be mediated by Thl cells, and where the alteration is an increase indicates the compound increases antigen-specific T cell activation, and the alteration is a decrease indicates the compound decreases antigen- specific T cell activation.
  • the SAP family member polypeptide is substantially identical to SEQ ID NO: 4 or SEQ ID NO: 6. In other embodiments, the SAP family member polypeptide is EAT-2.
  • the invention provides a method for identifying a compound as a SAP family member mimetic that includes the steps of: (a) providing a SLAM polypeptide, or a fragment or fusion thereof bearing non-phosphorylated tyrosine residues; (b) contacting the SLAM polypeptide with a SAP family member polypeptide; (c) contacting the SLAM polypeptide and the SAP family member polypeptide with a candidate compound; and (d) measuring the level of interaction of the SLAM polypeptide with the SAP family member polypeptide, where a decrease in the level in response to the compound relative to a level not contacted with the compound indicates that the compound is a SAP family member mimetic.
  • the SLAM polypeptide is bound to a solid state substrate.
  • the invention provides a method for identifying a compound as a SAP family member mimetic that includes the steps of: (a) providing a SLAM polypeptide, or a fragment or fusion thereof, the SLAM polypeptide bearing non-phosphorylated tyrosine residues; and (b) contacting the SLAM polypeptide with a candidate compound, where the candidate compound binding to the SLAM polypeptide indicates that the candidate compound is a SAP family member mimetic.
  • the SLAM polypeptide is bound to a solid state substrate.
  • the invention features a method for identifying a polypeptide that modulates SH2 domain-containing protein-mediated signal transduction that includes: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein that includes a SAP family member polypeptide covalently bonded to a binding moiety that is capable of specifically binding to the DNA-binding-protein recognition site; and (iii) a second fusion gene capable of expressing a second fusion protein that is selected from a library that includes a polypeptide covalently bonded to a gene activating moiety that is encoded by a cDNA of the library; and (b) measuring reporter gene expression in the cell, where an increase in the reporter gene expression identifies the presence of a polypeptide that modulates SH2 domain-containing protein-mediated signal transduction.
  • the cell is a yeast cell.
  • the SH2 domain-containing protein-mediated signal transduction is antigen-specific T cell activation, which may be mediated by Thl cells.
  • the library is constructed from a cell selected from a group consisting of a T cell, a B cell, a myeloid cell, a natural killer cell, a hepatocyte, a liver cell, a thymocyte, a hematopoietic progenitor cell, a fibroblast, a muscle cell, and a neuron.
  • the invention provides a method for modulating SH2 domain-containing protein-mediated signal transduction in a mammal that includes providing a transgene encoding a SAP family member polypeptide or fragment thereof to a cell of the mammal, where the transgene is positioned for expression in the cell.
  • the mammal is selected from a group consisting of a rodent (e.g., a mouse), a primate (e.g., a chimpanzee), a ruminant (e.g., a cow), a pig, a horse, a sheep, and a goat.
  • the invention provides a method for modulating SH2 domain-containing protein-mediated signal transduction in a mammal that includes administering to a cell of the mammal a compound which modulates SAP family member biological activity.
  • the mammal is selected from a group consisting of a rodent (e.g., a mouse), a primate (e.g., a chimpanzee), a ruminant (e.g., a cow), a pig, a horse, a sheep, and a goat.
  • the invention provides a method for increasing antigen- specific T cell activation in a mammal that includes providing a transgene encoding a SAP family member polypeptide, the transgene being positioned for expression in the cell.
  • the mammal is selected from a group consisting of a rodent (e.g., a mouse), a primate (e.g., a chimpanzee), a ruminant (e.g., a cow), a pig, a horse, a sheep, and a goat.
  • the antigen-specific T cell activation is mediated by Thl helper T cells.
  • the invention provides a method for increasing antigen- specific T cell activation in a mammal that includes administering to a cell of the mammal a compound which increases the biological activity of a SAP family member protein.
  • the mammal is selected from a group consisting of a rodent (e.g., a mouse), a primate (e.g., a chimpanzee), a ruminant (e.g., a cow), a pig, a horse, a sheep, and a goat.
  • the antigen-specific T cell activation is mediated by Thl helper T cells.
  • the invention features a transgenic mammal having a knockout mutation in an endogenous SAP family member protein-encoding nucleic acid sequence.
  • the SAP family member protein is a SAP protein or is an EAT-2 protein.
  • the mammal has altered SH2 domain-containing protein signal transduction relative to a mammal lacking an alteration in wild-type SAP family member-encoding nucleic acid sequences.
  • the mammal is selected from a group consisting of a rodent (e.g., a mouse), a primate (e.g., a chimpanzee), a ruminant (e.g., a cow), a pig, a horse, a sheep, and a goat.
  • a rodent e.g., a mouse
  • a primate e.g., a chimpanzee
  • a ruminant e.g., a cow
  • a pig e.g., a cow
  • the mammal has altered SH2 domain-containing protein signal transduction relative to a mammal lacking an exogenous SAP family member-encoding nucleic acid sequence operably linked to a promoter.
  • the mammal is selected from a group consisting of a rodent (e.g., a mouse), a primate (e.g., a chimpanzee), a ruminant (e.g., a cow), a pig, a horse, a sheep, and a goat.
  • SAP family member an isolated SH2 domain with SAP biological activity, particularly the ability to bind either a phosphorylated or a non- phosphorylated tyrosine residue.
  • a SAP family member polypeptide has fewer than 40 amino acids, preferably fewer than 35 amino acids, located N-terminally or C- terminally to the SH2 domain in the native polypeptide.
  • the non-SH2 domain amino acids have no enzymatic activity.
  • a fusion protein is made using, as one of the fusion partners an isolated SH2 domain excised from an SH2 domain-containing protein
  • the fusion protein is a SAP family member polypeptide if not more than 40 amino acids from the N-terminally and/or C- terminally located amino acids immediately adjacent to the SH2 domain in the native SH2 domain-containing protein are incorporated into the fusion protein.
  • a preferred SAP family member polypeptide modulates signal transduction pathways involving SH2 domain-containing proteins.
  • Exemplary SAP family member proteins are the SAP proteins and the EAT-2 proteins described herein.
  • SAP protein
  • SAP polypeptide a protein or polypeptide having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% overall identity with the amino acid sequence of human SAP (SEQ ID NO: 4) or murine SAP (SEQ ID NO: 6), as shown in Figs. 2B and 2D, respectively.
  • Polypeptide products from splice variants of SAP gene sequences are also included in this definition.
  • the SAP protein is encoded by nucleic acid having a sequence with hybridizes to the nucleic acid sequence of Figs. 2A or 2C under high stringency conditions. Proteins and polypeptides localized anywhere in a cell are included in the definition.
  • a SAP polypeptide may be nucleus-localized, or membrane attached through myristoylation or palmitoylation addition.
  • SAP family member specific antibody is meant an antibody (i.e., monoclonal or polyclonal) that binds a SAP family member polypeptide, such as the human or mouse SAP proteins or EAT-2 proteins described herein. Specifically excluded from the definition is an antibody that binds an SH2 domain-containing protein, as defined below.
  • the SAP family member specific antibody of the invention is a rabbit polyclonal antibody-containing antisera described below.
  • SH2 domain is meant a polypeptide domain which is defined by the presence of "blocks" found in many proteins initially identified as having sequence identity to a domain in src tyrosine kinase that defines the second src homology (SH2) domain.
  • Each of these "blocks” has a high degree (e.g., above 60%) sequence identity with a corresponding block in the SH2 domain of another SH2 domain-containing protein. Although as a entire family, the SH2 domain of one protein may show a moderate degree of identity with a second SH2 domain- containing protein; however the sequence identity across "blocks" identify an SH2 domain-containing protein.
  • SH2 domain-containing protein or "SH2 domain-containing polypeptide” is meant a protein or polypeptide which has an SH2 domain, as well as at least 50 amino acid residues located N-terminal or C-terminal to the SH2 domain.
  • SAP family member polypeptides specifically excluded from the definition of SH2 domain-containing polypeptides are SAP family member polypeptides.
  • the SH2 domain of an SH2-domain containing protein binds phosphorylated tyrosine residues, but does not bind non-phosphorylated tyrosine residues.
  • amino acids located N-terminal or C- terminal to the SH2 domain of an SH2 domain-containing protein may have functional activity (e.g., another SH2 domain, an SH3 domain, a kinase domain, or a phosphatase domain).
  • SLAM is meant the protein or polypeptide which is an isoform (i.e., a gene splice variant) of SLAM, a multifunctional 70 kDa glycoprotein member of the Ig superfamily.
  • SLAM is a high affinity self-ligand and is characterized by its rapid induction on naive T cells and B cells following activation of these cells.
  • the four SLAM isoforms currently known are SLAM 1 (or simply SLAM, the 70 kDa glycoprotein), SLAM2, SLAM3, and SLAM4.
  • SAP biological activity or "SAP family member biological activity” is meant any one or more of the biological activities described herein for any of the SAP family member polypeptides described herein, including, without limitation, the ability to bind to a non-phosphoryated tyrosine residue, the ability to bind to a phosphoryated tyrosine residue, and the ability to counteract signaling activity of an SH2 domain-containing polypeptide.
  • the non-phosphorylated tyrosine residue is preferably present in any polypeptide. Most preferably, the non-phosphorylated tyrosine residue is present in the cytoplasmic domain of the SLAM polypeptide.
  • SH2 domain-containing protein-mediated signal transduction is meant a signal transduction event or signal transduction pathway in which an SH2 domain- containing protein plays a role.
  • the platelet derived growth factor (PDGF) receptor-mediated signal transduction pathway is an SH2 domain-containing protein-mediated signal transduction because the pathway involves at least one SH2 domain-containing protein.
  • modulating SH2 domain-containing protein-mediated signal transduction is meant increasing (i.e., enhancing) or decreasing (i.e., inhibiting) the mtracellular signaling involving SH2 domain-containing proteins in a given cell population relative to a control cell population not exposed to a test compound.
  • the increase or decrease in the given cell population exposed to a test compound is a change of at least 25%, more preferably the change is at least 50%, and most preferably the change is at least one-fold, as compared to a control cell population.
  • SH2 domain-containing protein-mediated signal transduction may be measured by a variety of assays known in the art, including the assays described herein (e.g., SH2 domain-containing protein-mediated signal transduction-mediated kinase activity, phosphatase activity, or change in phosphotyrosine proteins). It will be appreciated that the degree of modulation provided by a SAP polypeptide or a modulating compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change or a therapeutically effective change in the level of SH2 domain-containing protein-mediated signal transduction which identifies a SAP family member polypeptide, or identifies a compound which modulates SAP family member polypeptide or is a SAP family member therapeutic.
  • modulating T cell activation or “altering T cell activation” is meant increasing (i.e., enhancing) or decreasing (i.e., inhibiting) the number of T cells that become stimulated by antigen via their antigen-specific receptors in a given cell population relative to a control cell population not exposed to a test compound.
  • the increase or decrease in the given cell population exposed to a test compound is a change of at least 25%>, more preferably the change is at least 50%), and most preferably the change is at least one-fold, as compared to a control cell population.
  • T cell activation may be measured by a variety of assays known in the art, including the assays described herein (e.g., T cell activation-mediated upregulation of T cell surface molecules CD69, CD25, and Fas Ligand).
  • the cell population is selected from a group including THl CD4 + T cells, TH2 CD4 + T cells, and/or CD8 + T cells.
  • the degree of modulation provided by a SAP polypeptide or a modulating compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change or a therapeutically effective change in the level of T cell activation which identifies a SAP polypeptide, or identifies a compound which modulates SAP or is a SAP therapeutic.
  • T cell activation or "antigen-specific T cell activation” is meant a T cell that exhibits an activated phenotype (e.g., increased expression of activation- dependent genes such as interleukin-2, CD69, CD25, ⁇ -interferon, Fas Ligand) in response to stimulation through the antigen-specific T cell receptor/CD3 complex.
  • Antigen-specific T cell activation may be by stimulation of the T cell with a syngeneic antigen-presenting cell presenting the antigen in context with MHC class I or class I.
  • an I-A b restricted ovalbumin pep tide-specific T cell may achieve antigen-specific T cell activation when stimulated with an H-2 antigen- presenting cell expressing on its cell surface the ovalbumin peptide in context with class II MHC.
  • Antigen-specific T cell activation may also be achieved by incubating the T cell with an antibody toward CD3 plus an antibody specific toward a costimulatory molecule (e.g., CD28).
