US20130344080A1 - PILR alpha Interactions and Methods of Modifying Same - Google Patents

PILR alpha Interactions and Methods of Modifying Same Download PDF

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US20130344080A1
US20130344080A1 US13/921,557 US201313921557A US2013344080A1 US 20130344080 A1 US20130344080 A1 US 20130344080A1 US 201313921557 A US201313921557 A US 201313921557A US 2013344080 A1 US2013344080 A1 US 2013344080A1
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binding
pilrα
ligand
pilr
cells
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Ali Zarrin
Yonglian Sun
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Genentech Inc
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Genentech Inc
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Priority to US13/921,557 priority Critical patent/US20130344080A1/en
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Priority to US14/598,870 priority patent/US9556269B2/en
Priority to US15/397,399 priority patent/US10160807B2/en
Priority to US16/181,246 priority patent/US20190276532A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • sequence listing comprising SEQ ID NOS: 1-63 is attached hereto. Each sequence provided in the sequence listing is incorporated herein by reference, in its entirety, for all purposes.
  • Described herein is a novel receptor-ligand interaction and agents that may modify and/or block the interaction.
  • Methods, uses, reagents and kits for the modulation of ligand activities related to its interaction with the novel receptor are disclosed.
  • therapeutic uses of reagents in treating inflammation-related disorders Specifically, this disclosure relates to PILR ⁇ , its ligands and methods of modulating their interaction.
  • methods of treating PILR ⁇ -mediated inflammation and/or diseases e.g., pathogenic infection, autoimmune arthritis, etc.
  • Immune function is regulated by the balance of activating and inhibitory signals mediated by immune cell-surface receptors (Lanier, Nature Immunol. (2001) 2:23-27; Smith et al., Immunol. Rev. (2001) 181:115-125; Moretta et al., Annu. Rev. Immunol. (2001) 19:197-223; Diefenbach & Raulet, Curr. Opin. Immunol. (2003) 15:37-44.).
  • these receptors there are families that harbor extracellular regions that have a highly conserved amino-acid sequence but have different intracellular domains responsible for activation and inhibitory signaling.
  • activating and inhibitory members of these families can generally recognize the same or very similar ligands with slightly different specificities, presumably resulting in fine-tuning of immune regulation. Since they are involved in both the activation and inhibition of immune function, they are generally referred to as paired receptor families. Paired immunoglobulin-like (Ig-like) type 2 receptors (PILRs) are one of the paired receptor families. Inhibitory receptors possess immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains and deliver inhibitory signals via tyrosine phosphatases, such as SHP-1.
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • the ITIM-bearing receptor PILR-alpha recruits SHP-1 via its amino-terminal SH2 domain and is likely to have cellular inhibitory potential.
  • the lack of a cytoplasmic tail and the presence of the transmembrane lysine residue in the second receptor, PILR-beta suggest its potential activating function. See Fournier et al., J. Immunol. (2000) 165:1197-1209; Mousseau et al., J. Biol. Chem. (2000) 275:4467-4474; Shiratori et al., J. Exp. Med. (2004) 199:525-533.
  • Herpes simplex virus type 1 (HSV-1) is a member of the alphaherpesvirus subfamily and can cause recurrent mucocutaneous lesions on the mouth, face, or genitalia and potentially meningitis or encephalitis.
  • Membrane glycoprotein B (gB) of HSV-1 is a second ligand for PILR-alpha (Satoh et al., Cell (2008)132:935-944). The interaction of HSV-1 gB with PILRa mediates viral entry and cell-cell fusion. Interestingly, expression of PILRa on cells enhances HSV-1 entry, whereas expression of PILRb does not (Fan and Longnecker, J. Virol. (2010) 84(17):8664-8672).
  • HSV-1 gB is not a ligand of PILRb, and subtle amino acid differences between a and b play a role in ligand selectivity.
  • binding of PILRa to HSV-1 gB also requires sialylated O-glycans (T53, T480) (Fan et al., J. Virol. (2009) 83(15):7384-7390).
  • PILRa specifically associates with HSV-1 gB, but not with other HSV-1 glycoproteins, although some other envelope proteins are known to be O-glycosylated (Fan et al., J. Virol. (2009) 83(15):7384-7390).
  • PILR-alpha and PILR-beta are abundantly expressed on myeloid cells, very little is known about their role in host defense against extracellular bacterial infection or in immune function. Thus, elucidating the role for PILR-alpha and PILR-beta is a necessary prerequisite for the prevention/treatment of diseases/conditions associated with PILR-alpha and/or PILR-beta dysfunction.
  • the present disclosure identifies sialidated glycans as necessary and sufficient for hPILRa binding.
  • the present disclosure also provides a method and compositions, such as selective binding agents, to modulate the interactions of the PILR and its ligands.
  • novel reagents and methods based on the interaction of infective microorganisms and/or endogenous ligands for the prevention/treatment of diseases/conditions associated with PILR-alpha and/or PILR-beta activity.
  • the present disclosure relates to various methods, uses, reagents, and kits based on modulation of the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • One embodiment of the invention relates to an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • compositions may comprise (1) an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA, and (2) a pharmaceutically acceptable carrier.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • Another embodiment of present invention further relates to a method for blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise the step of administering an effective amount of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • Another embodiment of the present invention provides an interaction occurring at the cell surface and a method for blocking the cell surface interaction between a PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA may comprise contacting cells (a cell expressing PILR-alpha and/or a cell expressing a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA) with an effective amount of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG
  • Another embodiment of the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • Another embodiment of the present invention relates to the use of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR
  • Another embodiment of the present invention relates to the use of an agent for treating an inflammatory disease/condition in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject, wherein the agent may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • Another embodiment of the present invention relates to a method for identifying a compound capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of an agent, wherein a lower binding of PILR-alpha to said ligand in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of blocking the interaction between PILR
  • Another embodiment of the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of the agent, wherein a lower binding of PILR-alpha to the ligand in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • Another embodiment of the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.
  • Another embodiment of the present invention relates to a use of an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for treating an inflammatory disease or condition in a subject.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • Another embodiment of the present invention relates to a use of an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament for treating an inflammatory disease or condition in a subject.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • compositions for treating an inflammatory disease or condition in a subject comprising (1) an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and (2) a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention relates to an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • compositions may comprise (1) an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and (2) a pharmaceutically acceptable carrier.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • a pharmaceutically acceptable carrier selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • Another embodiment of the present invention relates to a method for stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise the step of administering an effective amount of an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the interaction may occur at the cell surface and a method for stimulating the cell surface interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA may comprise contacting cells (a cell expressing PILR-alpha and/or a cell expressing a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA) with an effective amount of an agent that may stimulate the interaction between PILR-alpha and said ligand.
  • An additional embodiment of the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a further embodiment of the present invention relates to the use of an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, W
  • An embodiment of the present invention relates to the use of an agent for treating an inflammatory disease in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject wherein the agent may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • Another embodiment of the present invention relates to a method for identifying a compound capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of an agent, wherein an elevated binding of PILR-alpha and said ligand in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of stimulating the interaction between PILR-
  • Another further embodiment of the present invention relates to a method for identifying a compound capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA activity in the presence or absence of the agent, wherein an elevated PILR-alpha activity in the presence of the agent may be indicative that the agent is stimulating the interaction between PILR-alpha and said ligand.
