WO2018036947A1 - Methods of inhibiting biological responses induced by a receptor that uses pi3k to signal in a cell - Google Patents

Methods of inhibiting biological responses induced by a receptor that uses pi3k to signal in a cell Download PDF

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WO2018036947A1
WO2018036947A1 PCT/EP2017/070997 EP2017070997W WO2018036947A1 WO 2018036947 A1 WO2018036947 A1 WO 2018036947A1 EP 2017070997 W EP2017070997 W EP 2017070997W WO 2018036947 A1 WO2018036947 A1 WO 2018036947A1
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receptor
receptors
cell
fcyriib
inhibition
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PCT/EP2017/070997
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French (fr)
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Marc Daeron
Odile Malbec
Lydie Cassard
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université D'aix Marseille
Centre National De La Recherche Scientifique (Cnrs)
Institut Pasteur
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Publication of WO2018036947A1 publication Critical patent/WO2018036947A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [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
    • C07K16/283Immunoglobulins [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 against Fc-receptors, e.g. CD16, CD32, CD64
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/08Antiallergic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the present invention relates to methods of inhibiting biological responses induced by a receptor that uses PI3K to signal in a cell.
  • FcERI High-affinity IgE receptors
  • FcERI aggregation occurs when a plurivalent allergen binds to receptor-bound allergen-specific IgE antibodies.
  • FcERI oligomerization is sufficient to activate basophils and mast cells in vitro, as demonstrated using bivalent haptens (1), IgE dimers (2), or anti-receptor antibodies (3).
  • Mast cells and basophils of an individual carry, each, many receptor-bound polyclonal IgE directed against the various epitopes of the different proteins of an allergen.
  • allergic patients who are polysensitized have IgE antibodies against more than one allergen.
  • FcyRIIB inhibit IgE-induced mast cell and basophil activation. Inhibition requires that FcyRIIB be co-aggregated with FCERI (9). Co-aggregation enables the FcERI-associated Src kinase Lyn to phosphorylate both Immunoreceptor Tyrosine- based Activation Motifs (ITAMs) in FCERI, and the Immunoreceptor Tyrosine-based Inhibition Motif (ITIM) in FcyRIIB (12).
  • ITAMs Immunoreceptor Tyrosine-based Activation Motifs
  • ITIM Immunoreceptor Tyrosine-based Inhibition Motif
  • FcyRIIB recruit the Src-homology 2 (SH2) domain-containing inositol 5-phosphatase SHIP1 (13-15) into FCERI signalosomes where it hydrolyzes phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (15), generated by phosphoinositide 3-kinase (PI3K), thereby preventing the recruitment of Pleckstrin Homology (PH) domain-containing molecules that are critical for cell activation (16,17).
  • SH2 Src-homology 2
  • SHIP1 Src-homology 2
  • PI(3,4,5)P 3 phosphatidylinositol 3,4,5-trisphosphate
  • PI3K phosphoinositide 3-kinase
  • the present invention relates to methods of inhibiting biological responses induced by a receptor that uses PI3K to signal.
  • the present invention is defined by the claims.
  • Trans-inhibition affected high-affinity IgE receptor (FCERI)- dependent mouse mast cell, mouse basophil and human basophil activation, as well as growth factor receptor (Kit)-dependent normal mouse mast cell proliferation, and even the constitutive in vitro proliferation and the in vivo growth of oncogene (v-Abl)-transformed mastocytoma cells.
  • Trans- inhibition was induced by receptors that recruit SHIPl, whether inhibitory such as FcyRIIB, or activating such as FcERI. It was indeed due to the lipid phosphatase SHIPl which, by hydrolyzing PI(3,4,5)P 3 , induced a global unresponsiveness that affected biological responses triggered by receptors that use phosphoinositide 3-kinase (PI3K) to signal. Trans-inhibition can therefore control numerous processes, in physiology and pathology. It provides mechanistic grounds for unanticipated new therapeutic approaches, especially of allergic diseases.
  • the first aspect of the present invention relates to a method of inhibiting biological responses induced by a receptor that uses PI3K to signal in a cell that coexpresses receptors for the Fc portion of antibodies (FcRs) that recruit the lipid phosphatase SHIPl and/or SHIP2.
  • FcRs Fc portion of antibodies
  • the receptors for the Fc portion of antibodies may be the inhibitory low-affinity IgG receptors FcyRIIB.
  • the term "receptor for the Fc portion of antibodies” or "FcR” denote a protein found on the surface of certain cells - including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells - that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc (Fragment, crystallizable) region. Fc receptors bind to antibodies that are attached to infected cells or invading pathogens.
  • Fc receptors Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity.
  • Some viruses such as flaviviruses use Fc receptors to help them infect cells, by a mechanism known as antibody-dependent enhancement of infection.