  • Antigen-specific T cell activation may be mediated by helper T cells of the Thl, Th2, or ThO phenotype.
  • antigen is meant a protein or polypeptide capable of eliciting an immune response.
  • the antigen may be derived from any source.
  • antigen-presenting cell a cell that expresses on its cell surface MHC proteins.
  • a preferable antigen presenting cell expresses MHC class II proteins on its cell surface, and a most preferable antigen presenting cell expresses both MHC class I and MHC class II proteins on its cell surface.
  • nucleic acids or polypeptides sequences found to occur in nature. Included in the definition are naturally-occurring mutations, homologues, isoforms, truncations, splice variants, and other naturally-occurring variants of the nucleic acid or polypeptide.
  • high stringency conditions conditions that are commonly understood in the art as highly stringent.
  • Exemplary high stringency conditions include hybridizing conditions that employ low ionic strength and high temperature for washing.
  • One, high stringency condition may include hybridization at about 40°C in about 2XSSC and 1%SDS, followed by a first wash at about 65°C in about 2XSSC and /oSDS, and a second wash at about 65°C in about lXSSC.
  • Another preferred high stringency condition includes hybridizing at 2X SSC at 40°C with a probe length of at least 40 nucleotides.
  • protein or “polypeptide” is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
  • pharmaceutically acceptable carrier means a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
  • physiological saline is physiological saline.
  • physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences. (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.
  • substantially identical is meant a polypeptide or nucleic acid exhibiting at least 50%o, preferably at least 70%, more preferably at least 85%, still more preferably at least 90%), and most preferably at least 95% identity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 16 contiguous amino acids, preferably at least 20 contiguous amino acids, more preferably at least 25 contiguous amino acids, and most preferably at least 35 contiguous amino acids.
  • the length of comparison sequences will generally be at least 50 contiguous nucleotides, preferably at least 60 contiguous nucleotides, more preferably at least 75 contiguous nucleotides, and most preferably at least 110 contiguous nucleotides.
  • Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
  • a polypeptide substantially identical to a reference polypeptide differs only by conservative amino acid substitutions.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • substantially pure polypeptide is meant a polypeptide that has been separated from the components that naturally accompany it.
  • the polypeptide is substantially pure when it is at least 60%o, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the polypeptide is a SAP polypeptide that is at least 75%, more preferably at least 90%>, and most preferably at least 99%, by weight, pure.
  • a substantially pure SAP polypeptide may be obtained, for example, by extraction from a natural source (e.g., T lymphocytes); by expression of a recombinant nucleic acid encoding a SAP polypeptide in a cellular system different from the cell from which it naturally originates (e.g., SAP polypeptide expressed in bacteria); or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • substantially pure nucleic acid is meant nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic nucleic acid of a prokaryote or eukaryote; or which exists as a separate molecule (e.g. , a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • transgene any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell.
  • a transgene may include a gene which is partly or entirely exogenous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
  • transgenic any cell which includes a DNA sequence which is inserted by artifice into that cell and becomes part of the genome of the organism which develops from that cell.
  • the transgenic organisms are generally transgenic mammals (e.g., rodents such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome.
  • mutant mutation is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene.
  • the mutation may, without limitation, be an insertion, deletion, frameshift mutation, or a missense mutation.
  • the mutation is an insertion or deletion, or is a frameshift mutation that creates a stop codon.
  • transformation or “transfection” is meant any method for introducing foreign molecules into a cell. Methods for transformation and transfection include, without limitation, lipofection, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation.
  • positioned for expression is meant that the desired sequence (e.g., a cDNA) is operably linked to one or more regulatory sequences (e.g., a promoter) which directs transcription and translation of the desired sequence (i.e., facilitates the production of, e.g., a SAP polypeptide, a recombinant protein, or a sense or antisense RNA molecule).
  • regulatory sequences e.g., a promoter
  • operably linked is meant that a two nucleic acids (e.g.
  • a coding sequence and a gene promoter are connected such that the sequences have an effect upon each other, where the two sequences are not directly adjacent to each other in the naturally-occurring genome of an organism.
  • a gene promoter from a viral gene operably linked to a coding sequence from a mammalian gene will permit expression of the mammalian protein product encoded by the coding sequence when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the gene promoter sequence.
  • a fusion protein can be created by operably linking two coding sequences such that a polypeptide encoded by the first coding sequence is covalently bonded (via a peptide bond) to a polypeptide encoded by the second coding sequence.
  • promoter is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are cell type- specific, tissue-specific, or inducible by external signals or agents; such elements may naturally occur in the 5' or 3' or intron sequence regions of the native gene, or may naturally occur in the long terminal repeat (LTR) of a viral genome.
  • LTR long terminal repeat
  • reporter gene any gene which encodes a product whose expression is detectable.
  • a reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., luciferase or chloramphenicol acetyl transferase), toxicity (e.g., ricin), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably-labeled antibody).
  • detectably-labeled any means for marking and identifying a molecule (e.g., an oligonucleotide probe, a cDNA molecule, or an antibody), such that another molecule to which the detectably labeled molecule associates can be detected.
  • Methods for detectably-labeling molecules are well known in the art and include, without limitation, radioactive labeling (e.g., with 32 P or 3:> S) and nonradioactive labeling (e.g., labeling by biotinylation or chemiluminescence with, for example, fluorescein).
  • antisense as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene (e.g., a murine or human SAP gene).
  • Thl helper T cell is meant a helper T cell (i.e., a CD4 + T cell) which, when stimulated, for example, during an immune response, serves to activate cytotoxic T cells. Thl cells produce a number of characteristic cytokines including interleukin-2, interleukin-12, and interferon- ⁇ .
  • Th2 helper T cell is meant a helper T cell (i.e., a CD4 " T cell) which, when stimulated, for example, during an immune response, serves to activate antibody-producing B cells. Th2 cells produce a number of characteristic cytokines including interleukin-4.
  • Fig. 1 shows the nucleic acid sequence (SEQ ID NO: 1; above) and amino acid sequence (SEQ ID NO: 2; below) of the cytoplasmic domain of SLAM.
  • Figs. 2A-2D shows the DNA and amino acid sequences of human and murine SAP.
  • Fig. 2A shows the DNA sequence of human SAP (SEQ ID NO: 3).
  • Fig. 2B shows the amino acid sequence of human SAP (SEQ ID NO: 4).
  • Fig. 2C shows the cDNA sequence of murine SAP (SEQ ID NO: 5).
  • Fig. 2D shows the amino acid sequence of murine SAP (SEQ ID NO: 6).
  • Fig. 3 shows the DNA and protein sequences of the various SAP mutants described herein (SEQ ID Nos: 7-13). Note that in the exon 2 genomic mutant, G is a point mutation identified in Al patient. In normal individuals, it is a C. Upper case letters in the exon 2 genomic mutant represent exon2 sequence, while lower case letters represent intronic sequence.
  • Figs. 4A and 4B demonstrate that human SAP contains an SH2 domain.
  • Fig. 4A shows the blocks (in bold-face type) identifying the SH2 domain in the amino acid sequence of human SAP.
  • Fig. 4B shows the five individual SH2 domain-identifying blocks in human SAP.
  • Fig. 5 shows a comparison of the deduced amino-acid sequences of human SAP and murine SAP with other SH2 domain-containing proteins. Consensus ⁇ - helices and ⁇ -sheets are indicated in brackets. Exon/intron boundaries, as derived from the murine SAP gene, are demarcated by i arrows. Four truncation mutants were generated in the tail of human SAP (del 1 to 4)), and their locations in the amino acid sequence are shown with I arrows.
  • Figs. 6A-6D are a series of Western blotting (WB) analyses showing the characterization of the human and murine SAP proteins and their interactions with SLAM.
  • WB Western blotting
  • SLAM or vector transfected EL-4 cells were cell surface biotinylated and lysed in detergent.
  • Postnuclear lysates (1 mg/ml) were incubated with 5 ⁇ g of GST or GST- SAP for 1 hour in the presence of glutathione beads or 1 ⁇ g of anti-SLAM antibodies (2E7) for 3 hours in the presence of protein G beads. Bead associated proteins were extracted, SDS-PAGE resolved, and immunoblotted with streptavidin-HRP.
  • human peripheral blood lymphocytes PBL
  • PHA peripheral blood lymphocytes
  • lxl 0 8 /ml immunoprecipitated with an anti-SLAM monoclonal antibody
  • Immunoprecipitates were resolved on SDS-PAGE and immunoblotted with a anti-SLAM serum (upper panel) or with a rabbit anti-SAP antibody (lower panel).
  • Figs. 7A-7C are Northern blotting analyses showing SAP expression using a 32 P radiolabelled human SAP probe.
  • Fig. 7A shows a Northern blotting analysis of poly A+ mRNA isolated from various human tissues (Clontech). A human ⁇ -actin probe was used as loading control.
  • Fig. 7B shows a Northern blotting analysis of total RNA from different human and mouse cell lines (20 ⁇ g/lane). A human 18S rRNA probe was used as a loading control.
  • Fig. 7C shows a Northern blotting analysis of human T cell subsets and two EBV+ human B cell lines. A human ⁇ -actin probe was used as loading control.
  • Figs. 8 A and 8B show SAP mRNA analyses of XLP patients.
  • Fig. 8 A is a photograph of a 10% polyacrylamide gel in which are resolved cDNAs isolated using RT PCR (reverse transcriptase-PCR) from PBMC (peripheral blood mononuclear cells) from XLP patients and healthy donors.
  • Samples Al, Bl and B2 are from XLP patients; sample B3 is from a healthy brother of Bl and B2; and samples CT-1 and CT-2 are from healthy controls.
  • RNA 8B is a Northern blotting analysis of total RNA from human T cell tumor cells (Jurkat), from a subset of CD45RA Hlgh cells from a healthy donor, and from PBMC from patient B2 (20 ⁇ g/lane). Specific RNA were detected using a 32 P radiolabelled human SAP probe; ⁇ -actin probe was used as a loading control.
  • Figs. 9A and 9B show the nucleic acid and amino acid sequences of SAP isolated from a patient with X-linked proliferative disease (XLP). DNA products were cloned in the pCR2.1 vector, and the nucleotide sequence determined on two cDNA clones in both directions using an ABI prism 377 DNA sequencer.
  • Fig. 9A shows a comparison of the nucleic acid sequence of two cDNA clones isolated by RT- PCR from an XLP patient (Al - 1 and Al -2) with those of human SAP (hS AP and hSAP ⁇ 55).
  • Fig. 9B shows a comparison of the predicted amino acid sequences of SAP protein isolated from an XLP patient and of SAP protein isolated from a normal human. SAP.
  • Figs. 10A-10C show genomic analysis of patients with X-linked proliferative disease.
  • Fig. 10A depicts the location of the point mutation near exon 2 of XLP patient Al, as well as a mechanism to explain the generation of the variant form of SAP, hSAP ⁇ 55, found in all healthy individuals.
  • Fig. 10B is a photograph of a 10%) polyacrylamide gel in which are resolved Mnl I-digested PCR amplified products of Exon 2 sequence from genomic DNA from patient Al, three healthy individuals (CT- 1, CT-2, and B3), and two human cell lines (Raji and Jurkat). In addition, PCR products from 78 healthy women and 30 healthy men were analyzed.
  • Fig. 10A depicts the location of the point mutation near exon 2 of XLP patient Al, as well as a mechanism to explain the generation of the variant form of SAP, hSAP ⁇ 55, found in all healthy individuals.
  • Fig. 10B is a photograph of a 10%) polyacrylamide gel in which are
  • 10C is a photograph of a 2% agarose gel in which are resolved PCR amplified products of hSAP Exon 1, Exon 2, Exon 3, Exon 4, and BRCA1 Exon 2 from genomic DNA from patient Al, Bl, B2, B3, and from the cell line Raji.
  • Figs. 11 A and 1 IB show the CD8SLAM fusion proteins.
  • Fig. 11 A shows schematic diagrams of SLAM3, SLAM4, CD8-SLAM3 fusion protein, CD8-SLAM4 del 1 fusion protein, and CD8-SLAM4 fusion protein. Tyrosine residues in the cytoplasmic domains of these proteins are as indicated.
  • Fig. 1 IB shows the amino acid sequences of the cytoplasmic domains of SLAM3 and SLAM4, as well as the amino acid sequence of a truncated cytoplasmic domain of the SLAM4 del 1 protein.
  • Figs. 12A-12D are a series of Western blotting (WB) analyses showing that the SAP SH2-domain binds to a specific sequence in the cytoplasmic domain of SLAM.