  • Another embodiment of the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of the agent, wherein an elevated binding of PILR-alpha and said ligand in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • Yet another embodiment of the present invention provides a method of identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA activity in the presence versus the absence of the agent, wherein an elevated PILR-alpha activity in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA activity
  • a further embodiment of the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.
  • Another embodiment of the present invention relates to a use of an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for treating an inflammatory disease or condition in a subject.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • Another embodiment of the present invention relates to a use of an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament for treating an inflammatory disease or condition in a subject.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA
  • compositions for treating an inflammatory disease or condition in a subject comprising an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and a pharmaceutically acceptable carrier.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and a pharmaceutically acceptable carrier.
  • the ligand may be any one of NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the variant comprises an alteration at a residue corresponding to R133 in SEQ ID NO:1.
  • the alteration may be the addition, deletion or substitution of the residue.
  • the variant may comprise a mutation corresponding R133A.
  • the variant comprises an alteration at a residue corresponding to R126 in SEQ ID NO:3.
  • the alteration may be the addition, deletion or substitution of the residue.
  • the variant may comprise a mutation corresponding R126A.
  • the variant comprises an alteration that abrogates binding of PILRa to a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the variant comprises an alteration that inhibits or decreases binding of PILRa to a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • An additional embodiment of the present invention relates to a binding motif for ligands of PILR-alpha.
  • the ligands comprise a sialidated glycan domain.
  • the human PILR-alpha binds to a ligand is via a sialoadhesin homology domain.
  • At least one ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA interacts with PILR-beta.
  • the ligand binds to PILR-beta but with lower affinity than to PILR-alpha.
  • FIGS. 1A to 1E illustrate that PILR ⁇ binds to mouse but not human CD99 and that transfer of mouse CD99 PKAPT motif to human CD99 restores PILR ⁇ binding.
  • 1 A 293T cells were transfected with mouse and human CD99 expression vectors and the transfectants were stained with hPILR ⁇ -Fc (black line), mouse PILR ⁇ -Fc (black line) or control Ig (grey area). CD99 expressing cells were gated and their binding to PILR ⁇ -Fc was shown.
  • 1 B Comparison of profiles of O-glycans from human and mouse CD99-Fc fusion proteins. O-glycans were released by reductive ⁇ -elimination and permethylated then analyzed by MALDI-TOF MS.
  • 1 C shows the glycosylation motifs in mouse CD99 and the variant human CD99 where the mouse PKAPT motif was inserted. See FIG. 5A in Shiratori et al., J. Exp. Med. (2004) 199(4):525-533 for the mouse CD99 sequence.
  • 1 D illustrates the recovery of binding of human CD99 with the PKAPT motif inserted (i.e., the PKAPT motif from mouse CD99 was inserted into the human CD99) to both mouse and human PILRa. Both human and mouse PILRa bind mouse CD99 (second row) but fail to bind human CD99 (third row).
  • 293T cells were transfected with mouse, human CD99 or human CD99 with mouse PKAPT motif inserted after T41 and the transfectants were stained with hPILR ⁇ -Fc (black line), mouse PILR ⁇ -Fc (black line) or control Ig (grey area).
  • FIGS. 2A to 2E illustrate novel PILR ⁇ ligands NPDC1 and COLEC12.
  • 2 A COS7 cells were transfected with hNPDC1 and hCOLEC12 expression vectors, stained with hPILR ⁇ -AP, mPILR ⁇ -AP and control supernatant followed with AP substrate.
  • 2 B 293T cells were transfected with mCD99, hNPDC1 and hCOLEC12, and the transfectants were stained with isotype control, hPILR ⁇ -Fc or mouse PILR ⁇ -Fc (black line). Binding to mock transfectants (grey area) represents background binding. Transfected ligand expressing cells were gated and their binding to PILR ⁇ -Fc was shown.
  • 2 C 293T cells were transfected with human and mouse PILR ⁇ , and the transfectants were stained with hNPDC1-Fc or hCOLEC12-his (black line). Binding to mock transfectants (grey area) represents background binding. PILR ⁇ positive cells were gated and ligand fusion staining was shown.
  • 2 D Radioligand assay was used to determine the equilibrium binding affinity of hPILR ⁇ -Fc to hNPDC1 transiently expressed on 293T cells. 125 I labeled hPILRa ⁇ -Fc was allowed to bind to cells in the presence of increasing amounts of unlabeled hPRILa-Fc. The average equilibrium K D from two replicate assays was 49 nM.
  • 2 E SPR equilibrium binding analysis of hPILR ⁇ -Fc binding to immobilized hCOLEC12 is shown. The equilibrium K D for hPILR ⁇ /hCOLEC12 was 1.1 uM.
  • FIG. 3 shows that human NPDC1 and COLEC12 are glycosylated with sialylated glycans. Comparison of MALDI-TOF MS spectra of permethylated O-glycans, released by reductive ⁇ -elimination, from human NPDC1-Fc and human COLEC12-his. Molecular ions of permethylated glycans (glycan alditols) were detected in positive ion mode, as sodium adducts (M+Na) + .
  • FIG. 4 shows that sialylated glycans on NPDC1 or COLEC12 are required for their binding to PILR ⁇ .
  • the binding of selected proteins to hPILR ⁇ was determined by surface plasmon resonance.
  • Human PILR ⁇ -Fc (25 ug/ml) was immobilized to a CM5 sensor chip. Fusion proteins with and without Sialidase A treatment were used as analytes (1 ⁇ M). The difference between the responses of negative control protein and the testing proteins was shown.
  • FIGS. 5A to 5G illustrate that a conserved Arginine site in PILR ⁇ is required for its binding to ligand.
  • 5 A Amino acid sequence alignment of PILR ⁇ from various species, PILR ⁇ and N-terminal of SIGLEC1. The positions of Ig fold residues are designated by an arrow above the sequence ( ⁇ ) based on comparison with Igk/1 and TCRb V set Ig domains. conserveed non-Ig PILR ⁇ residues are designated by an ⁇ above the sequence. SIGLEC1 residues involved in the sialic acid-binding are boxed (dashed lines). Conserved Siglec residues across the family are boxed (solid lines).
  • Asterisks represent PILR ⁇ amino acids that are important for sialic acid interaction.
  • Solid arrows beneath the sequence denote the positions corresponding to active sites of SIGLEC 1 crystal structure (and PILR ⁇ homology model).
  • the underlined segments designate beta strands in PILR.
  • Black circles represent the mutational binding analysis that have been done on PILR ⁇ .
  • the pairwise-percentage-residue identity between PILR ⁇ and SIGLEC1 was 23%.
  • 293T cells were transfected with wild type human and mouse PILR ⁇ (grey line), or human PILR ⁇ R126A and mouse PILR ⁇ 133A (black line) expression constructs, and the transfectants were stained with mCD99-Fc, hNPDC1-Fc or hCOLEC12-his, their binding to mock transfectants (grey area) was shown as background binding.
  • PILR ⁇ positive cells were gated and ligand fusion staining was shown.
  • 293T cells were transfected with mouse CD99, human NPDC1, human COLEC12, or HSV1 gB expression vectors, and the transfectants were stained with hPILR ⁇ -Fc or mPILR ⁇ -Fc (grey line), hPILR ⁇ R126A-Fc or mPILR ⁇ R133A-Fc (black line), their binding to mock transfectants (grey area) was shown as background binding.
  • Transfected ligand expressing cells were gated and PILR ⁇ -Fc staining was shown.
  • 5 D 1 to 5 D 2 293T cells were infected with HSV1. Twenty-four hours later ( FIG.