  • Fc receptors There are several different types of Fc receptors, which are classified based on the type of antibody that they recognize. For example, those that bind the most common class of antibody, IgG, are called Fc-gamma receptors (FcyR), those that bind IgA are called Fc-alpha receptors (FcaR) and those that bind IgE are called Fc-epsilon receptors (FcsR).
  • the receptors for the Fc portion of antibodies may be a Fc-gamma receptor like FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD 16), FcyRIIIB (CD 16b), a Fc-alpha receptor like FcaRI (CD89) or Fc-epsilon receptors like FcsRI or FcsRII (CD23).
  • Fc-gamma receptor like FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD 16), FcyRIIIB (CD 16b), a Fc-alpha receptor like FcaRI (CD89) or Fc-epsilon receptors like FcsRI or FcsRII (CD23).
  • FcyRIIB has its general meaning in the art and refers to receptors for the Fc portion of IgG encoded by FCGR2B gene (Gene ID: 2113).
  • the term is also known as CD32; FCG2; CD32B; FCGR2; IGFR2.
  • the receptor has a low affinity for monomeric IgG but it binds IgG immune complexes with a high avidity.
  • the encoded protein is involved in the endocytosie of soluble immune complexes, the phagocytosis of particulate immune complexes, the regulation of cell activation induced by ITAM-containing receptors (an example of which is the regulation of antibody production initiated by B cells), the regulation of cell proliferation induced by Receptor Tyrosine Kinase Receptors.
  • An exemplary human amino acid sequence is represented by the NCBI reference sequence NP 001002273.1, NP 001002274.1, NP_001002275.1, NP_001177757.1 or NP 003992.3.
  • PI3K has its general meaning in the art and refers to a phosphoinositide 3-kinase or variant thereof, which is capable of phosphorylating the inositol ring of PI in the D-3 position.
  • PI3K variant is intended to include proteins substantially homologous to a native PI3K, i.e., proteins having one or more naturally or non-naturally occurring amino acid deletions, insertions, or substitutions ⁇ e.g., PI3K derivatives, homologs, and fragments), as compared to the amino acid sequence of a native PI3K.
  • PI3K examples include, but are not limited to pl l0-a, ⁇ 110- ⁇ , ⁇ 110- ⁇ , ⁇ 110- ⁇ , PI3K-C2a, PI3K-C2P, PI3K-C2y and Vps34.
  • PI3Ks are classified into at least four classes. Class I includes pi 10-a, pi 10- ⁇ , pi 10- ⁇ , and pi 10- ⁇ . Class I PI3Ks are responsible for the production of phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), and phosphatidylinositol (3,4,5)- trisphosphate (PI(3,4,5)P3).
  • PI(3)P phosphatidylinositol 3-phosphate
  • PI(3,4)P2 phosphatidylinositol
  • PI(3,4,5)P3 phosphatidylinositol 3-phosphat
  • the PI3K is activated by G protein-coupled receptors and tyrosine kinase receptors.
  • Class II includes PI3K-C2a, PI3K-C2p, and PBK-C2y.
  • Class III includes Vps34.
  • Class IV includes mTOR, ATM, ATR, and DNA-PK.
  • the PI3K is a Class I kinase.
  • the PI3K is selected among pi 10-a, pi 10- ⁇ , pi 10- ⁇ , and pi 10- ⁇ .
  • the expression "receptor that uses PI3K to signal" refers to any receptor that activate a PI3K and in particular a class I PI3K.
  • the receptor that uses PI3k to signal is a G protein-coupled receptor.
  • G protein-coupled receptor or “GPCR” has its general meaning in the art and refers to a large protein family of receptors, that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. The term is also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptor, and G protein-linked receptors (GPLR).
  • GPCRs can be grouped into 6 classes based on sequence homology and functional similarity: Class A (or 1) (Rhodopsin-like), Class B (or 2) (Secretin receptor family), Class C (or 3) (Metabotropic glutamate/pheromone), Class D (or 4) (Fungal mating pheromone receptors), Class E (or 5) (Cyclic AMP receptors) and Class F (or 6) (Frizzled/Smoothened).
  • GPCRs include but are not limited to Chemokine (C-C motif) receptor 1 (CCRl, CKRl); Chemokine (C-C motif) receptor 2 (CCR2, CKR2); Chemokine (C-C motif) receptor 3 (CCR3, CKR3); Chemokine (C-C motif) receptor 4 (CCR4, CKR4); Chemokine (C-C motif) receptor 5 (CCR5, CKR5); Chemokine (C-C motif) receptor 8 (CCR8, CKR8); Chemokine (C-C motif) receptor-like 2 (CCRL2, CKRX); chemokine (C motif) receptor 1 (XCR1, CXC1) InterPro: IPR005393; chemokine (C-X3-C motif) receptor 1 (CX3CR1, C3X1) InterPro: IPR005387; GPR137B (GPR137B, TM7SF1); Chemokine receptor InterPro: IPR000355; Chemokine
  • the receptor that uses PI3K to signal is a Receptor Tyrosine Kinase
  • RTK refers to a transmembrane receptor that contains a tyrosine kinase in its intracytoplasmic domain n.