  • WB Western blotting
  • postnuclear mouse thymocytes lysates (1 mg/ml) were incubated for 1 hour with the indicated peptides coupled to beads (7 ⁇ M) or control beads in the absence or presence of free peptides (280 ⁇ M). Bead- associated proteins were extracted and immunoblotted with anti-human SAP rabbit serum (1/1000 dilution).
  • Fig. 12C postnuclear mouse thymocytes lysates (1 mg/mL) were incubated for 1 hour with SLAM Yl peptide coupled to beads (7 mM) in the absence or presence of free peptides (70 ⁇ M).
  • COS-7 cells were co-transfected with CD8-SLAM3 construct and one of the following SAP deletion mutants cloned into the pCMV2-FLAG construct: Del 1, Del 2, Del 3 or Del 4 (see Fig. 5). Cell surface proteins were biotinylated, and cells were lysed 48 hours after transfection.
  • Postnuclear lysates were immunoprecipitated with 1 ⁇ g of anti-CD8 antibody (OKT8) and immunoprecipitates were immunoblotted with anti-FLAG antibody (KODAK, 1/1000 dilution) (upper panel) or streptavidin (lower panel).
  • Figs. 13A-13D are a series of Western blotting (WB) analyses showing that SAP blocks recruitment of the tyrosine phosphatase SHP-2 to the phosphorylated cytoplasmic domain of SLAM.
  • COS-7 cells were transfected as indicated, cell surface biotinylated and lysed.
  • Fig. 13 A postnuclear lysates were immunoprecipitated with anti-CD8 antibodies to isolate the CD8-SLAM3 chimera, and immunoprecipitates were immunoblotted with either streptavidin (upper panel) or anti-FLAG antibody (lower panel).
  • streptavidin upper panel
  • anti-FLAG antibody lower panel
  • FIGs. 13B postnuclear lysates were incubated for 1 hour with the SLAM Yl peptide coupled to beads (7 ⁇ M). Bead- associated proteins were extracted and immunoblotted with anti-FLAG antibody (1/1000 dilution).
  • Figs. 13C and 13D COS-7 cells were co-transfected with a combination of SLAM, SAP, and c-fyn constructs. Cell surface expressed proteins were biotinylated 48 hours after transfection, and cells were lysed.
  • Postnuclear lysates were immunoprecipitated with anti-SLAM antibodies and immunoprecipitates were immunoblotted with anti-Phosphotyrosine-HRP (Zymed Laboratories, San Francisco, CA), streptavidin-HRP (Zymed), rabbit anti-SHP2 (Santa Cruz Biotech., Santa Cruz, CA) or anti-human SAP rabbit sera.
  • SLAM is seen as a broad band because of its extensive glycosylation (Cocks et al, supra).
  • Fig. 14 is a bar graph demonstrating that SAP has a positive affect in the SLAM co-stimulatory pathway.
  • Jurkat human T cells were transfected with an IL-2- promoter luciferase reporter construct plus SLAM, SAP, SLAM+SAP constructs, or vector only (pCDNA3). Luciferase activity was measured after stimulation with antibodies as indicated (hatched bar, no antibody; black bar, anti-SLAM, white bar; anti-CD3 plus anti-SLAM, gray bar).
  • Fig. 15 shows the DNA sequence of the 5' region exonl/intronl of the murine
  • Fig. 16 shows the DNA sequence of exon2/intron2 of the murine SAP gene (SEQ ID NO: 15).
  • Fig. 17 shows the DNA sequence of intron2/exon3/intron3/exon4/intron4 of the murine SAP gene (SEQ ID NO: 16).
  • Fig. 18 is a schematic diagram showing the map locations of the four murine SAP exons on the murine X chromosome.
  • Fig. 19 is a schematic diagram showing putative transcription factor binding sites on the human and murine SAP gene sequences.
  • Fig. 20 is a Northern blotting analysis using as a probe radiolabelled murine
  • SAP cDNA showing the level of murine SAP mRNA species (the 0.9 kB band) in murine Thl helper T cells (as identified by the presence of IFN- ⁇ mRNA) or Th2 helper T cells (as identified by the presence of IL-4 mRNA) following 6 hours of stimulation with anti-CD3 antibody plus human IL-2. Equivalent loading of the lanes is shown in the equivalent amounts of the 2.2 kB ⁇ -actin mRNA per lane.
  • Fig. 21 is a Western blotting analysis of anti-CD8 immunoprecipitations from externally biotinylated COS cells that had been transiently transfected with CD8SLAM3 + empty vector; CD8SLAM3 + Flag-tagged EAT-2, and CD8SLAM3 + Flag-tagged SAP.
  • the upper panel shows the results obtained by Western blotting with anti-FLAG antibody; the middle panel shows the results obtained by blotting with anti-SAP antibody; and the lower panel shows blotting with streptavidin.
  • SAP X-linked proliferative disease
  • SAP family member protein Binding of a SAP family member protein to the cytoplasmic domain of SLAM blocks the recruitment of SHP-2. Therefore two modes of SLAM signaling are likely to exist: one in which the inhibitor SAP family member acts as a negative regulator and another in which the SHP-2 dependent signal transduction pathway becomes operational. A switch between these two signalling scenarios could occur upon T cell activation, because the level of expression of the SAP protein-encoding gene was observed to diminish rapidly after triggering of the T cell receptor. It is also possible that upon activation of T cells, SAP is released from SLAM and subsequently binds to other protein(s).
  • SAP family members act as an inhibitory molecules of SH2-domain interactions can, in general, be extended to molecules other than SLAM.
  • SAP protein i.e., one of the two members of the SAP family
  • SAP protein is found predominantly in thymus derived lymphocytes
  • mutations in SAP protein will most likely affect SLAM induced signal transduction events in T lymphocytes.
  • Many XLP patients display impaired interferon- ⁇ production by helper T cells, suggesting a Th2 like phenotype.
  • engagement of SLAM during antigen-specific T-cell stimulation has been shown to induce IFN- ⁇ production and to redirect the Th2 phenotype to a Thl/ThO phenotype (Carballido et al, J. Immunol.
  • the SLAM/SLAM binding in T/B lymphocyte interactions might serve the same purpose as CD40/CD40L interactions in activation of dendritic cells by helper T cells.
  • SAP Family Member Proteins' Biological Activity SAP protein was identified by virtue of its ability to bind to the cytoplasmic tail of SLAM4 in a yeast two-hybrid screen. Interestingly, although tyrosine residues which are normally phosphorylated in mammalian cells are not phosphorylated in yeast cells, an SH2 domain is present in SAP protein. However, as the SH2 domain in SAP protein was also found to be able to bind phosphorylated tyrosine residues, SAP binds to both non-phosphorylated and phosphorylated tyrosine residues.
  • Tyrosine phosphorylation of SLAM gives rise to its being bound by the protein tyrosine phosphatase, SHP2 (also known as SHPTP-2 or syp-2).
  • SHP2 protein tyrosine phosphatase
  • the SLAM- SHP2 complex can act as a negative regulator of signal transduction cascades (Marengere et al, Science 272: 1 170-1173, 1996). Binding of non-tyrosine phosphorylated SLAM by SAP protein prevents SHP2 from binding and, hence, enhances SLAM's contribution to antigen-specific T cell activation.
  • SAP protein as the prototype, we also identified another member of the SAP family, namely EAT-2 (Thompson et al, Oncogene 13: 2649-2658, 1996) which also binds the cytoplasmic tail of SLAM and, thus, modulated SH2 domain- containing protein-mediated signal transduction.
  • EAT-2 Thimpson et al, Oncogene 13: 2649-2658, 1996) which also binds the cytoplasmic tail of SLAM and, thus, modulated SH2 domain- containing protein-mediated signal transduction.
  • While antigen-specific T cell activation is enhanced by the upregulation of the expression of a SAP family member protein, given the unique ability of a SAP family member protein to bind both phosphorylated tyrosine residues and non- phosphorylated tyrosine residues, expression of SAP family member protein in a non- T cell may either enhance or inhibit SH2 domain-containing protein-mediated signal transduction in that cell. Whether expression of a SAP family member enhances or inhibits SH2 domain-containing protein-mediated signal transduction in any particular cell depends upon which SH2 domain-containing proteins are involved in any particular signalling pathway.
  • SAP family member protein or polypeptide fragment thereof
  • administration of a SAP family member protein, or polypeptide fragment thereof may enhance antigen-specific T cell activation, as measured by antigen-specific T cell activation assays known in the art and described herein.
  • An antigen-specific T cell activation- inhibiting amount of a SAP reagent e.g., a compound that reduces the biological function of a SAP family member, such as an anti-SAP protein neutralizing antibody or SAP protein antisense nucleic acid
  • SAP reagent e.g., a compound that reduces the biological function of a SAP family member, such as an anti-SAP protein neutralizing antibody or SAP protein antisense nucleic acid
  • Such assays may be carried out in a cell which either expresses endogenous SAP family member proteins, or a cell into which is introduced an ectopic SAP family member polypeptide.
  • the cell is a T cell that is capable of undergoing antigen- specific T cell activation.
  • SAP family member e.g., SAP protein
  • TCR-mediated signal transduction the role of a SAP family member protein in NFAT activation may be readily elucidated in various assays known to the skilled artisan.
  • one method of rapidly measuring NFAT activity is through the use of a reporter gene whose expression is directed by a NFAT binding site containing promoter (Rooney et al. , Mol. Cell. Biol. 15: 6299-6310 1995; Luo et al, J. Exp. Med. 184: 141-147, 1996).
  • the expression vector is preferably inserted by artifice into a cell capable of undergoing antigen-specific T cell activation or is responsive to TCR-mediated signal transduction.
  • a change in the level of expression of the reporter gene an NF AT-inducing or inhibiting ability of a SAP family member polypeptide or reagent may be readily assessed.
  • SAP family members' biological activity may be assayed in any cell type that has an SH2 domain-containing protein-mediated signal transduction pathway.
  • PDGF platelet derived growth factor
  • An effect of an introduced SAP family member polypeptide on PDGF receptor-mediated signaling can be assessed by a number of different ways, including changes in fibroblast cell cycle, and changes in the association of a number of SH2 domain-containing proteins with the phosphorylated PDGF receptor, such as phosphatidylinositol 3-kinase (PI-3 kinase) and phospholipase C- ⁇ (PLC- ⁇ ).
  • the ability of SAP family member proteins to modulate SH2 domain- containing protein-mediated signal transduction can be defined in in vitro systems in which alterations SH2 domain-containing protein-mediated signal transduction can be detected.
  • SH2 domain-containing protein-mediated signal transduction can be induced in that cell by standard methods, for example, by addition of ligand to a cell whose SH2 domain-containing protein-mediated signal transduction pathway is initiated by that the ligand receptor on the cell surface (e.g., addition of PDGF to a PDGF receptor-expressing fibroblast).
  • ligand receptor on the cell surface e.g., addition of PDGF to a PDGF receptor-expressing fibroblast.
  • cells are cultured under the same conditions as those induced to undergo SH2 domain-containing protein-mediated signal transduction, but either not transfected, or transfected with a vector that lacks a SAP family member-encoding insert.
  • each SAP family member encoding construct to enhance or inhibit SH2 domain-containing protein-mediated signal transduction upon expression can be quantified by calculating the activation profiles of the cells, i.e., the ratio of the increase in the number of transfected cells to the increase in the number of control cells. These experiments can confirm the presence of SH2 domain-containing protein-mediated signal transduction modulating activity of a full length SAP family member protein protein. These assays may also be performed in combination with the application of additional compounds in order to identify compounds that modulate SH2 domain-containing protein-mediated signal transduction via expression of the SAP family member protein.
  • SAP family member polypeptides may be synthesized by introducing the polypeptide-encoding nucleic acid sequences, or fragments thereof, into various cell types or using in vitro extracellular systems. The function of SAP family member proteins may then be examined under different physiological conditions. For example, a SAP family member polypeptide-encoding cDNA sequence may be manipulated to characterize the expression of a given SAP family member in a particular cellular compartment. Alternatively, cell lines may be produced which over-express the SAP family member gene product allowing purification of SAP family member polypeptides for biochemical characterization, large-scale production, antibody production, and patient therapy (e.g., therapy of XLP patients).
  • patient therapy e.g., therapy of XLP patients.