  • 5D-1 glycoprotein B expression in HSV1 (black line) or mock (grey area) infected cells was shown;
  • FIG. 5D-2 hPILR ⁇ -Fc or mPILR ⁇ -Fc (grey line), hPILR ⁇ R126A-Fc or mPILR ⁇ R133A-Fc (black line) binding to HSV1 infected cells was shown, their binding to mock transfectants (grey area) was shown as background binding.
  • 5 E WT hPILRa-Fc and hPILRa R126-Fc were immobilized on a chip and their binding to hNPDC1, hCOLEC12, and mCD99 was compared.
  • FIG. 6 illustrates the binding of PILRa-Fc to human NPDC1 and human COLEC12 expressing cells.
  • HEK293T cells were transfected with human NPDC1, human COLEC12, human CD99 or vector.
  • Transfected 293T cells were incubated with control immunoglobulins or the indicated the PILR constructs indicated on the top followed by an incubation with a second antibody and analyzed by flow cytometry.
  • FIG. 7 illustrates the PILRa-Fc binding to ligand transfectants.
  • FIGS. 8A and 8B illustrate the binding of (A) mouse and (B) human PILRa and the variant PILRa fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands—COLEC12, CLEC4G and FceRII.
  • FIG. 8C also shows the binding of mPILRb to the same ligands. Note that the variant mPILRa does not bind and that mPILRb binds to a lesser degree than mPILRa for CLEC4G and FceRII.
  • FIGS. 9A and 9B illustrate the binding of (A) mouse and (B) human PILRa and the variant PILRa fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands—MAG, FceRII and LRRTM4.
  • FIG. 9C also shows the binding of mPILRb to the same ligands. Note that the variant mPILRa does not bind and that mPILRb binds to a lesser degree than mPILRa for FcRII.
  • FIGS. 10A and 10B illustrate the binding of (A) mouse and (B) human PILRa and the variant PILRa fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands—BR3, IL2Ra and PSS8.
  • FIG. 10C also shows the binding of mPILRb to the same ligands. Note that the variant mPILRa does not bind and that mPILRb binds to a lesser degree than mPILRa for BR3.
  • FIG. 11 is an extension of FIGS. 6-10 and illustrates the binding of anti-ragweed (control), hPILR ⁇ , and the variant hPILRa (R126A) fusion proteins to 2 different human ligands—hNPDC1 and mCD99.
  • Neo indicates cells transformed with a neomycin containing vector used as a negative control. Note that the variant hPILRa does not bind any ligands tested or to the control.
  • FIG. 12 is a table showing the various tissue and/or cell types that express each of the listed ligands.
  • Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Practitioners are particularly directed to Sambrook et al., 1989, and Ausubel F M et al., 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.
  • nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • PILR refers to paired immunoglobulin-like receptors (PILR) alpha and beta. They are related type I transmembrane receptors bearing a highly similar (83% identity) extracellular Ig-like variable (V)-type domain but divergent intracellular signaling domains. When only one of the members is being referenced, it will be designated as either PILR ⁇ or PILR ⁇ .
  • Mouse PILR-alpha refers to the protein having the amino acid sequence of SEQ ID NO: 1, which includes a potential signal sequence.
  • Human PILR-alpha refers to the protein having the amino acid sequence of SEQ ID NO: 3, which includes a potential signal sequence.
  • PILR-alpha may also be referred to as PILRa or PILR ⁇ herein.
  • Mouse PILR-beta refers to the protein having the amino acid sequence of SEQ ID NO:33
  • Human PILR-beta refers to the protein having the amino acid sequence of SEQ ID NO:35, which includes a potential signal sequence.
  • Paired immunoglobulin like type 2 receptor beta may play a role in transmembrane receptor protein tyrosine kinase signaling pathway. Increased expression of the gene encoding the protein is associated with acute lymphoblastic leukemias.
  • PILR-beta may also be referred to as PILRb or PILR ⁇ herein.
  • NPDC1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 5, which includes a potential signal sequence.
  • Neural proliferation, differentiation and control, I (NPDC1) is specifically expressed in neural cells when they stop to divide and begin to differentiate. It may also regulate transcription, cell proliferation, neuron differentiation, and organ morphogenesis. Its expression is developmentally regulated and persists in the adult; it increases in the embryonic brain, in distinct, defined regions, and is correlated with growth arrest and terminal differentiation.
  • NPDC1 has long hydrophobic stretch of amino acids (residues 13-29), a coiled-coil region (amino acids 93-120), a transmembrane domain (amino acids 191-207), an acidic domain (amino acids 277-307), and MAP-kinases consensus sites (amino acids 234-244) (Evrard and Rouget, (2005) J. Neuro. Res. 79:747-755). It may be clipped and exist in a soluble form.
  • COLEC12 refers to the protein having the amino acid sequence of SEQ ID NO: 7.
  • Collectin subfamily member 12 (COLEC12) is a type II transmembrane glycoprotein that binds bacteria through its lectin domain and may play a role in host defense.
  • ETBR refers to the protein having the amino acid sequence of SEQ ID NO: 8, which includes a potential signal sequence.
  • Endothelin receptor type B regulates angiogenesis, smooth muscle contraction, and cell migration; gene mutation correlates with melanoma, Waardenburg syndrome, and Hirschsprung disease, aberrantly expressed in asthma, atherosclerosis, and several cancers.
  • CLEC4G refers to the protein having the amino acid sequence of SEQ ID NO: 10, which includes a potential signal sequence.
  • C type lectin superfamily 4 member G (CLEC4G) is a homodimerizing protein that functions as a pathogen associated molecular pattern receptor, may play a role in cell-cell adhesion, antigen processing, and presentation. See also, Liu et al., (2004) J. Biol. Chem. 279(18):18748-58.
  • BR3 refers to the protein having the amino acid sequence of SEQ ID NO: 12.
  • BR3 is a 184-residue type III transmembrane protein expressed on the surface of B cells (Thompson, et al., (2001) Science 293:2108-2111; Yan, et al., (2001) Curr. Biol. 11:1547-1552).
  • BR3 is expressed in a variety of disease tissue including multiple myeloma and non-Hodgkin Lymphoma (Novak, A J (2004) Blood 104:2247-2253; Novak, A J (2004) Blood 103:689-694).
  • BR3 mediates NF-kappaB activation and IL10 production, elevated mRNA levels are associated with systemic lupus erythematosus and multiple sclerosis.
  • MAG as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 13, which includes a potential signal sequence.
  • Myelin associated glycoprotein (MAG, also called SIGLEC4A) may play a role in cell adhesion.
  • An increase in autoimmune antibody correlates with autism, demyelinating diseases, and polyneuropathies associated with paraproteinemias.
  • Gene polymorphism is associated with schizophrenia.
  • IL-2Ra refers to the protein having the amino acid sequence of SEQ ID NO: 15, which includes a potential signal sequence.
  • Interleukin 2 receptor alpha plays a role in regulation of T cell mediated immune response, expression is altered in several neoplasms, immune system and inflammatory diseases, Parkinson disease, asthma, and type I diabetes mellitus.
  • FceRII refers to the protein having the amino acid sequence of SEQ ID NO: 17.
  • Fc fragment of IgE low affinity II receptor acts in thymocyte maturation, histamine secretion, and TNF production, regulates NO production in monocytes, upregulated in hypogammaglobulinaemia, Kawasaki disease, Graves thyrotoxicosis, and chronic uremia.