  • the RTKs have been divided into a number of classes as follows: RTK class I (EGF receptor family); II (insulin receptor family); III (PDGR receptor family); IV (FGF receptor family); V (VEGF receptor family); VI (HGF receptor family); VII (Trk receptor family); VIII (AXL receptor family); IX (AXL receptor family); X (LTK receptor family); XI (TIE receptor family); XII (ROR receptor family); XIII (DDR receptor family); XV (KLG receptor family); XVI (RYK receptor family); arid XVII (MuSK receptor family).
  • RTKs that depend upon cytosolic receptors include integrins, interferon receptors, interleukin receptors, GP130 associated proteins, etc.
  • RTKs include but are not limited to EPOR, GHR, CFSR, PRLR, MPL; IFN Family: IFNAR1 , 2, IFNGR1 ,2; yC Family: IL2RA, B, G, IL4R, IL2RG (Type 1 receptor), IL4R-IL13RAl(Type II receptor), IL7R, IL2RG, IL9R, IL15RA, IL2RB, IL10RA, B, IL12RB 1 , 2, IL13RA1; IL3 Family: IL3RA, CSF2RA, B, IL5RA, GP130 Family: IL6R, IL6ST, IL1 1RA, LIFR, OSMR, IL6GT, CNTFR, IL6ST, and LIFR.
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • FGFR fibroblast growth factor receptor
  • HGFR hepatocyte growth factor receptor
  • NGFR nerve growth factor receptor
  • the RTK is a member of the EGFR family such as EGFR or erbB-1 , erbB-2, erbB-3, or erbB-4.
  • the RTK is EGFR, which is a 170 kDa membrane-spanning glycoprotein that binds to, for example, EGF, TNF-a, amphiregulin, heparin-binding EGF (HB-EGF), betacellulin, epiregulin, and NRG2-a.
  • the RTK is HER2, a proto-oncogene that encodes a transmembrane receptor protein of 185 kDa.
  • the RTK may also be a member of the VEGF receptor (VEGFR) family, which includes VEGFR-1, VEGFR-2, VEGFR-3, neuropilin-1 and neuropilin-2.
  • VEGFR VEGF receptor
  • Ligands that bind to VEGFR-1 and VEGFR- 2 include isoforms of VEGF (VEGF121, VEGF 145, VEGF 165, VEGF 189 and VEGF206).
  • the RTK is a member of t the type III family of receptor tyrosine kinase.
  • type III family of receptor tyrosine kinases or "type III RTKs” is intended to include receptor tyrosine kinases which typically contain five immunoglobulin like domains, or Ig-like domains, in their ectodomains.
  • type III RTKs include, but are not limited to PDGF receptors, the M-CSF receptor, the FGF receptor, the Flt3-receptor (also known as Flk2) and the KIT receptor.
  • the type III RTK is KIT (also known in the art as the SCF receptor).
  • KIT like other type III RTKs is composed of a glycosylated extracellular ligand binding domain (ectodomain) that is connected to a cytoplasmic region by means of a single transmembrane (TM) domain (reviewed in Schlessinger (2000) Cell 103: 211- 225).
  • KIT type III RTKs
  • PTK cytoplasmic protein tyrosine kinase
  • At least two splice isoforms of the KIT receptor are known to exist, the shorter making use of an in- frame splice site. All isoforms of KIT, and the other above described RTKs, are encompassed by the present invention.
  • KIT KIT and KIT receptor
  • RTK transmembrane receptor tryosine kinase
  • SCF Stem Cell Factor
  • KIT is composed of an extracellular domain that includes five Ig-like domains (designated D1-D5), a single transmembrane domain, a juxtamembrane region , a tyrosine kinase domain split by a kinase insert and a C-terminal tail.
  • the KIT is human KIT.
  • KIT is also intended to include recombinant human KIT (rhKIT), which can be prepared by standard recombinant expression methods.
  • the receptor that uses PI3K to signal is an immunoreceptor.
  • Immunoreceptors comprise, among others, B Cell receptors (BCR), T Cell Receptors (TCR), Fc Receptors (FcR).
  • the cell is a B cell.
  • B cell has its general meaning in the art. B cells are lymphocytes that play a large role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by T cells).
  • the cell is a mast cell or a basophil.