  • eukaryotic and prokaryotic expression systems may be generated in which SAP family member encoding nucleic acid sequences are introduced into a plasmid or other vector which is then used to transform living cells. Constructs in which SAP family member encoding nucleic acids containing the entire open reading frames are inserted in the correct orientation into an expression plasmid may be used for protein expression. Alternatively, portions of the SAP family member gene sequences, including wild-type or mutant SAP family member sequences, may be inserted. Prokaryotic and eukaryotic expression systems allow various important domains of the SAP family member proteins to be recovered as fusion proteins and then used for binding, structural and functional studies, and also for the generation of appropriate antibodies.
  • Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the inserted SAP family member-encoding nucleic acid in the plasmid bearing cells. They may also include eukaryotic or prokaryotic origin of replication sequences allowing for their autonomous replication within the host organism, sequences that encode genetic traits that allow vector- containing cells to be selected for in the presence of otherwise toxic drugs, and sequences that increase the efficiency with which the synthesized mRNA is translated.
  • Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of, for example, viruses (e.g. , the OriP sequences from the Epstein Barr Virus genome).
  • Cell lines may also be produced which have integrated the vector into the genomic DNA, and in this manner the nucleic acid product is produced on a continuous basis.
  • Expression of foreign sequences in bacteria requires the insertion of the SAP family member encoding nucleic acid sequence into a bacterial expression vector.
  • This plasmid vector contains several elements required for the propagation of the plasmid in bacteria, and expression of inserted DNA of the plasmid by the plasmid-carrying bacteria. Propagation of only plasmid-bearing bacteria is achieved by introducing in the plasmid selectable marker-encoding sequences that allow plasmid-bearing bacteria to grow in the presence of otherwise toxic drugs.
  • the plasmid also bears a transcriptional promoter capable of producing large amounts of mRNA from the cloned DNA.
  • Such promoters may or may not be inducible promoters which initiate transcription upon induction.
  • the plasmid also preferably contains a polylinker to simplify insertion of the DNA in the correct orientation within the vector.
  • T7 late- promoter expression system in E. coli (see standard protocol in, e.g., Ausubel et al, Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, 1994). Using this system, large amounts of mRNA corresponding to the cloned SAP family member encoding DNA can be produced, and the resulting mRNA or protein can be radioactively labeled according to standard techniques.
  • sequences encoding SAP family member proteins may be introduced into a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., S. cerevisiae, insect cells such as Sf9 cells, or mammalian cells such as COS, NIH 3T3, Daudi, Jurkat, or HeLa cells).
  • a prokaryotic host e.g., E. coli
  • a eukaryotic host e.g., S. cerevisiae, insect cells such as Sf9 cells, or mammalian cells such as COS, NIH 3T3, Daudi, Jurkat, or HeLa cells.
  • the method of transformation and the choice of expression vehicle will depend on the host system selected.
  • the baculovirus system may be used (as commercially available from, e.g., Clontech, Palo Alto, CA); for expression in mammalian cells, the vaccinia virus system may be used (as described, e.g., in Ausubel et al, Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, 1994).
  • the expression system may be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Mol. Cell Biol. 5:3610-3616, 1985).
  • Eukaryotic expression systems permit appropriate post-translational modifications to expressed SAP family member proteins, or mutants or fragments thereof.
  • expression in eukaryotic cells enables the study of the function of the normal complete protein, specific portions of the protein, or of naturally-occurring polymo ⁇ hisms and artificially produced mutated proteins.
  • Eukaryotic cell expression also allows the identification of regulatory elements located in the 5', 3', or intronic regions of SAP family member genes, and their roles in tissue regulation of protein expression.
  • SAP family member proteins may be produced by a transiently-transfected or stably-transfected mammalian cell line.
  • Eukaryotic cells expressing SAP family member proteins may also be used to test the effectiveness of pharmacological agents on, for example, SAP family member- associated antigen-specific T cell activation, or as means by which to study SAP family member proteins as components of other SH2 domain-containing protein- mediated signal transduction systems.
  • the recombinant protein can be isolated from the expressing cells by cell lysis followed by protein purification techniques, such as affmity chromatography.
  • an anti-SAP family member antibody which may be produced by the methods described herein, can be attached to a column and used to isolate the recombinant SAP family member proteins. Lysis and fractionation of SAP protein-harboring cells prior to affinity chromatography may be performed by standard methods (see e.g., Ausubel et al, supra).
  • the recombinant protein can, if desired, be purified further by e.g. , by high performance liquid chromatography (HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology. Work and Burdon, Eds., Elsevier, 1980).
  • SAP family member-encoding nucleic acid sequences can be altered using procedures known in the art, such as restriction endonuclease digestion, DNA polymerase fill-in, exonuclease deletion, terminal deoxynucleotide transferase extension, ligation of synthetic or cloned DNA sequences, and site-directed sequence alteration using specific oligonucleotides together with PCR.
  • Polypeptides of the invention particularly short SAP family member polypeptide fragments, such as the fragment corresponding to the SAP protein SH2 domain of approximately 100 amino acids in length, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis. 2nd ed., 1984, The Pierce Chemical Co., Rockford, IL). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful SAP family member polypeptide fragments or analogs, as described herein.
  • Polypeptide fragments including various portions of SAP family member proteins are useful in identifying the domains important for the biological activities of SAP family member proteins. Such fragments may be generated by expression of SAP family member polypeptide fragment encoding nucleic acid fragments, generated using the nucleotide sequences provided herein, or by chemical synthesis.
  • SAP family member polypeptide fragments are useful, for example, in evaluating the portions of the SAP family member protein (e.g., the SH2 domain) involved in SH2 domain-containing protein-mediated signal transduction.
  • polypeptide fragments of SAP family members are used to induce or prevent activation-dependent gene expression with or without antigen stimulation through the T cell receptor.
  • T cell activation assays include, without limitation, expression of activation-dependent genes, such as the cell surface receptors CD25, CD69, and FAS ligand, and production of cytokines, such as interleukin-2 (IL-2) and interferon- ⁇ (IFN- ⁇ ).
  • SAP family member polypeptide fragments may include the SAP:SLAM or the EAT2:SLAM binding domain, or the SAP protein SH2 domain.
  • polypeptide fragments of SAP family members are used to induce or inhibit an SH2 domain-containing protein-mediated signal transduction event in a non-lymphoid cell.
  • SAP family member polypeptide fragment activities are assessed using one or more known assays which depend upon the cell type and the pathway in which the SH2 domain-containing protein-mediated signal transduction event plays a role.
  • a SAP fragment-mediated modulation of cell proliferation induction by an SH2 domain-containing protein-mediated signal transduction event may be readily assayed in epidermal cell proliferation assays by modifying known cell proliferation techniques (see, e.g., Ausubel et al, supra).
  • SAP family member polypeptide fragments may also be used to inhibit normal activities of an endogenous full-length SAP family member in a cell exhibiting an inappropriately high level of activity of a SAP family member protein (e.g., inappropriately high level of SAP protein activity in a T cell of a patient with an autoimmune disease).
  • SAP Family Member Antibodies In addition to the rabbit polyclonal antibodies that recognize human SAP protein and murine SAP described below, monoclonal antibodies directed toward SAP family member proteins (e.g., human EAT-2) may be produced by using as antigen a SAP family member protein isolated from SAP family member-expressing cultured prokaryotic or eukaryotic cells or a SAP family member isolated from expresing cells (e.g., EAT-2 from Ewing's sarcoma cells). Methods for generating monoclonal antibodies are well known in the art and are described, for example, in Kohler et al, Nature 256: 495, 1975; Kohler et al, Eur. J. Immunol. 6:511, 1976; Kohler et al, Eur. J. Immunol. 6: 292, 1976; Hammerling et al., Monoclonal
  • Antibodies that specifically recognize SAP family member proteins may be produced using amino acid sequences that do not reside within highly conserved regions, and that appear likely to be antigenic, as analyzed by criteria such as those provided by the PeptideStructure Program (Genetics Computer Group Sequence Analysis Package, Program Manual for the GCG Package, Version 7, 1991) using secondary structure according to either the Chou-Fasman method (Adv. in Enzymol. 47: 45-148, 1978) or the Garnier-Osgutho ⁇ e-Robson method (Gamier et al, J. Mol. Biol. 120: 97, 1978); hydrophilicity according to either the Kyte-Doolittle method or Hopp-Woods method (Proc. Natl. Acad. Sci.
  • SAP sequences preferably reside in the short cytoplasmic tail of SAP (i.e., the final 26 amino acid residues of human SAP, the final 24 amino acid residues of murine SAP, or the final 32 amino acids of murine EAT-2).
  • SAP sequences preferably reside in the short cytoplasmic tail of SAP (i.e., the final 26 amino acid residues of human SAP, the final 24 amino acid residues of murine SAP, or the final 32 amino acids of murine EAT-2).
  • SAP amino acid sequences that reside within highly conserved regions For example, amino acid sequences from the SH2 domain of SAP may be used as antigen to generate antibodies specific toward human or murine SAP.
  • antibodies In addition to intact monoclonal and polyclonal anti-SAP family member antibodies, various genetically engineered antibodies, humanized antibodies, and antibody fragments, including F(ab')2, Fab', Fab, Fv, and sFv fragments, may generated that specifically recognize human or murine SAP protein, human or murine EAT-2 protein, or fragments thereof.
  • Antibodies can be humanized by methods known in the art (e.g. , by expression in transgenic animals, as described in Green et al, Nature Genetics 7: 13-21, 1994).
  • monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA).
  • Ladner U.S. Patent Nos. 4,946,778 and 4,704,692 describes methods for preparing single polypeptide chain antibodies.
  • Ward et al. (Nature 341 : 544-546, 1989) describe the preparation of heavy chain variable domains, which they term "single domain antibodies," which have high antigen-binding affinities.
  • McCafferty et al. (Nature 348: 552-554, 1990) show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen, and that rare phage (one in a million) can be isolated after affinity chromatography.
  • Boss et al. U.S.
  • Patent 4,816,397 describe various methods for producing immunoglobulins, and immunologically functional fragments thereof, which include at least the variable domains of the heavy and light chain in a single host cell.
  • Cabilly et al. U.S. Patent 4,816,567) describe methods for preparing chimeric antibodies.
  • the screening methods of the invention involve screening any number of compounds for therapeutically active agents by employing any number of in vitro or in vivo experimental systems.
  • the methods of the invention simplify the evaluation, identification, and development of active agents for the treatment and prevention of conditions involving an inappropriate level of SH2 domain-containing protein-mediated signal transduction, which may be excessive or insufficient, depending upon the condition.
  • Screening methods provide a facile means for selecting natural product extracts or compounds of interest in a large population, which are further evaluated and condensed to a few active and selective materials. Constituents of this pool are then purified and evaluated to determine their SH2 domain-containing protein-mediated signal transduction inhibiting or inducing activities.
  • novel drugs for the treatment of conditions involving an inappropriate level of SH2 domain-containing protein-mediated signal transduction are identified from large libraries of both natural product or synthetic (or semi- synthetic) extracts or chemical libraries according to methods known in the art.
  • test extracts or compounds are not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic-, or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
  • Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • Biotics Systemussex, UK
  • Xenova Sahalose, UK
  • Harbor Branch Oceangraphics Institute Ft. Pierce, FL
  • PharmaMar, U.S.A. Click-Time, MA
  • any library or compound may be readily modified using standard chemical, physical, or biochemical methods.
  • SAP family member protein encoding DNAs may be used to facilitate the identification of compounds that increase or decrease SAP family member protein expression.
  • candidate compounds are added, in varying concentrations, to the culture medium of cells expressing SAP family member mRNA.
  • the SAP mRNA expression is then measured, for example, by Northern blotting analysis (Ausubel et al, supra) using a SAP family member protein encodmg DNA, cDNA, or RNA fragment as a hybridization probe.
  • the level of SAP family member protein encoding mRNA expression in the presence of the candidate compound is compared to the level of mRNA expression of a SAP family member protein in the absence of the candidate compound, all other factors (e.g. , cell type and culture conditions) being equal.
  • the effect of candidate compounds on SAP family member protein modulation of SH2 domain-containing protein-mediated signal transduction may, instead, be measured at the level of translation by using the general approach described above with standard protein detection techniques, such as Western blotting or immunoprecipitation with an antibody specific toward a SAP family member protein (for example, the SAP protein-specific antibody described herein).
  • candidate compounds may be tested for an ability to regulate a reporter gene whose expression is directed by a SAP family member-encoding gene promoter.
  • a cell which does not express a SAP family member may be transfected with a expression plasmid, such as a luciferase reporter gene operably linked to the SAP family member-encoding gene promoter.
  • Candidate compounds may then be added, in varying concentrations, to the culture medium of the cells.
  • Luciferase expression levels may then be measured, for example, using the luciferase assay system kit used herein that is commercially available from Promega (Madison, WI), and rapidly assessing the level of luciferase activity on a luminometer.