  • LRRTM4 refers to the protein having the amino acid sequence of SEQ ID NO: 18, which includes a potential signal sequence.
  • Leucine rich repeat transmembrane neuronal 4 may stimulate beta-secretase mediated processing of beta-amyloid-precursor protein, may play a role in brain development and is associated with Alzheimer disease.
  • LRRTM4 contains nine leucine rich repeats.
  • DAG1 refers to the protein having the amino acid sequence of SEQ ID NO: 20, which includes a potential signal sequence.
  • Dystroglycan 1 or dystrophin-associated glycoprotein 1 (DAG1) is an extracellular matrix glycoprotein that acts in muscle contraction, may be involved in synaptic transmission and establishment of cell polarity, aberrant protein expression correlates with muscular dystrophies and several neoplasms.
  • APLP1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 22, which includes a potential signal sequence.
  • Amyloid beta precursor like protein 1 (APLP1) is an alpha 2A adrenergic receptor binding protein that regulates proteolysis of amyloid precursor proteins, negatively regulates endocytosis; map position of corresponding gene correlates with Alzheimer disease.
  • PTPRN refers to the protein having the amino acid sequence of SEQ ID NO: 24, which includes a potential signal sequence.
  • Protein tyrosine phosphatase receptor-type N (PTPRN) is a putative transmembrane receptor protein tyrosine phosphatase that acts as an autoantigen in type 1 diabetes mellitus.
  • WDR31 refers to the protein having the amino acid sequence of SEQ ID NO: 26, which includes a potential signal sequence.
  • WD repeat domain 31 contains five WD domain G-beta repeats, has low similarity to S. pombe Cpc2p, which is required for normal mating, sporulation, and protein translation and is a putative receptor for S. pombe Pck2p during cell wall synthesis and morphogenesis.
  • PSS8 refers to the protein having the amino acid sequence of SEQ ID NO: 27, which includes a potential signal sequence.
  • Protease serine 8 or prostasin (PSS8) is a serine protease that plays a role in regulation of the amiloride-sensitive epithelial sodium channel, overexpressed in ovarian cancer cells; corresponding gene expression is downregulated in prostate cancers.
  • SIGLEC7 refers to the protein having the amino acid sequence of SEQ ID NO: 29, which includes a potential signal sequence.
  • SIGLEC7 Sialic acid binding Ig-like lectin 7 (SIGLEC7) is a disialoganglioside-binding inhibitory receptor in natural killer cells that may act in myelopoiesis. It interacts with the lipooligosaccharide (LOS) on the surface of Campylobacter jejuni.
  • LOS lipooligosaccharide
  • IL15-RA refers to the protein having the amino acid sequence of SEQ ID NO: 31, which includes a potential signal sequence.
  • Interleukin 15 receptor alpha IL15-RA
  • IL15 interleukin-15
  • functions in signal transduction, cell proliferation, and natural killer cell survival, may be involved in the cellular defense response to various pathogens.
  • antibody herein is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, mutant, variant or derivation thereof which so long as they exhibit the desired biological activity (e.g., epitope binding activity).
  • Ig immunoglobulin
  • Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra).
  • the “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.
  • references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system (e.g., see U.S. Provisional Application No. 60/640,323, Figures for EU numbering).
  • multispecific antibody is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity.
  • Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (V H ) and a light chain variable domain (V L ), where the V H V L unit has polyepitopic specificity, antibodies having two or more V L and V H domains with each V H V L unit binding to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments that have been linked covalently or non-covalently.
  • “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). “Monospecific” refers to the ability to bind only one epitope. According to one embodiment the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 ⁇ M to 0.001 pM, 3 ⁇ M to 0.001 pM, 1 ⁇ M to 0.001 pM, 0.5 ⁇ M to 0.001 pM, or 0.1 ⁇ M to 0.001 pM.
  • PILR is a functional receptor for sialidated glycan ligands.
  • the ligand is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • PILR gene or “PILR nucleic acid molecule” or “polynucleotide” refers to a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a specific PILR polypeptide.
  • Exemplary nucleotide sequences are set forth in FIG. 1A of Fournier et al., J. Immunol. (2000) 165:1197-1209 and NM — 013439 for human PILR ⁇ ; multiple cDNAs have been identified for PILR ⁇ (Wilson et al., (2006) Physiol. Genomics 27:201-18.) and annotated by NCBI, e.g., NM — 178238.1, NM — 178238.2, for human PILRb.
  • polypeptide allelic variant refers to the polypeptide encoded by one of several possible naturally occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms.
  • polypeptide derivatives refers to a polypeptide that has been chemically modified.
  • polypeptide fragment refers to a polypeptide that comprises a truncation at the amino terminus (with or without a leader sequence) and/or a truncation at the carboxy terminus of the polypeptide whose sequence is as defined herein.
  • Polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity.
  • preferred fragments include soluble forms such as those lacking a transmembrane or membrane-binding domain.
  • truncations comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids.
  • the polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids.
  • Such polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to the polypeptides.
  • PILR fusion polypeptide refers to a fusion of one or more amino acids (such as a heterologous peptide or polypeptide) at the amino or carboxy terminus of a PILR polypeptide.
  • heterologous polypeptide is an Fc region of on IgG.
  • polypeptide ortholog refers to a polypeptide from another species that corresponds to an polypeptide as defined herein.
  • mouse and human PILRa polypeptides are considered orthologs of each other.
  • human and mouse PILRa polypeptide sequences are aligned and shown in FIG. 1C of Fournier et al., (2000) supra.
  • polypeptide variants refers to, for example, PILRa polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or PILRa polypeptide fragments), and/or additions (such as internal additions and/or PILRa fusion polypeptides) as compared to the PILR ⁇ polypeptide as defined above.
  • Variants may be naturally occurring (e.g., PILRa polypeptide allelic variants, PILRa polypeptide orthologs and PILRa polypeptide splice variants) or may be artificially constructed.
  • Such PILRa polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as defined above for the PILRa gene.
  • the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.
  • a polypeptide “variant” i.e. a variant of any polypeptide disclosed herein
  • a variant means a biologically active polypeptide having at least about 80% amino acid sequence identity with the corresponding native sequence polypeptide.
  • variants include, for instance, polypeptides wherein one or more amino acid (naturally occurring amino acid and/or a non-naturally occurring amino acid) residues are added, or deleted, at the N- and/or C-terminus of the polypeptide.
  • a variant will have at least about 80% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% or more amino acid sequence identity with the native sequence polypeptide.
  • Variants also include polypeptide fragments (e.g., subsequences, truncations, etc.), typically biologically active, of the native sequence.
  • Percent (%) amino acid sequence identity herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087, and is publicly available through Genentech, Inc., South San Francisco, Calif.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, e.g., digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • Essentially pure protein means a composition comprising at least about 90% by weight of the protein, based on total weight of the composition, preferably at least about 95% by weight, more preferably at least about 90% by weight, even more preferably at least about 95% by weight.
  • Essentially homogeneous protein means a composition comprising at least about 99% by weight of protein, based on total weight of the composition.
  • Antagonist when used herein refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a protein of the invention including its binding to one or more receptors in the case of a ligand or binding to one or more ligands in case of a receptor.
  • Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like.
  • Antagonists also include small molecule inhibitors of a protein of the invention, and fusions proteins, receptor molecules and derivatives which bind specifically to protein thereby sequestering its binding to its target, antagonist variants of the protein, antisense molecules directed to a protein of the invention, RNA aptamers, and ribozymes against a protein of the invention.
  • blocking antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of each antigen.
  • An antigen may have one or more epitopes.
  • active and “biologically active” polypeptides refer to polypeptides having at least one activity characteristic of the reference polypeptide.
  • the enzymatic activity associated with a protease is proteolysis and, thus, an active protease has proteolytic activity.
  • the biological activity of a given protein refers to any biological activity typically attributed to that protein by those of skill in the art. as defined above.
  • immunoadhesin designates antibody-like molecules that combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
  • the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence.
  • the adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand—such as a PILRa, for example.
  • the immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.
  • immunoglobulin such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.
  • the present invention relates to an agent that may block the interaction between a ligand (as defined herein) and a PILR family member. In another aspect, the present invention relates to an agent that may modulate the interaction between a ligand (as defined herein) and a PILR family member.
  • an “agent” that may block the interaction between a ligand (as defined herein) and PILRa may be a protein.
  • a protein may be an (isolated) antibody, or antigen-binding fragment (portion) thereof, that may specifically bind to a ligand (as defined herein) and/or PILRa.
  • the antibody may be, for example, a monoclonal antibody and/or a polyclonal antibody.
  • Monoclonal antibodies (MAbs) may be made by one of several procedures available to one of skill in the art, for example, by fusing antibody producing cells with immortalized cells and thereby making a hybridoma.
  • the general methodology for fusion of antibody producing B cells to an immortal cell line is well within the province of one skilled in the art.
  • Another example is the generation of MAbs from mRNA extracted from bone marrow and spleen cells of immunized animals using combinatorial antibody library technology.
  • MAbs derived from animals or from derived cell lines are that although they may be administered to a patient for diagnostic or therapeutic purposes, they are often recognized as foreign antigens by the immune system and are unsuitable for continued use.
  • Antibodies that are not recognized as foreign antigens by the human immune system have greater potential for both diagnosis and treatment. Methods for generating human and humanized antibodies are now well known in the art.
  • Polyclonal antibodies may be obtained by immunizing a selected animal with a protein or polypeptide (for example without limitation a ligand or PILRa or PILRb). Serum from the animal may be collected and treated according to known procedures. Polyclonal antibodies to the protein or polypeptide of interest may then be purified by affinity chromatography. Techniques for producing polyclonal antisera are well known in the art.
  • Antibodies may originate for example, from a mouse, rat or any other mammal.
  • the antibody may also be a human antibody which may be obtained, for example, from a transgenic non-human mammal capable of expressing human immunoglobulin genes.
  • the antibody may also be a humanized antibody which may comprise, for example, one or more complementarity determining regions of non-human origin. It may also comprise a surface residue of a human antibody and/or framework regions of a human antibody.
  • the antibody may also be a chimeric antibody which may comprise, for example, variable domains of a non-human antibody and constant domains of a human antibody.
  • Suitable antibodies may also include, for example, an antigen-binding fragment, a Fab fragment; a F(ab′)2 fragment, and Fv fragment; or a single-chain antibody comprising an antigen-binding fragment (e.g., a single chain Fv).
  • An antibody encompassed in the present invention may be an antibody binding specifically to PILRa.
  • an antibody encompassed in the present invention may be an antibody binding specifically to a ligand (as described herein).
  • Anti-PILR agents may be experimentally tested and validated using in vivo and in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays.
  • the activity of a ligand interaction with PILR may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann, et al., Proc. Natl. Acad. Sci. USA 78:2488-2492 (1981); Herrmann, et al., J. Immunol. 128:1968-1974 (1982); Handa, et al., J. Immunol.
  • T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033 (1990); and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544 (1995); Inaba et al., J. Exp. Med. 173:549-559 (1991); Macatonia, et al., J. Immunol. 154:5071-5079 (1995); Porgador, et al., J. Exp. Med. 182:255-260 (1995); Nair, et al., J.
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808 (1992); Gorczyca, et al., Leukemia 7:659-670 (1993); Gorczyca, et al., Cancer Res. 53:1945-1951 (1993); Itoh, et al., Cell 66:233-243 (1991); Zacharchuk, J. Immunol. 145:4037-4045 (1990); Zamai, et al., Cytometty 14:891-897 (1993); Gorczyca, et al., Int. J. Oncol. 1:639-648 (1992).
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica, et al., Blood 84:111-117 (1994); Fine, et al., Cell. Immunol. 155:111-122, (1994); Galy, et al., Blood 85:2770-2778 (1995); Toki, et al., Proc. Nat. Acad. Sci. USA 88:7548-7551 (1991).
  • a (protein) agent may also be a “soluble protein”.
  • Soluble proteins (purified) of the invention may be obtained from any techniques well known in the art.
  • a soluble protein may be obtained by transfecting a recombinant DNA molecule expressing solely the extracellular region of a molecule and/or portion thereof followed by purification.
  • a protein and/or a portion of a protein may be fused to a constant domain (Fc portion) of an immunoglobulin.
  • a (purified) soluble protein of the present invention may be soluble PILR and/or portion thereof.
  • portion of soluble protein for example
  • An agent of the present invention may be soluble PILRa.
  • An agent of the present invention may be portions of soluble PILR ⁇ .
  • Human PILRa (SEQ ID NO: 3) is a 303 amino acid protein. Its extracellular domain is approximately 170 amino acids in length.
  • a (purified) soluble human PILRa may have a sequence that may consist from about residue 20 to residue 207 of SEQ ID NO:3. The present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges therein whatsoever.
  • any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.
  • the term “block” or “inhibit” refers to a decrease in one or more given measurable activity by at least 10% relative to a reference and/or control. Where inhibition is desired, such inhibition is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%, i.e., complete inhibition or absence of the given activity. As used herein, the term “substantially inhibits/blocks” refers to a decrease in a given measurable activity by at least 50% relative to a reference.
  • substantially inhibits refers to a decrease in a given measurable activity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to and including 100% relative to a reference.
  • blocks/prevents/inhibits/impairs/lowers the interaction with reference to the binding of a ligand that binds to a receptor refers to a decrease in binding by at least 10% relative to a reference.
  • An agent may block the binding of a ligand to a receptor-expressing cells.
  • “Inhibits the interaction” and/or “block the binding” preferably refers to a decrease in binding of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%.
  • a “receptor” as provided for herein means PILRa or PILRb.
  • a “ligand” as provided for herein is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • a general feature of a ligand is glycan modification, e.g., sialidated glycans.
  • compositions of the invention including an agent may be manufactured in a conventional manner.
  • a pharmaceutically acceptable diluent or carrier e.g., water or a saline solution such as phosphate buffer saline.
  • a diluent or carrier is selected on the basis of the mode and route of administration, as well as standard pharmaceutical practice.
  • Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of compositions may be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • an agent of the invention may be administered in a time release formulation, for example in a composition which includes a slow release polymer.
  • the active agents may be prepared with carriers that will protect the agent against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the relevant art.
  • the present invention relates to compositions that may comprise an agent capable of modulating PILR (e.g., PILRa or PILRb) activity and a pharmacologically acceptable carrier.
  • such compositions include an agent that may block the interaction between a ligand and PILRa to treat an PILRa-related disease (for example an immune-related disease and/or inflammatory disease and/or microbial infection).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for parenteral administration.
  • the carrier may be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media is incompatible with the active agent, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.