  • the level of luciferase expression in the presence of the candidate compound is compared to the level of luciferase expression in the absence of the candidate compound, all other factors (e.g., cell type and culture conditions) being equal.
  • Compounds that modulate the level of expression of a SAP family member protein may be purified, or substantially purified, or may be one component of a mixture of compounds such as an extract or supernatant obtained from cells, from mammalian serum, or from growth medium in which mammalian cells have been cultured (Ausubel et al, supra).
  • expression of a SAP family member protein is tested against progressively smaller subsets of the compound pool (e.g., produced by standard purification techniques such as HPLC or FPLC) until a single compound or minimal number of effective compounds is demonstrated to modulate expression of a SAP family member protein.
  • Compounds may also be screened for their ability to affect the ability of a SAP family member protein to modulate, for example, SH2 domain-containing protein- mediated signal transduction.
  • the degree of SH2 domain-containing protein-mediated signal transduction in the presence of a candidate compound is compared to the degree of SH2 domain-containing protein-mediated signal transduction in its absence, under equivalent conditions.
  • the screen may begin with a pool of candidate compounds, from which one or more useful modulator compounds are isolated in a step-wise fashion.
  • the level of an SH2 domain- containing protein mediated signal transduction pathway may be measured by any standard assay.
  • the SH2 domain-containing protein-mediated antigen- specific T cell activation signaling pathway may be measured using T cell activation assays, many of which are described herein.
  • Another method for detecting compounds that modulate the SH2 domain- containing protein mediated signal transduction modulating activity of a SAP family member protein is to screen for compounds that interact physically with a given SAP family member polypeptide in a yeast two-hybrid system, as described below.
  • Another method for detecting protein interactions is an in vitro binding assay.
  • one protein which may be recombinantly produced and may be a fusion protein, is used like an antibody in an immunoprecipitation reaction to bind to and remove its interacting protein partner from a sample, such as a cell lysate.
  • a sample such as a cell lysate.
  • the GST-SAP fusion protein described below may be recombinantly produced in bacteria and used to isolate a compound capable of binding a SAP family member. Resolution of the bead-bound proteins by SDS-PAGE will detect a SAP family member interacting protein, which may then be sequenced by N-terminal peptide sequencing.
  • a compound that promotes an increase in the expression or biological activity of the SAP family member protein is considered particularly useful in the invention; such a molecule may be used, for example, as a therapeutic to increase cellular levels of a SAP family member and thereby exploit the ability of SAP family member polypeptides to modulate SH2 domain-containing protein mediated signal transduction.
  • a compound that can increase the level of a SAP family member protein and thereby induce the SAP family member protein-mediated enhancement of antigen-specific T cell activation would be advantageous in the treatment of diseases involving insufficient antigen-specific T cell activation (e.g., the XLP disease, tuberculosis, AIDS, schistosomiasis) or cancer (e.g., breast cancer, prostate cancer, leukemia).
  • a compound that decreases the activity of a SAP family member protein may be used to decrease the ability of a SAP family member protein to modulate SH2 domain-containing protein mediated signal transduction.
  • a compound that decreases a SAP family member protein, thereby decreasing antigen-specific T cell activation would be advantageous in the treatment of diseases involving an excessive level of antigen-specific T cell activation, such as autoimmune diseases.
  • Preferred screens for compounds that affect a SAP family member protein's modulation of SH2 domain-containing protein-mediated signal transduction are rapid and high through-put.
  • SAP protein syngeneic antigen-presenting cells
  • APCs syngeneic antigen-presenting cells expressing, for example, an ovalbumin peptide
  • a multiwell e.g., a 96 well microtiter
  • T cells that specifically recognize ovalbumin peptide in context with the MHC expressed on the cultured syngeneic antigen presenting cells may be transfected with DNA that includes the promoter of an gene whose expression is upregulated upon antigen-specific T cell activation (e.g., the promoter from the gene encoding IL-2) operably linked to a reporter gene, such as green fluorescent protein (GFP).
  • the T cells are added to each peptide plus APC-containing well, followed by addition to each well of a compound, or combination thereof, being screened for an ability to modulate SAP protein-mediated enhancement of antigen- specific T cell activation.
  • the compound-treated plate is then subjected to analysis on a 96 well fluorescent plate reader for the presence of GFP.
  • a well with increased GFP expression compared to a control well not treated with any compound indicates a compound with an ability to modulate SAP protein-mediated enhancement of antigen- specific T cell activation.
  • Molecules that are found, by the methods described above, to effectively modulate gene expression of a SAP family member protein or biological activity of such a protein may be tested further in animal models. If they continue to function successfully in an in vivo setting, they may be used as therapeutics to either inhibit or enhance SH2 domain-containing protein-mediated signal transduction, as appropriate. c Screens for Additional Reagents which Mimic SAP Family Member Proteins
  • Non-structural mimetic reagents may also have this capability. Utilizing the SH2 domain-containing protein-mediated signal transduction assays described herein, such reagents may be identified. d Identification and Generation of SAP Family Member Protein SH2 Domain
  • One efficient method to treat patients (suffering from, for example, XLP disease) with the SH2 domain of a SAP family member is to generate mimetics which possess the non-phosphorylated tyrosine residue-binding abilities of the SAP family member proteins.
  • Peptide mimetics of the SH2 domain of a SAP family member, or DNA encoding these peptides may be used in concert. For example, two or more different peptides which correspond to different regions of the SAP protein SH2 domain may be introduced into the same population of cells. Likewise, a peptide that corresponds to the SH2 domain of an SAP protein may be introduced with a peptide that corresponds to the SH2 domain of an EAT-2 protein.
  • Synthetic peptides corresponding to the SH2 domain or a SAP family member protein may purchased from commercially available sources (such as the peptide generating facility at the Department of Biochemistry and Molecular Pharmacology at Harvard Medical School, Boston, MA), and tested for an ability to affect SH2 domain-containing protein mediated signal transduction events, such as the ability of a SAP family member to inhibit antigen-specific T cell activation, in various signal transduction detecting assays known in the art and described herein.
  • Such peptides may be taken up directly by cells in culture or delivered to the cells by a variety of methods, including lipid vesicles or electroporation.
  • nucleic acid sequences encoding these peptides may be subcloned into the cloning site of an expression cloning vector and the plasmid DNA introduced to the cell of interest by various transfection methods known in the art (e.g., electroporation, DEAE-dextran, calcium phosphate).
  • DNA encoding peptides corresponding to the SH2 domain of a SAP family member may also be inco ⁇ orated into coding sequences of fusion proteins and the mimetic delivered by transfection of the fusion protein encoding expression vector or fusion protein encoding viral vectors.
  • Peptides, or combinations thereof, may be screened for efficacy and effective dose requirements using the various SH2 domain-containing protein-mediated signal transduction assays well known in the art.
  • SH2 domain-containing protein-mediated signal transduction assays include in vitro kinase and in vitro phosphatase assays of SH2 domain-containing or phosphotyrosine residue-containing proteins with kinase and phosphatase activities, respectively.
  • various concentrations of peptide may be introduced with the SAP family member promoter- luciferase plasmid described herein.
  • DNA encoding potential peptide mimetics of the SH2 domain of a SAP family member may also be identified by hybridization of the DNA to the nucleotide sequences encoding SAP protem (SEQ ID NOs: 3 and 5) provided on Figs. 2A and
  • DNAs may be identified by an ability to hybridize to the SAP family member-encoding nucleotide sequences under high stringency conditions.
  • High stringency conditions may include hybridization at about 40°C in about 2XSSC and 1%SDS, followed by a first wash at about 65°C in about 2XSSC and 1%>SDS, and a second wash at about 65°C in about lXSSC.
  • a DNA which hybridizes to the nucleotide sequences encoding a SAP family member may be used to generate a polypeptide product by standard techniques.
  • the DNA may be subcloned into the pCDNA3.1 expression plasmid (commercially available from Clontech) and the resulting plasmid transfected into, for example, COS cells (commercially available from the ATCC), to produce recombinant polypeptide.
  • This polypeptide product may then be screened for SAP biological activity using the various SH2 domain-containing protein-mediated signal transduction assays described herein. The identification of minimal peptide killing sequences allows the generation of non-peptidic mimetics.
  • Therapies may be designed to circumvent or overcome a SAP gene defect or inadequate SAP family member gene expression, and thus modulate and possibly alleviate conditions involving an inappropriate amount of SH2 domain-containing protein-mediated signal transduction.
  • such therapies may be targeted at any tissues demonstrated to express SAP family member protem (e.g., SAP protein in T cells).
  • SAP family member protem e.g., SAP protein in T cells.
  • therapies to inhibit human SAP family member gene expression are useful in reducing antigen-specific T cell activation in autoreactive T lymphocytes.
  • therapies to enhance SAP family member gene expression are useful in enhancing antigen- specific T cell activation in, for example, XLP patients.
  • SH2 domain-containing protein-mediated signal transduction-modulating SAP family member reagents may include, without limitation, full length or fragment SAP family member polypeptides, SAP family member antisense nucleic acid, or any compound which increases a SAP family member's SH2 domain-containing protein- mediated signal transduction-modulating activity.
  • a) Protein Therapy Treatment or prevention of inappropriate SH2 domain-containing protein- mediated signal transduction can be accomplished by replacing mutant or su ⁇ lus SAP family member protein with normal protein, by modulating the function of mutant protein, or by delivering normal SAP family member protein to the appropriate cells. It is also be possible to modify the pathophysiologic pathway (e.g., a signal transduction pathway involving SH2 domain containing proteins) in which the SAP protein participates in order to correct the physiological defect.
  • pathophysiologic pathway e.g., a signal transduction pathway involving SH2 domain containing proteins
  • SAP family member encoding gene is introduced into T cells to successfully encode for normal and abundant protein in cells which undergo inappropriately low levels of T cell activation.
  • nucleic acids encoding SAP family member antisense RNA may be introduced into T cells to inhibit an inappropriately high level of T cell activation.
  • SAP family member-encoding sequences or SAP family member antisense RNA may also be introduced into non-lymphoid cells, to modulate inappropriate levels of SH2 domain-containing protein-mediated signal transduction.
  • the SAP family member-encoding sequence (or antisense SAP family member RNA- encoding sequence) must be delivered to those cells in a form in which it can be taken up and encode for sufficient protein to provide effective function.
  • sequences are operably linked to the endogenous SAP family member gene promoter.
  • endogenous SAP family member gene promoter it may be possible to promote normal levels of SH2 domain-containing protein-mediated signal transduction by introducing another copy of the homologous gene bearing a second mutation in that gene or to alter the mutation, or use another gene to block any negative effect.
  • Transducing retroviral vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and expression.
  • the targeted cells however must be able to divide and the expression levels of normal protein should be high.
  • the full length SAP family member gene, or portions thereof can be cloned into a retroviral vector and driven from the endogenous SAP family member gene promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest (e.g., the CD2 promoter in T cells).
  • Other viral vectors which can be used include adenovirus, adeno-associated virus, vaccinia virus, bovine papilloma virus, or a he ⁇ es virus such as Epstein-Barr Virus.
  • Retroviral vectors may be used as a gene transfer delivery system for a therapeutic SAP family member gene construct.
  • Numerous vectors useful for this pu ⁇ ose are generally known (Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis and Anderson, BioTechniques 6: 608-614, 1988; Tolstoshev and Anderson, Curr. Opin. Biotech.
  • Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N. Engl. J. Med 323: 370, 1990; Anderson et al, U.S. Patent No. 5,399,346).
  • Gene transfer could also be achieved using non-viral means requiring transfection in vitro.
  • Transplantation of normal genes into the affected cells of a patient can also be useful therapy.
  • a normal SAP family member gene is transferred into a cultivatable cell type, either exogenous or endogenous to the patient. These cells are then injected serotologically into the targeted tissue(s).
  • SAP family member-encoding DNA may be introduced into a T cell by lipofection (Feigner et al, Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al, Neurosci. Lett. 117: 259, 1990; Brigham et al, Am. J. Med. Sci.
  • a therapeutic SAP family member DNA construct is preferably applied to the site of the desired normal SH2 domain-containing protein-mediated signal transduction (for example, by injection). However, it may also be applied to tissue in the vicmity of the affected SH2 domain-containing protein-mediated signal transduction event or to a blood vessel supplying the cells with an inappropriate level of SH2 domain- containing protein-mediated signal transduction.
  • SAP family member cDNA expression can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or, preferably, the endogenous SAP gene promoter), and regulated by any appropriate mammalian regulatory element.