  • administering of a composition may be performed by any suitable routes. Such routes may include parenteral, pulmonary, nasal and/or oral routes.
  • the pharmaceutical composition may be intra-muscular (IM), subcutaneous (SC), intra-dermal (ID), intra-venous (IV) and/or intra-peritoneal (IP) routes using any suitable means.
  • IM intra-muscular
  • SC subcutaneous
  • ID intra-dermal
  • IV intra-venous
  • IP intra-peritoneal
  • an effective amount is intended to mean an amount of an agent sufficient to substantially block the interaction between a ligand (as defined herein) and PILR (e.g., PILRa or PILRb).
  • An effective amount may also encompass either “therapeutically effective amount” and/or “prophylactically effective amount”.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction in disease progression and/or alleviation of the symptoms associated with a disease.
  • a therapeutically effective amount of modulators of a PILR activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing and/or inhibiting (reducing) the rate of disease onset or progression.
  • a prophylactically effective amount may be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering of the compositions.
  • inflammatory mediators may be, for example and without limitation, amines, eicosanoids, growth factors, reactive oxygen species, enzymes (for example a proteinase), chemokines, cytokines, etc.
  • Measuring the binding of a ligand (as defined herein) to PILRa may be performed using (without limitation) such suitable assays as quantitative comparisons comparing kinetic and equilibrium binding constants.
  • the kinetic association rate (k on ) and dissociation rate (k off ), and the equilibrium binding constants (k d ) may be determined using surface plasmon resonance on a BIAcoreTM instrument following the standard procedure in the literature. Binding properties of these interactions may also be assessed by flow cytometry and/or by solid phase binding assay.
  • the present invention also relates to a method of identifying a compound capable of blocking the interaction between a ligand and PILRa; the method may comprise measuring a ligand-mediated PILRa activity in the presence or absence of the agent, wherein a lower PILRa activity in the presence of the agent may be indicative that the agent is blocking the interaction between a ligand and PILRa.
  • an activity mediated by a ligand” or “a ligand-mediated PILRa activity” is an activity involving or resulting from the binding of a ligand to PILRa, and includes, but is not limited to, binding to PILRa, the induction of T cells to produce and secrete cytokines (for example IL-2, IL-10, IFN- ⁇ and TNF- ⁇ ), the synthesis of inflammatory molecules (inflammatory mediators) such as IL-6, IL-8 and metalloproteinases and T-cell proliferation (or inhibition thereof), etc.
  • cytokines for example IL-2, IL-10, IFN- ⁇ and TNF- ⁇
  • inflammatory mediators such as IL-6, IL-8 and metalloproteinases and T-cell proliferation (or inhibition thereof
  • the ligand-mediated activity may depend on the specific ligand, e.g., NPCD1, BR3, etc., being evaluated.
  • the use may be for the treatment or prevention of inflammatory-related diseases or condition, or a microbial infection in the subject.
  • RNA adult tissue panel was purchased from Zyagen (San Diego, Calif.). Recombinant human cytokines were from Peprotech (Rocky Hill, N.J.).
  • anti-mouse antibodies were used for flow cytometry: anti-mPILRa, anti-mCD99 and anti-mCOLEC12 are all from R&D Systems (Minneapolis, Minn.), anti-FLAG (M2) monoclonal antibody was purchased from Sigma-Aldrich and labeled with Alexa Flour-647 monoclonal antibody labeling kit from Invitrogen, and anti-mouse IgG2a-FITC was from BD Biosciences (San Jose, Calif.).
  • This example illustrates preparation of potentially glycosylated forms of the desired ligands or PILR proteins (either of which is referred to in this example as a desired protein) by recombinant expression in mammalian cells. Preparations of PILR antibodies are also described.
  • the vector, pRK5 (see EP 307,247, published Mar. 15, 1989), was employed as the expression vector in all instances.
  • DNA encoding the desired protein was ligated into pRK5 with selected restriction enzymes to allow insertion of such DNA using ligation methods such as described in Sambrook et al., supra.
  • Epitope-tagged variants of the desired protein may also be expressed in cells.
  • the DNA encoding the desired protein was ligated into pRK5 containing the desired epitope tag (poly-His, FLAG, human IgG 1 Fc) in frame with the desired epitope tag.
  • the predicted extracellular domains of human and mouse PILR were cloned without the transmembrane domain into the pRK5 vector containing a C-terminal human IgG 1 -Fc or 8 ⁇ -His tag, or with the transmembrane domain into the pRK5 vector containing a C-terminal GFP tag.
  • Soluble forms of these proteins were produced in a CHO cell transient transfection and purified by affinity chromatography using anti-FLAG (M2) agarose affinity gel (Sigma-Aldrich) for FLAG-tagged proteins, Ni-NTA agarose (Qiagen) for 8 ⁇ -His-tagged proteins, or protein-A Sepharose (Amersham Pharmacia) for IgG 1 -Fc fusion proteins. Proteins were further separated from aggregates and contaminants with a Superdex 200 gel-filtration column and/or MonoQ/S ion exchange columns (Amersham). Protein purity was assessed by SDS-PAGE followed by SimplyBlue Safe Stain (Invitrogen) and purified proteins were aliqouted and frozen at ⁇ 80° C. until needed.
  • the selected host cells may be HEK293T cells.
  • Human 293 cells (ATCC CCL 1573) were grown to 50-80% confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. 1-10 ⁇ g of DNA encoding the desired protein ligated into pRK5 was introduced into HEK293T cells using commercially available transfection reagents Superfect® (Qiagen), Lipofectamine® (Invitrogen) or Fugene® (Roche) according to manufacturer's instructions. 18-24 hours after the transfections, the culture medium was removed and tested in selected bioassays or cells were harvested using 10 mM EDTA in 20 mM Na phosphate buffer, pH7.4, and tested in selected bioassays.
  • Stable expression of the desired protein was achieved in HEK293T cells by cloning DNA encoding the desired protein into pRK5 vector with a selection marker that confers resistance to the antibiotic Geneticin®.
  • pRK5 vector with a selection marker that confers resistance to the antibiotic Geneticin®.
  • desired proteins cells were transfected as described and allowed to grow in DMEM with a concentration of Geneticin® that would permit growth of cells in which the desired vector had integrated into the genome (1-0.5 ⁇ g/ml).
  • the epitope tagged versions of the desired protein can be expressed in host CHO cells. Twelve micrograms of the desired plasmid DNA was introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Qiagen), Dosper®, Lipofectamine® (Invitrogen) or Fugene® (Boehringer Mannheim) according to manufacturer's instructions. The cells are grown as described in Lucas et al. ( Nucl. Acids Res. (1996) 24:9 1774-1779). Approximately 3 ⁇ 10 ⁇ 7 cells are frozen in an ampule for further growth and production as described below.
  • the ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing.
  • the contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes.
  • the supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 ⁇ m filtered PS20 with 5% 0.2 ⁇ m diafiltered fetal bovine serum).
  • the cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C.
  • spinners After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3 ⁇ 10 5 cells/mL.
  • the cell media was exchanged with fresh media by centrifugation and resuspension in production medium.
  • any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used.
  • a 3 L production spinner was seeded at 1.2 ⁇ 10 6 cells/mL. On day 0, the cell number and pH was determined. On day 1, the spinner was sampled and sparging with filtered air was commenced.
  • the spinner was sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH was adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture was harvested by centrifugation and filtering through a 0.22 ⁇ m filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.