  • CMV human cytomegalovirus
  • SV40 simian virus 40
  • endogenous SAP gene promoter any suitable mammalian regulatory element.
  • enhancers known to preferentially direct gene expression in neural cells, lymphocytes, or muscle cells may be used to direct expression of a SAP family member protein.
  • the enhancers used could include, without limitation, those that are characterized as tissue- or cell-specific in their expression.
  • regulation may be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
  • Antisense based strategies may be employed to explore SAP family member gene function and as a basis for therapeutic drug design. Antisense strategies may use a variety of approaches, including the use of antisense oligonucleotides and injection of antisense RNA. For our analysis of SAP family member gene function, a method of transfection of antisense RNA expression vectors into targeted cells is employed. Phenotypic effects induced by antisense effects are based on changes in criteria such as protein levels, protein activity measurement, and target mRNA levels.
  • SAP family member gene therapy may also be accomplished by direct administration of antisense SAP family member mRNA to a cell that is expected to undergo an undesirably high level of T cell activation (e.g., an autoreactive T cell).
  • the antisense SAP family member mRNA may be produced and isolated by any standard technique, but is most readily produced by in vitro transcription using an antisense SAP family member cDNA under the control of a high efficiency promoter (e.g., the T7 promoter).
  • Administration of antisense SAP family member nucleic acid to cells can be carried out by any of the methods for direct nucleic acid administration described above.
  • SAP polypeptide recombinant SAP polypeptide
  • administration of recombinant SAP polypeptide either directly to the site of a desired inhibition of SH2 domain-containing protein-mediated signal transduction event (for example, by injection) or systemically (for example, by any conventional recombinant protein administration technique).
  • the dosage of SAP family member depends on a number of factors, including the size and health of the individual patient, but, generally, between O.l mg and 100 mg inclusive are administered per day to an adult in any pharmaceutically acceptable formulation.
  • any of the above therapies may be administered before the occurrence of the disease phenotype.
  • the therapies may be provided to an XLP patient who does not yet show EBV-infection induced non-Hodgkin's lymphoma.
  • compounds shown to increase SAP family member expression or SAP family member biological activity may be administered to patients diagnosed with infectious diseases or cancer by any standard dosage and route of administration (see above).
  • gene therapy using a antisense SAP family member mRNA expression construct may be undertaken to reverse or prevent the T cell defect prior to the development of an autoimmune disease.
  • the methods of the instant invention may be used to reduce or diagnose the disorders described herein in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated or diagnosed, the SAP family member polypeptide, nucleic acid, or antibody employed is preferably specific for that species.
  • a SAP family member protein, gene, or modulator may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer neutralizing SAP family member-specific antibodies or SAP family member-inhibiting compounds (e.g., antisense SAP family member or a SAP family member dominant negative mutant) to patients suffering from a disease characterized by an excessive level of T cell activation (e.g., an autoimmune disease).
  • a SAP family member protein, a cDNA encoding a SAP family member protein, or a mimetic thereof may be administered to a patient suffering from a disease characterized by an insufficient level of T cell activation (e.g., XLP or cancer). Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, by suppositories, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • SAP family member modulatory compounds include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • treatment with a SAP family member protein, gene, or modulatory compound may be combined with more traditional therapies for the disease involving excessive or insufficient levels of SH2 domain-containing protein-mediated signal transduction.
  • SAP family member polypeptides and nucleic acid sequences find diagnostic use in the detection or monitoring of conditions or diseases involving signal transduction pathways which include SH2 domain-containing proteins. For example, increased expression of a SAP family member may be correlated with diseases hallmarked by decreased SH2 domain-containing protein-mediated signal transduction in humans. Accordingly, a decrease or increase in the level of SAP family member protein production may provide an indication of a deleterious condition. Levels of expression of SAP family member proteins may be assayed by any standard technique. For example, SAP family member expression in a biological sample (e.g.
  • a biopsy may be monitored by standard Northern blot analysis or may be aided by PCR (see, e.g., Ausubel et al, supra; PCR Technology: Principles and Applications for DNA Amplification. H. A. Ehrlich, Ed. Stockton Press, NY; Yap et al, Nucl. Acids. Res. 19: 4294, 1991).
  • a biological sample obtained from a patient may be analyzed for one or more mutations in SAP nucleic acid sequences using a mismatch detection approach.
  • these techniques involve PCR amplification of nucleic acid from the patient sample, followed by identification of the mutation (i.e., mismatch) by either altered hybridization, aberrant electrophoretic gel migration, binding or cleavage mediated by mismatch bmding proteins, or direct nucleic acid sequencing. Any of these techniques may be used to facilitate mutant SAP family member detection, and each is well known in the art; examples of particular techniques are described, without limitation, in Orita et al. (Proc. Natl. Acad. Sci. USA 86: 2766- 2770, 1989) and Sheffield et al. (Proc.
  • immunoassays are used to detect or monitor SAP family member protein expression in a biological sample.
  • SAP family member-specific polyclonal or monoclonal antibodies produced as described above may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA) to measure SAP family member polypeptide levels. These levels would be compared to wild-type SAP family member polypeptide levels. For example, an increase in SAP production may indicate a condition involving insufficient antigen-specific T cell activation. Examples of immunoassays are described, e.g., in Ausubel et al, supra.
  • Immunohistochemical techniques may also be utilized for SAP family member protein detection.
  • a tissue sample may be obtained from a patient, sectioned, and stained for the presence of SAP protein using an anti-SAP antibody and any standard detection system (e.g. , one which includes a secondary antibody conjugated to horseradish peroxidase).
  • any standard detection system e.g. , one which includes a secondary antibody conjugated to horseradish peroxidase.
  • a combined diagnostic method may be employed that begins with an evaluation of SAP family member protein production (for example, by immunological techniques or the protein truncation test (Hogerrorst et al, Nature Genetics 10: 208-212, 1995) and also includes a nucleic acid-based detection technique designed to identify more subtle SAP family member mutations (for example, point mutations). As described above, a number of mismatch detection assays are available to those skilled in the art, and any preferred technique may be used. Mutations in SAP family member-encoding nucleic acids may be detected that either result in loss of expression of SAP family member or loss of normal SAP family member biological activity. In a variation of this combined diagnostic method, SAP family member biological activity is measured as antigen-specific T cell activation-inhibiting activity using any appropriate antigen-specific T cell activation assay system (for example, those described herein).
  • Mismatch detection assays also provide an opportunity to diagnose a SAP family member-mediated predisposition to diseases caused by inappropriate SH2 domain-containing protein-mediated signal transduction. For example, a patient heterozygous for a SAP family member mutation that induces a reduced level of SAP family member expression may show no clinical symptoms and yet possess a higher than normal probability of developing serious disease. Given this diagnosis, a patient may take precautions to minimize their exposure to adverse environmental factors (for example, UV exposure or chemical mutagens) and to carefully monitor their medical condition (for example, through frequent physical examinations). This type of SAP family member diagnostic approach may also be used to detect SAP mutations in prenatal screens.
  • adverse environmental factors for example, UV exposure or chemical mutagens
  • SAP family member diagnostic assays described above may be carried out using any biological sample (for example, any biopsy sample or other tissue) in which the SAP family member is normally expressed. Identification of a mutant SAP family member-encoding gene may also be assayed using these sources for test samples.
  • a SAP family member mutation particularly as part of a diagnosis for predisposition to SAP family member-associated cancer, may be tested using a DNA sample from any cell, for example, by mismatch detection techniques.
  • the DNA sample is subjected to PCR amplification prior to analysis.
  • Standard techniques such as the polymerase chain reaction (PCR) and DNA hybridization, may be used to clone additional SAP family member homologues in other species.
  • additional SAP family member sequences may be readily identified using low stringency hybridization.
  • region of high homology to a SAP family member may be used to screen databases for partial SAP family member sequences and SAP family member-specific primers may be used to clone additional SAP family member related genes by RT-PCR.
  • the murine and human SAP family member genes i.e., SAP protein encoding genes and EAT-2 protein encoding genes
  • the model is a mammalian animal, most preferably a mouse.
  • the murine SAP family member genomic DNA described herein enables the development of a mouse lacking an endogenous SAP family member gene.
  • such a mouse with a SAP protein encoding gene knock-out may be used as a murine model of XLP disease.
  • an animal model of SAP family member ove ⁇ roduction or an animal model of mutant SAP family member expression may be generated by integrating one or more SAP family member-encoding sequences into the genome operably linked to functional promoter sequences, according to standard transgenic techniques.
  • a replacement-type targeting vector which would be used to create a knockout model, can be constructed using an isogenic genomic clone, for example, from a mouse strain such as 129/Sv (Stratagene Inc., LaJolla, CA).
  • the targeting vector will be introduced into a suitably-derived line of embryonic stem (ES) cells by electroporation to generate ES cell lines that carry a profoundly truncated form of a SAP family member encoding gene.
  • ES embryonic stem
  • the targeted cell lines will be injected into a mouse blastula stage embryo. Heterozygous offspring will be interbred to homozygosity.
  • Knockout mice would provide the means, in vivo, to screen for therapeutic compounds that modulate SH2 domain-containing protein- mediated signal transduction via a SAP family member-dependent pathway. Making such mice may require use of loxP sites due to the multiple copies of SAP family member encoding genes on the chromosome (see Sauer and Henderson, Nucleic Aids Res. 17: 147-61,1989).
  • cDNA clones were also isolated from a human T cell library (Jurkat). We submitted the human SAP cDNA and protein sequence to the GenBank database, and it has been assigned GenBank Accession No. AF073019 (NID No. g3695070).
  • Mouse SAP cDNA (Figs. 2C and 2D) was thus cloned by the screening of a mouse (C57BL6) thymus cDNA library in the Zap Express vector using as a probe a cDNA amplified from mouse thymic mRNA by RT-PCR (reverse transcriptase PCR), using specific primers flanking the 5' and 3' of the EST sequence AA255258.
  • RT-PCR reverse transcriptase PCR
  • the predicted SAP protein contains a single SH2 domain (residues 6-102) followed in human SAP by a short 26 amino acid tail (residues 103-128 in human SAP) and in murine SAP by a short 24 amino acid tail (residues 103-126 in murine SAP).
  • SAP was determined to contain an SH2 domain based on the close identity of five "blocks" of sequences within the putative SH2 domain with "blocks" found in SH2 domain-containing proteins using the position-based method of Henikoff and Henikoff, J. Mol. Biol. 243: 574-578, 1994; Henikoff and Henikoff, Genomics 19: 97-107, 1994. Shown on Fig. 4A is the human SAP amino acid sequence with the positions of the five SH2 domain-hallmarking "blocks" shown in bold. In Fig. 4B, the sequences of these blocks is shown. Each of these "blocks" has a high degree
  • sequence identity with a corresponding block in the SH2 domain of an SH2 domain-containing protein.
  • SAP was isolated using a two-hybrid screen in yeast, which do not phosphorylate tyrosine residues, we do not define SAP as an SH2 domain- containing protein.
  • Human and murine SAP are highly homologous (96%> identical) both in the SH2 and tail domains.
  • human and murine SAP amino acid sequences were compared with the SH2-domains which were found most similar in a computer aided search, namely human SHIP, murine EAT-2 (GenBank Accession No. AF020263; Thompson et al, Oncogene 13: 2649-2658, 1996), and human Abl.
  • Rabbits antisera were obtained using standard methods (see, for example, Ausubel et al, Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, 1994).
  • the anti-human SAP antibodies were used to prepared immunoprecipitates of whole lysates prepared from human T cell tumor cells (Jurkat) or human peripheral blood leukocytes (PBL) (Fig. 6A).
  • the anti-murine SAP antibodies were used to prepare immunoprecipitates of whole lysates prepared from murine (C57BL/6) thymocytes (Fig. 6B).
  • the crude lysate was first precleared using 50 ⁇ l of protein G-agarose beads (GIBCO BRL) and 5 ⁇ l of normal mouse semm or normal rabbit semm for 1 hour. Immunoprecipitations were carried out using the indicated antibodies and 30 ⁇ l of protein G-agarose beads for 3 hours at 4°C.
  • Antibodies directed at human SAP tail sequences detected a 15 kD protein in anti-SAP immunoprecipitates of detergent cell lysates made either from a human T cell tumor (Jurkat) or human peripheral blood leukocytes (PBL) (Fig. 6A).
  • antibodies directed at murine SAP tail sequences detected a 15 kD protein in detergent lysates made from murine thymocytes (Fig. 6B). Because the observed molecular weight was consistent with the predicted molecular mass, the human and mouse SAP cDNAs are unlikely to encode a fragment of a larger protein.
  • El-4 cells were stably transfected by electroporation with a cDNA encoding SLAM4 or vector alone. Cells then were selected by growth in media with 600 mg/ml neomycin for three weeks. Cells then were stained with anti-SLAM PE conjugated antibodies (A12) and positive cells were sorted.
  • SLAM and SAP interact in T lymphocytes, as shown by the ability of a human SAP-GST fusion protein to specifically precipitate SLAM from detergent lysates made from a murine T cell (EL-4) transfected with human SLAM (Fig. 6C). In contrast, no interaction was detected in lysates made with untransfected EL4 cells (Fig. 6C).
  • SAP as detected by Western blotting with anti-SAP antibody, was also co- immunoprecipitated with anti-SLAM antibodies in detergent lysates prepared from PHA-activated human peripheral blood lymphocytes (as described in Isomaki et al, J. Immunol. 159: 2986-2993, 1997) (top panel, Fig. 6D). Immunoprecipitations with anti-SLAM antibodies were performed as described above for anti-SAP immunoprecipitation.
  • the level of human SAP mRNA expression was found to be highest in the thymus and lower in spleen and peripheral blood lymphocytes (Fig. 7A). SAP was expressed in all major subsets of human T cells (CD4+, CD45RO+, CD45RA+, and CD8+) (Fig. 7C) and in the T cell tumor, Jurkat, and the EBV+ Burkitt lymphoma line, Raji.
  • SAP is encoded by the X-linked Lymphoproliferative disease fXLP) gene
  • the SAP gene was localized within band A5.1 of the murine X chromosome. Because of synteny between murine band A5.1 and human Xq25, the locus at which the immune deficiency X-linked Lymphoproliferative disease (XLP) had been mapped (Lamartine et al, Eur. J. Hum. Genet. 4: 342-351, 1996; Porta et al, Genome Research. 7: 27-36, 1997; Lanyi et al, supra; Purtilo, D.T., supra), it was plausible that the SAP gene was involved in this disease.
  • XLP immune deficiency X-linked Lymphoproliferative disease
  • XLP patient Al developed hypogammaglobunemia and recurrent pulmonary infections a few months after severe EBV induced infectious mononucleosis (Rousset et al, supra). His family history and the persistence of an unbalanced vims-host relationship after EBV infection are consistent with the main characteristics of XLP syndrome (Rousset et al, supra).
  • RT-PCR reverse transcriptase-PCR
  • RT-PCR was performed using GenAmp RT-PCR kit (Perkin Elmer Co ⁇ ., Norwalk, CT) using the following primers: forward primer 5'-GCC TGG CTG CAG TAGCAG CGG CAT CTC CC-3' (SEQ ID NO: 17); and reverse primer 5'-ATG TAC AAA AGT CCA TTT CAG CTT TGA C-3' (SEQ ID NO: 18).
  • the RT-PCR products were then subjected to 10% polyacrylamide gel electrophoresis at 200 volts for 7 hr. The gels were stained with CYBR Green (Molecular Probes Inc., Eugene, OR) for 20 min., according to manufacturer's procedures, and visualized and photographed using a UV illuminator.
  • RT-PCR products of T cells from patient Al were analyzed by polyacrylamide gel electrophoresis (Fig. 8A), initially three products were found with the sizes: 629 bp (the full length SAP coding sequence); 565 bp; and 520 bp.
  • mRNAs were identified: (i) full length hSAP; (ii) a coding sequence, ⁇ E2, lacking the 64 nucleotides of exon 2 (Al-1 in Figs. 3, 9A, and 9B); (iii) E3 ⁇ 55 with a 55 nucleotide deletion in exon 3 (hSAP ⁇ 55 in Figs.
  • T cells from patient Al had four mRNA species coding for different forms of SAP, two of which were detected in healthy individuals (629 and 574 bp) and two that were specific for the patient (Al-1 of 565 bp and Al -2 of 520 bp). (Note that the 574 and 565 bp species cannot be separated on PAGE in Fig. 8A.)
  • the predicted amino acid sequences suggested two tmncated proteins in which essentially only the first exon of SAP was intact.
  • RT PCR products from peripheral blood mononuclear cells of 60 healthy individuals contained two mRNA species (629 and 574 bp), as judged by polyacrylamide gel electrophoresis and nucleotide sequence analyses (Figs. 8A and 9B). These represented full length hSAP and E3 ⁇ 55 (hSAP ⁇ 55 in Figs. 3, 9A and 9B), in which part of exon 3 had been deleted.
  • the E3 ⁇ 55 coding sequence started at the beginning of exon 3 at nucleotide position 288 and ended at nucleotide 342 (Fig. 9A).
  • the predicted protein sequence was identical to that of the first two exons of SAP (amino acids 1-67) followed by a nine amino acid sequence and a stop codon (Fig. 9B).
  • the mRNAs of patient Al either had a deletion of exon 2, or a deletion of exon 2 in addition to a deletion of exon3, which was a normal variant.
  • These mutant forms derived from patient Al were estimated to represent 90%> of the patient's SAP mRNA.
  • each of the four exons of human SAP gene was amplified by PCR using the following primers: forward primer 5'-GCC CTA CGT AGT GGG TCC ACA TAC CAA CAG-3' (SEQ ID NO: 19), and reverse primer 5'-GCA GGA GGC CCA GGG AAT GAA ATC CCC AGC-3' (SEQ ID NO: 20) for exon 1; forward primer 5'-GGA AAC TGT GGT TGG GCA GAT ACA ATA TGG-3' (SEQ ID NO: 21), and reverse primer 5'-GGC TAA ACA GGA CTG GGA CCA AAA TTC TC-3' (SEQ ID NO: 22) for exon 2; forward primer 5'-GCT CCT CTT GCA GGG AAA TTC AGC CAA CC-3' (SEQ ID NO: 23), and reverse primer 5'-GCT ACC TCT CAT TTG ACT TGC TGG CTA CAT C-3' (SEQ ID NO: 24) for exon 2
  • PCR primers were designed based on the genomic sequence of human SAP released by the EMBL database (Accession No. AL022718). The PCR products were visualized on 2% agarose gels, and subcloned into pCR2.1 vector (commercially available from Invitrogen Co ⁇ ., Carlsbad, CA followed by sequencing.
  • exon 2 PCR products were gel-purified (using reagents commercially available from Qiagen Inc., Santa Clarita, CA) and digested with restriction enzyme Mnl I (commercially available from New England Biolabs Inc., Beverly, MA), followed by 10% polyacrylamide gel electrophoresis at 200 volts for 3 hr. As shown in Fig.
  • the first patient, Bl was a 23 year old male with a history of recurrent pulmonary infections, dysgammaglobulinemia (elevated IgA and IgM), poor specific antibody responses to tetanus toxoid antigen, and depressed T cell proliferative response to mitogens. He developed fever, pneumonia, and hilar adenopathy, which quickly progressed to fulminant infectious mononucleosis with hemophagocytosis. EBV infection was documented by PCR and serology.
  • B2 One of Bl's male siblings, B2, also suffered from the XLP syndrome, which manifests itself as pancytopenia, splenomegaly, dysgammaglobulinemia, and depressed T cell proliferative responses to mitogens.
  • Example IV The SH2-domain of SAP binds to a specific sequence in the cytoplasmic domain of SLAM
  • SAP as the gene product altered in XLP raised the question of how this SLAM interactive T cell protein could account for the immunologic disturbances associated with disease.
  • the SLAM cytoplasmic domain contains three Tyr residues (Y281, Y307, and Y327) that are surrounded by consensus SH2-domain- binding-sequences (Cocks et al, supra), suggesting the possibility of an interaction with the SH2-domain of SAP.
  • SAP was found to bound to SLAM in a yeast two-hybrid system, a system in which tyrosine residues are not phosphorylated.
  • the SAP-SH2 domain had to bind to SLAM in a more complex fashion than through a classical interaction with a short phospho-tyrosine containing peptide.
  • constmcts encoding the CD8 extracellular and transmembrane domains
  • tmncated cytoplasmic domains of SLAM were generated, and schematic diagrams of which are shown on Fig. 11 A. Shown in Fig. 1 IB are the amino acid sequences of the cytoplasmic domains of SLAM3 and SLAM4 that were fused to CD8 extraceullar and transmembrane domains.
  • a truncation of the cytoplasmic domain of SLAM4 was generated by PCR from a full-length SLAM4 template, and the PCR product used to generate the CD8-SLAM del 1 fusion protein.
  • the CD8-SLAM constmcts were cloned in pCDNA3 (commercially available from Invitrogen Co ⁇ .).
  • Human SAP cDNA was cloned in expression vector pCMV2-FLAG (commercially available from KODAK), thus generating a Flag- tagged version of human SAP.
  • the CD8-SLAM constmcts were co-expressed with Flag-tagged human SAP in COS-7 cells by transforming the cells using the DEAE-dextran method (Ausubel et al, supra).
  • cell lysates which were prepared, clarified by centrifugation at 14,000 x g for 15 min. at 4°C, and the cmde lysate precleared using 50 ⁇ l of protein G-agarose beads (GIBCO BRL, Gaithersburg, MD) and 5 ⁇ l of normal mouse semm or normal rabbit serum for 1 hour.
  • Anti-CD8 (OKT3) and anti-SLAM immunoprecipitations were next performed using the antibodies and 30 ⁇ l of protein G-agarose beads for 3 hours at 4°C.
  • CD8-SLAM3 derived from a natural variant of SLAM having a short cytoplasmic domain containing only Y281
  • CD8-SLAM dell containing Y281 and Y307
  • two control chimeric proteins comprising the CD8 extracellular and transmembrane domains, and either the CD3-6 or the CD3- ⁇ cytoplasmic domain, did not co-precipitate SAP (Fig. 12A). This suggested the presence of a specific SAP binding site around the most membrane proximal tyrosine residue (Y281 ) of the SLAM cytoplasmic tail.
  • SLAM Yl CVEKKSLTIYAQVQK (SEQ ID NO: 27); SLAM pYl : CVEKKSLTIpYAQVQK (SEQ ID NO: 28); SLAM FI : CVEKKSLTIFAQVQK (SEQ ID NO: 29); SLAM Y2: CTTIYVAATEPVPESVQE (SEQ ID NO: 30); and SLAM Y3: CTVYASVTLPES (SEQ ID NO: 31).
  • peptides were coupled to beads (sulfolink coupling gel, Pierce Chemical Co.). Coupled peptides were incubated for 1 hour with lysates from mouse thymocytes or from CD8SLAM3 transiently transfected cells. Where indicated in Figs. 12B and 12C, the coupled peptides were incubated with lysate in the presence of different concentrations of soluble peptides. Following incubation, the beads were washed three times and bead-bound proteins resolved by SDS-PAGE.
  • SAP blocks recmitment of the tyrosine phosphatase SHP-2 to the phosphorylated cytoplasmic domain of SLAM
  • SLAM and c-fyn were co-transfected with or without SAP into COS-7 cells, which contain endogenous SHP-2. If SLAM was phosphorylated by c-fyn, SHP-2 was co-precipitated with anti-SLAM (Fig. 13C), but in the absence of c-fyn SLAM did not bind SHP-2.
  • a similar result was obtained with the Al deletion mutant.
  • recmitment of SHP-2 to phosphorylated SLAM was blocked by the binding of SAP to the sequence surrounding the most membrane proximal tyrosine residue Y281 of SLAM.
  • SAP was over-expressed by transient transfections into the Jurkat T cells, together with an IL-2-promoter- luciferase reporter constmct and SLAM.
  • cDNAs coding for SLAM and SAP were transiently transfected with an IL-2 promoter-luciferase constmcts into Jurkat-Tag cells, as described (Martinez-Martinez, S. et al, Mol Cell Biol. 17: 6437-6447, 1997).
  • transfected cells were stimulated with anti-CD3 and/or anti-SLAM monoclonal antibodies, as described (Cocks et al, supra). Postnuclear lysates were analyzed in a Berthold luminometer 8 hours later.