  • 10% antifoam e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion
  • the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated at 4° C., in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • Ni-NTA column Qiagen
  • the purified protein was then run over a Superdex S200 gel filtration column and/or a MonoQ/S ion exchange column (Applied Biosystems) to remove aggregated or proteolysed protein or any contaminants and subsequently concentrated and dialyzed into PBS.
  • the homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. Proteins were stored at ⁇ 80° C. until used in bioassays.
  • the proteins are purified using an anti-FLAG (M2) agarose column (Sigma).
  • the conditioned media was pumped onto a 6 ml anti-FLAG column equilibrated at 4° C. with 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ L of 1 M Tris buffer, pH 9. The highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.
  • Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows.
  • the conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ L of 1 M Tris buffer, pH 9.
  • the highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.
  • mice 4 to 6-week-old Armenian hamsters (Cytogen) or mice were immunized with 2 ⁇ g/injection each murine and human recombinant PILR ⁇ proteins.
  • the immunogens are resuspended in monophosphoryl lipid A/trehalose dicorynomycolate adjuvant and injected via footpad or I.P. at 3 to 4 day intervals for a total of 10 boosts.
  • lymphocytes from immunized hamster spleens and lymph nodes were harvested for fusion with SP2/0 myeloma cells (American Type Culture Collection) by using the Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences).
  • Cytofusion Medium C (Cyto Pulse Sciences)
  • the isolated lymphocytes and SP2/0 cells were mixed at a 1:1 ratio and then resuspended at 10 million cells/ml in Cytofusion Medium C
  • electrofusion was performed according to manufacturer's guidance.
  • Fused cells were cultured in ClonaCell-HY Medium C (StemCell Technologies) overnight at 37° C. in a 7% CO 2 incubator.
  • fused cells were centrifuged and resuspended in 10 ml ClonaCell-HY Medium C and then gently mixed with 90 ml Methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies) containing HAT components.
  • the fused cells were plated into 100 mm Petri dishes (Becton Dickinson) and allowed to grow in 37° C. in a 7% CO 2 incubator. After 7-10 days incubation, the single hybridoma clones were picked by ClonePix (Genetix, United Kingdom) and transferred into 96-well cell culture plates (Becton Dickinson) with 200 ⁇ L/well ClonaCell-HY Medium E (StemCell Technologies). Hybridoma culture media was changed prior to ELISA screening. All ELISA positive clones were further screened by FACS. After at least 2 rounds of single cell subcloning by limiting dilution, final clones were scaled up and the supernatants were collected for antibody purification.
  • the hybridoma supernatants were purified by Protein A affinity chromatography, then sterile filtered (0.2 ⁇ m pore size, Nalgene Nunc International, NY, USA) and stored at 4° C. in PBS.
  • the purified mAbs were confirmed by ELISA and FACS before testing on functional assays.
  • the isotypes of purified mAbs were determined by the mouse monoclonal antibody isotyping kit from Roche Diagnostics Corporation.
  • the isotypes of purified hamster mAbs were determined by ELISA.
  • This example uses one PILRa ligand, mCD99, to illustrate the importance of sialidation on the ligand for PILRa binding.
  • Mouse PILR ⁇ binds to mCD99 with relatively low affinity, however, it is unclear whether PILR ⁇ can bind human CD99. Shiratori et al. (2004) J Exp Med 199, 525-533; Tabata et al. (2008) J Biol Chem 283, 8893-8901. To test this, we expressed mouse or human CD99 in 293T cells to see whether they can bind to PILR ⁇ -mIgG2a (PILR ⁇ -Fc) fusion proteins. We found both mouse and human PILR ⁇ -Fc fusions bound mCD99 transfectants.
  • PILR ⁇ -Fc PILR ⁇ -Fc
  • the differential binding of mouse and human CD99 to PILR ⁇ may be the result of the low sequence identity between CD99 homologs (about 41% in the extracellular domain) or the differences in glycosylation (type and/or a number of glycans). It has been shown that sialylated O-linked glycans in mCD99 play an essential role in PILR ⁇ binding to mCD99. Wang et al. (2008) J Immunol 180, 1686-1693. hCD99 has been reported to be O-glycosylated, and similarly to mCD99, does not have any potential N-glycosylation sites. Gelin et al. (1989) EMBO J 8, 3253-3259.
  • O-glycans The composition of observed O-glycans is consistent with the presence of sialylated Gal ⁇ 1-3GalNAc core 1 structures. North et al. (2010) J Biol Chem 285, 5759-5775; Olson et al. (2005) Glycobiology 15, 177-191.
  • Each glycopeptide was glycosylated with a single NeuAc 1-2 Hex 1 HexNAc 1 O-glycan.
  • the presence of two sialylated glycopeptides was consistent with the LC-MS analysis of reduced hCD99-Fc.
  • the detected sialoglycopeptide APDGGFLDL S DALPDNENKKP T 41 AIPK contained Thr-41 corresponding to Thr-45 O-glycosylation site of mCD99 and a Ser residue which potentially could be O-glycosylated. However, the exact site of O-glycosylation could not have been sequenced through the performed type of LC-MS experiment.
  • This example demonstrates the identification of novel ligands for PILRa.
  • PILR ⁇ ligands CD99 is expressed on T-cells, B-cells, NK cells, monocytes and neutrophils.
  • hPILRa is expressed predominately in cells of the myelomonocytic lineage, including monocytes/macrophages, granulocytes, and dendritic cells (DC).
  • Discordant PILRa-Fc binding and CD99 expression as well as a low affinity interaction with CD99 (2.2 mM) suggested that there may be additional ligands for PILRa.
  • PILRa Alkaline phosphatase
  • hPILR ⁇ (Met1-196Thr) and mPILR ⁇ (Met1-197Val) were cloned into the expression vector pRK5 as fusions to C-terminal alkaline phosphatase (AP) tag.
  • 293T cells were transfected with PILR ⁇ -AP-pRK5 constructs and Fugene 6 Transfection Reagent (Roche) according to the manufacturer's instructions. Three days after transfection, supernatants were collected for screening.
  • COST cells were plated in a 24-well format and transfected with 10 ul of Origene library DNA (Origene, human DNA library containing 20 k genes) per well with Fugene 6 Reagent.
  • the resulting positive clones were identified and further analyzed using FACS.
  • the ligands that bound hPILRa are NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA. See FIGS. 6-11 .
  • the expression of these newly identified PILRa ligands on various cell types are summarized in FIG. 12 .
  • NPDC1 is a type I transmembrane protein and has been identified as a neural-specific gene involved in the control of cell proliferation and differentiation. Galiana et al. (1995) Proc Natl Acad Sci USA 92, 1560-1564.
  • COLEC12 is a type II transmembrane collectin family member as also known as collectin placenta 1 (CL-P1) and Scavenger Receptor with C-type Lectin (SRCL) type I.
  • CL-P1 collectin placenta 1
  • SRCL Scavenger Receptor with C-type Lectin
  • N-terminal flag-tagged hNPDC1 or C-terminal his tagged hCOLEC12 were expressed in 293T cells, and then tested for mPILR ⁇ -Fc and hPILR ⁇ -Fc binding. Mock transfected cells were used as a negative control and mCD99 transfected cells were used as positive control. Both mPILR ⁇ -Fc and hPILR ⁇ -Fc bound to mCD99, hNPDC1 and hCOLEC12 expressing cells ( FIG. 2B ). Similar results were also obtained by hNPDC1-Fc or hCOLEC12-his in PILR ⁇ transfectants by FACS analysis.
  • Neutral glycans were mostly of the high-mannose type (predominantly Man-5) and complex afucosylated or core-fucosylated bi-, tri- and tetra-antennary glycans, galactosylated to various degrees. Negatively charged glycans were sialylated (0-4 NANA), complex type bi-, tri- and tetra-antennary N-glycans, core-fucosylated and mostly fully galactosylated.
  • NPDC1 has been identified as a neuronal-specific gene involved in the control of cell proliferation and differentiation. Galiana et al. (1995) Proc Natl Acad Sci USA 92, 1560-1564. Our results suggest that PILR ⁇ recognizes several ligands, some of which are not conserved between mouse and human. The identification of multiple PILR ⁇ ligands in the neuronal system (PANP and NPDC1) suggests that PILR ⁇ might play a role in the CNS.
  • COLEC12 is the only member of the Collectin scavenger receptor family that is expressed as a cell surface transmembrane protein and its ECD contains coiled-coil, collagen-like, and C-type lectin/carbohydrate domains.
  • COLEC12 is expressed in vascular endothelia cells and monocytes to mediate the uptake of oxidized low density lipoprotein and microbes.
  • PILR ⁇ is emerging as a receptor that recognizes a specific group of ligands in cellular or pathogenic sources with unique sialic acid pattern.
  • human and mouse PILR ⁇ -Fc fusions bind to mouse thymocytes, peripheral CD8+ T and activated CD4+ T, B, NK, NKT cells, granulocytes and monocytes, as well as a majority of human PBMC.
  • This example demonstrates a conserved domain on PILRa critical for its binding to sialylated ligands.
  • Sialylated glycan modifications appear to be a general feature of all identified PILR ⁇ ligands and coincidentally few other receptors such as Siglecs.
  • Kogure et al. (2011) Biochem Biophys Res Commun 405, 428-433; Wang et al. (2008); Crocker et al. (2007) Nat Rev Immunol 7, 255-266; Wang et al. (2009).
  • a conserved Arg site in the ECD of Siglecs plays a critical role in their binding to sialic acid. May et al. (1998) Mol Cell 1, 719-728; Ikehara et al. (2004) J Biol Chem 279, 43117-43125; Vinson et al.
  • the PILR ⁇ ECD has two, similar Arg sites (human Arg 96, mouse arginine 103; human R126, mouse R133) that are highly conserved across species ( FIG. 5 A).
  • the second Arg site corresponds to the critical Arg site that is required for the binding of Siglecs to sialic acid in the ligands. May et al. (1998) Mol Cell 1, 719-728.
  • the hPILR ⁇ R126 is not an Ig fold stabilizing residue ( FIG. 5A ). We examined whether mutation of this Arg site in mouse or human PILR ⁇ affects their binding to ligands.
  • ligand fusion proteins including mCD99-Fc, hNPDC1-Fc and hCOLEC12-his to cell surface-expressed WT or Arg mutated hPILR ⁇ R126A and mPILR ⁇ R131A.
  • the Arg mutation did not affect cell surface expression of human and mouse PILR ⁇ .
  • PILR ⁇ -ligand interactions require a conserved Arg motif on PILR ⁇ and specific sialylated decorations on the ligands. Since sialylation occur on many cell surface proteins, we predict PILR ⁇ ligands should be broadly expressed in various cells and hPILR ⁇ R126A/mPILR ⁇ 133A should not bind to the natural ligands expressed on the surface of primary cells. To test this, we stained mouse and human hematopoietic cells with human or mouse PILR ⁇ -Fc. We found that both human and mouse PILR ⁇ -Fc bound to the majority of human PBMC ( FIG. 5F-1 ). PILR ⁇ -Fc highly bound to human T cells and monocyte subsets ( FIG.
  • the PILR ⁇ Sequence was aligned with the N terminal of the mouse Sialoadhesion from 1QFO.PDB using MOE2010.10 Protein Align application. Blosum62 was used as the alignment matrix, with tree-based build-up, Gap Start penalty of 7 and Gap Extend penalty of 1, Iteration limit of 100 and Failure limit of 10.
  • the aligned structures were used to build a homology model using the Homology Model application in MOE2010.10.
  • the crystal structure of the first chain in the SIGLEC 1 was used as the template, while the ligand atoms were used as the ‘environment’ during model building. May et al. (1998) Mol Cell 1, 719-728; Munday and Crocker (1999) J Leukoc Biol 66, 705-711.
  • the C, N-terminal outgaps were not built. A total of 25 models were built with fine minimization, and the final model was put through the Protonate3D procedure to detect the correct protonation states and was finely minimized at the end.
  • the Merck force field (MMFF94x) with Born solvation method was used to reproduce the small molecule interactions of the active site.
  • the active site-ligand interactions of SIGLEC1 and those of the PILR ⁇ model were rendered using the Ligand Interaction Diagram application of MOE2010.10. Clark and Labute (2007) J Chem Inf Model 47, 1933-1944.
  • the active site-ligand contacts are coded according to Table 1.
  • SIGLEC1.SER103 and PILR ⁇ .THR131 both accept a hydrogen bond via their backbone carboxylate oxygens from the ligand Sialic acid O4 (contact C05).
  • the peptidic backbone of SIGLEC1.LEU107 and the corresponding PILR ⁇ .GLN140 have identical contacts with the ligand; their amino group contacts the ligand sialic acid O8 (coded as C11) and their carboxylate oxygen can accept a hydrogen bond from the ligand Sialic acid 09 (coded as C 12).
  • the side chain amide group of PILR ⁇ .GLN140 is able to contact ligand galactose O6, which in SIGLEC1, the non-polar side chain of LEU108 has a non-favorable interaction with the ligand. This contact is coded as C 13.
  • PILR ⁇ active site is very similar to the SIGLEC1, with at least two additional hydrogen bonding contacts (Arg132, Gln140). These residues are exposed to solvent, even in the complex, suggesting possible contacts with the protein domains of other ligands.
  • Arg97 in SIGLEC1 contacts the carboxylate group of sialic acid and two tryptophans that interact with the N-acetyl and glycerol moieties of N-acetyl neuraminic acid. May et al. (1998) Mol Cell 1, 719-728; Munday et al. (1999) J Leukoc Biol 66, 705-711.
  • Our functional data with arginine-mutated PILR ⁇ is consistent with a model in which Siglecs and PILR ⁇ have some similarity in their ligand interaction domains.
  • arginine 126 as a key contact residue in hPILR ⁇ (Arg 133 in mPILR ⁇ ) and its location appears to be critical to mediate PILR ⁇ interaction with sialic acid on gB as well as other ligands. Further support of this model is provided by recent data that the Tryptophan-139 residue is also critical for human PILR ⁇ binding to HSV1 gB. Fan et al. (2010) J Virol 84, 8664-8672.
  • the homology model of the structure of PILR ⁇ identifies active site residues that are almost identical between the Siglecs and PILR ⁇ .
  • high similarities among the active site-ligand contacts between PILR ⁇ and SIGLEC1 allows us to confide in the homology model, and provides us with an expanded hypothesis for ligand binding of PILR ⁇ which can further analyzed via additional mutation studies.
  • Structural resolution of PILR ⁇ in the context of sialylated proteins will be necessary to define the interaction domains.
  • SIGLEC1 and multiple CD33-related Siglecs can interact with sialic acids on pathogens such as N. meningitidis, C.

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