  • Figs. 15-18 are shown the DNA sequences of the 5' region / exonl / intronl of the murine SAP gene (SEQ ID NO: 14), the DNA sequences of exon2 / intron2 of the murine SAP gene (SEQ ID NO: 15), and the DNA sequences of intron2 / exon3 / intron3 / exon4 / intron4 of the murine SAP gene (SEQ ID NO: 16), respectively. From these sequences, we constructed a schematic diagram showing the locations of the four exons of the murine SAP gene on the X chromosome. This information allows the production of a SAP knock-out animal, or a transgenic animal expressing a mutant or tmncated SAP protein (see above). Methods for generating such animals are well known (see for example, Fung-Leung et al, Cell 65:443-9,
  • Fig. 19 we identified a number of putative binding sites for transcription factors in the non-coding regions of both human and murine SAP (i.e., the 5' untranslated regions and the introns) which may regulate the expression of SAP. By specifically dismpting one or more of these binding sites, the level of expression of SAP may be altered in a patient suffering from a disease characterized by an inappropriate amount of SH2-domain containing protein mediated signal transduction.
  • Thl and Th2 helper T cells were isolated Thl and Th2 cells from mice according to standard techniques. Briefly, CD4 + T cells from mice expressing the transgene for the
  • DO11.10 ⁇ -TCR which recognizes residues 323-339 of chicken ovalbumin (OVA) in association with I-A d , were cultured in complete RPMI 1640 media with OVA 323- 339 peptide (1 ⁇ M) and mitomycin-treated splenocytes.
  • OVA ovalbumin
  • recombinant murine IL-12 (10 ng/ml), neutralizing anti-IL-4 monoclonal antibody (clone 1 IB 11, 40 ⁇ g/ml, commercially available from R&D Ssytems, Minneapolis, MN) was added.
  • Th2 phenotype T cell development recombinant murine IL-4 (10 ng/ml) and neutralizing polyclonal anti-murine IL-12 (clone TOSH-2, 3 ⁇ g/ml, commercially available from Endogen, Cambridge, MA) was added.
  • Cells were cultured for three rounds of antigenic stimulations under polarizing conditions, and then stimulated on plate-bound anti-CD3 (clone 2C11, commercially available from PharMingen, San Diego, CA) in the presence of human IL-2 (commercially available from Endogen) for 6 hours.
  • polyA+ RNA was prepared from the cells (using the FastTrack mRNA kit commercially available from Invitrogen, Carlsbad, CA), electrophoretically resolved, and subjected to Northern blotting analyses using as probes the following radiolabelled murine DNAs: Interferon-gamma (IFN- ⁇ ; to identify Thl cells), interleukin-4 (IL-4; to identify Th2 cells), mSAP (which identifies a 0.9 kB band), mSLAM (which identifies a 2.1 kB band) and ⁇ -actin (control; identifies a 2.2 kB band).
  • IFN- ⁇ Interferon-gamma
  • IL-4 interleukin-4
  • mSAP which identifies a 0.9 kB band
  • mSLAM which identifies a 2.1 kB band
  • ⁇ -actin control; identifies a 2.2 kB band.
  • the 2.1 kB SLAM mRNA band increased in both stimulated (vs. non-stimulated) Thl and Th2 cells.
  • the 0.9 kB SAP mRNA decreased in both Thl and Th2 cells following 6 hours of stimulation (note that the decrease was less noticeable in the Thl cell population).
  • the level of SAP mRNA species was higher in Thl cells than in Th2 cells.
  • EAT-2 as a SAP family member protein
  • the murine EAT-2 protein was identified as an SH2 aberrantly expressed in
  • the EAT2-FLAG encoding expression vector was co-transfected with the CD8SLAM3 encoding vector into COS cells. Following cell surface biotinylation using the EZ-Link Sulfo-NHS-Biotin reagent (Pierce Chemical Co.), the COS cells were lysed and immunoprecipitated with anti-CD8 antibody.
  • the immunoprecipitates were resolved by SDS-Page and subj ected to Western blotting analyses with anti-FLAG antibody, anti-SAP antibody, or streptavidin.
  • both Flag tagged SAP and Flag tagged EAT2 can be pulled down with CD8SLAM3 (Fig. 21, top panel).
  • EAT2 like SAP, binds the cytoplasmic tail of SLAM3, which bears only the one tyrosine residues, Y281.
  • Equivalent loading of the lanes is shown at the bottom panel of Fig. 21, with equivalent binding of streptavidin to all three lanes.
  • a plasmid which encodes the GAL4 DNA-binding domain fused to the entire coding domain of human or murine SAP is constructed by inserting DNA encoding the coding region of human or SAP into a GAL4 DNA binding domain fusion protein expression vector pGBT9 (Clontech, Palo Alto, CA). GAL4 DNA binding domain- SAP fusion protein is then used as "bait" (DNA-binding domain hybrid) in yeast two- hybrid screens of a human cDNA "prey” library of PH A- stimulated peripheral blood leukocytes (PBL) or a murine cDNA "prey” library of a T cell lymphoma (both commercially available from Clontech). The yeast two-hybrid assay and isolation of positive clones and subsequent interaction analyses are carried out as has been previously described (see, for example, PCT Publication WO 95/28497).
  • DNA sequencing of positive SAP-interacting clones is performed on an Applied Biosytems model 373 A automated DNA sequencer.
  • Full length cDNA encoding polypeptide fragments of SAP-interacting proteins are isolated by using the cDNA clone encoding the SAP-interacting polypeptide fragment to screen other libraries (e.g., longer cDNA libraries or genomic libraries) using standard hybridization techniques (see Ausubel et al, supra; Sambrook et al, supra).
  • SH2 Domain-Containing Protein-Mediated Signal Transduction Assays Numerous assays are known which evaluate SH2 domain-containing protein mediated signal transduction. Such signal transduction pathways occur in, for example, epidermal cells through the Epidermal Growth Factor (EGF) receptor (Vogel et al, Science 259: 1611-1614, 1993), fibroblasts through the Platelet Derived Growth Factor (PDGF) receptor (Fantl et al, Cell 69: 413-423, 1992), and B cells (Ono et al, Cell 90: 293-301, 1997).
  • EGF Epidermal Growth Factor
  • PDGF Platelet Derived Growth Factor
  • SAP activity on antigen-specific T cell activation can be assessed using a variety of antigen-specific T cell activation assays are known in the art. Most are based upon the increased production of activation dependent T cell genes including, without limitation, the following cell surface molecules: Fas ligand, MHC class II, CD25, and CD69; as well as the following cytokines: interleukin-2 (IL-2), and ⁇ -interferon. All of the following examples may be easily modified to identified compounds which affect SAP expression and biological activities, as well as peptide and non-peptide SAP mimetics which have SAP biological activity.
  • IL-2 interleukin-2
  • a T cell specific for, for example, a viral peptide may be stimulated with a syngeneic antigen presenting cell presenting that viral peptide on its cell surfaces in context with MHC.
  • An activated T cell will express Fas ligand, MHC class II, CD25, and CD69 on its cell surface (as opposed to a resting, unactivated T cell which expresses none of these cell surface molecules).
  • an activated T cell unlike a resting T cell, will produce IL-2 and ⁇ -IFN.
  • the cell surface activation markers can be detected by antibodies (commercially available from, for example, Becton Dickinson, San Jose, CA).
  • Binding of these antibodies may be rapidly assessed by binding of a secondary labeled antibody, followed by detection of that bound secondary antibody (by, for example, FACS analysis or addition of colorimetric substrate for the label).
  • the cytokines released by activated T cells may be detected from the supernatants of cells by commercially available ELISA kits (commercially available from, for example, Endogen Inc., Woburn, MA).
  • Antigen-specific T cell activation may also be determined by the production of activation-dependent cytokines, such as IL-2, in a proliferation assay of IL-2 dependent cells.
  • IL-2 activation-dependent cytokines
  • Jurkat human T cells may be stimulated with anti-CD3 plus anti-CD28 antibodies (commercially available from, for example, Pharmingen, San Diego, CA).
  • anti-CD3 plus anti-CD28 antibodies commercially available from, for example, Pharmingen, San Diego, CA.
  • CTLL-20 available from the ATCC.
  • the CTLL-20 cells are then returned to culture for 48-72 hours total, with the addition of 3 H-thymidine to the cells in the final 6-20 hours of culture.
  • Non-radioactive cell proliferation assays are also known in the art (e.g., the CellTiter 96® AQ ueous Non-Radioactive Cell Proliferation Assay commercially available from Promega Co., Madison, WI).
  • antigen-specific T cell activation assays are also provided in the following references: increased interleukin-2 (IL-2) production (Aversa et al, J. Immunol. 1588: 4036-4044, 1997); increased interferon- ⁇ (IFN- ⁇ ) production (Aversa et al, J. Immunol. 1588: 4036-4044, 1997); increased CD69 expression (Zubiaur et al, J.
  • IL-2 interleukin-2
  • IFN- ⁇ interferon- ⁇
  • the invention includes any protein which is substantially identical to the human SAP polypeptide provided in Fig. 2B (SEQ ID NO: 4), murine SAP polypeptide provided in Fig. 2D (SEQ ID NO: 6), or to GenBank Accession Nos. AF020263 and AF020264; such homologues include other substantially pure naturally-occurring mammalian SAP family member proteins as well as splice variants, allelic variants; natural mutants; induced mutants; DNA sequences which encode proteins and also hybridize to the SAP family member encoding DNA sequence of Fig. 2A (SEQ ID NO: 3), Fig. 2C (SEQ ID NO: 5), GenBank Accession No.
  • AF020263 or GenBank Accession No. AF020264, under high stringency conditions (e.g., hybridizing at 2X SSC at 40°C with a probe length of at least 40 nucleotides) or, less preferably, under low stringency conditions (e.g. , hybridizing at 5X SSC at 25°C with a probe length of at least 80 nucleotides); and proteins specifically bound by antisera directed to a SAP family member polypeptide.
  • high stringency conditions e.g., hybridizing at 2X SSC at 40°C with a probe length of at least 40 nucleotides
  • low stringency conditions e.g., hybridizing at 5X SSC at 25°C with a probe length of at least 80 nucleotides
  • the invention further includes analogs of any naturally-occurring SAP family member polypeptides.
  • Analogs can differ from the naturally-occurring SAP family member proteins by amino acid sequence differences, by post-translational modifications, or by both.
  • Analogs of the invention will generally exhibit at least 50%), more preferably 70%, and most preferably 90% or even 95% identity with all or part of a naturally-occurring SAP family member amino acid sequence.
  • the length of sequence comparison is at least 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues.
  • Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes.
  • Analogs can also differ from the naturally-occurring SAP family member polypeptide by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook et al, supra; or Ausubel et al, supra).
  • cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally-occurring or synthetic amino acids, e.g., B or Y amino acids.
  • the invention also includes SAP family member polypeptide fragments.
  • fragment means at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids.
  • Polypeptide fragments of SAP family members can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
  • Preferable fragments or analogs according to the invention are those which facilitate specific detection of a SAP family member nucleic acid or amino acid sequence in a sample to be diagnosed.
  • Particularly useful SAP family member polypeptide fragments for this pu ⁇ ose include, without limitation, the amino acid fragments corresponding to the SH2 domain.

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Abstract

L'invention porte sur de nouvelles protéines SAP et sur des séquences d'acide nucléique. Cette invention identifie également la famille SAP dont les membres sont de nouveaux régulateurs de transduction de signaux. L'invention porte également sur des polypeptides de la famille SAP, sur des anticorps spécifiques des polypeptides de la famille SAP, et sur des procédés de modulation de la transduction de signaux induite par une protéine contenant le domaine SH2, notamment l'activation des lymphocytes T spécifiques de l'antigène. De plus, ces procédés permettent de détecter des composés en vue de moduler la transduction de signaux induite par une protéine contenant le domaine SH2.
PCT/US1998/024976 1997-11-21 1998-11-19 Reactifs et procedes d'utilisation de proteines de la famille sap, nouveaux regulateurs de transduction de signaux WO1999026980A1 (fr)

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Cited By (10)

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US6828428B2 (en) 1996-12-05 2004-12-07 Kyowa Hakko Kogyo Co., Ltd. IGA nephropathy-related genes
US6962984B2 (en) 1996-12-05 2005-11-08 Nihon University IgA nephropathy-related DNA
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WO2005015205A2 (fr) * 2003-08-08 2005-02-17 Novartis Ag Utilisation de sap
WO2005015205A3 (fr) * 2003-08-08 2005-06-23 Novartis Ag Utilisation de sap
WO2006122401A1 (fr) * 2005-05-16 2006-11-23 Institut De Recherches Cliniques De Montreal/I.R.C.M. Regulation progressive de fonctions cellulaires au moyen de eat-2, d'un regulateur lie a sap exprime dans des cellules immunitaires innees
CN107037219A (zh) * 2017-06-12 2017-08-11 首都医科大学附属北京友谊医院 X‑连锁淋巴增殖综合征诊断试剂盒及其应用

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