  • the term “mast cell” refers to a granulocyte that contains granules with histamine and heparin. In some embodiments, the term “mast cell” refers to a mastocyte. In some embodiments, the term “mast cell” refers to a basophil. In some embodiments, the term “mast cell” refers to an inactivated mast cell.
  • the term “mast cell” refers to an activated mast cell. In some embodiments, the term “mast cell” refers to a mast cell residing in the bone marrow, in the systemic circulatory system, and/or in organ tissues. In some embodiments, the organ tissue is the lung, the skin, the heart, the brain, the eye, the gastrointestinal tract, the thymus, the spleen, the ear, the nose or combinations thereof.
  • the term “basophil” refers to a basophil granulocyte. In some embodiments, the term “basophil” refers to a human basophil progenitor. In some embodiments, the term “basophil” refers to a basophil lineage-committed progenitor.
  • the term “basophil” refers to a human common myeloid progenitor (hCMP). In some embodiments, the term “basophil” refers to any combination of a basophil granulocyte, a human basophil progenitor, a basophil lineage-committed progenitor, and a human common myeloid progenitor (hCMP). In some embodiments, the term “basophil” refers to a basophil residing in the bone marrow, in the systemic circulatory system, and/or in organ tissues. In some embodiments, the organ tissue is the lung, the skin, the heart, the brain, the eye, the gastrointestinal tract, the thymus, the spleen, the ear, the nose or combinations thereof.
  • the cell is a malignant cell.
  • the malignant cell expresses FcRs. These cells are primarily, but not exclusively, cells of the myeloid lineage. Malignant cells can also be a transformed B cell. Non-hematopietic cells can also express FcRs. One example is malignant melanoma cells that express FcRIIB.
  • trans-inhibition is a consequence of SHIPl/2-mediated cis-inhibition.
  • SHIP 1/2 is recruited by receptors that are phosphorylated by a Src family tyrosine kinase and that SHIP 1/2 inhibits signals generated by receptors that use PI3K, by hydro lyzing PIP3.
  • SHIP 1/2 can therefore inhibit PIP3 -dependent activation/proliferation signals that depend on all receptors which use PI3K and which are co-expressed with receptors capable of recruiting SHIP 1/2.
  • Trans-inhibition therefore comprises two phases:
  • SHIP 1/2 can be recruited either: • by activating receptors themselves that use it as an auto-regulation mechanism.
  • These can be ITAM-containing immunoreceptors such as FcRs, BCRs, TCRs, activating NK receptors, etc., RTKs including most growth factor receptors such as KIT, Insulin receptors, EGFR, VEGFR, hormone receptor etc...
  • FcyRIIB Prototypic SHIPl/2-recruiting inhibitory receptors are FcyRIIB.
  • FcyRIIB need to be co- aggregated with activating receptors that use a Src kinase that phosphorylates the FcyRIIB ITIM.
  • FcyRIIB can therefore recruit SHIP 1/2 when co-engaged with a variety of activating receptors.
  • trans-inhibition can be induced by all ligands that co-engage activating receptor or inhibitory receptor like FcyRIIB with activating receptors that use a Src kinase to signal.
  • Such a co-engagement can be induced by bi-specific ligands that can bind to FcyRIIB and to activating receptors.
  • FcyRIIB are low-affinity IgG receptors, they can be engaged very efficiently by the Fc portion of antibodies that recognize activating receptors expressed on the same membrane. Antibodies are therefore appropriate tools to induce trans-inhibition.
  • the invention relates to a molecule which is capable of co-engaging a FcR like the FcyRIIB with an activating receptor that use a Src kinase to signal.
  • an activating receptor that use a Src kinase to signal may be an immunoreceptor such as activating FcRs, BCRs, TCRs, RTKs including most growth factor receptors such as KIT, Insulin receptors, - EGFR or VEGFR.
  • an immunoreceptor such as activating FcRs, BCRs, TCRs, RTKs including most growth factor receptors such as KIT, Insulin receptors, - EGFR or VEGFR.
  • activating FcRs may be FcyRI (CD64), FcyRIIA (CD32a), FcyRIIIA (CD 16), FcyRIIIB (CD 16b), a Fc-alpha receptor like FcaRI (CD89) or Fc-epsilon receptors like FcsRI or FcsRII (CD23).
  • the molecule which is capable of co-engaging a FcR like the FcyRIIB with an activating receptor may be a antibody or a plurispecific molecule like a bispecific antibody.
  • the molecule which is capable of co-engaging the FcR like the FcyRIIB with an activating receptor is a plurispecific antibody comprising at least one binding site that specifically binds to the FcR like the FcyRIIB receptor, and at least one binding site that specifically binds to the activating receptor.
  • Exemplary formats for the plurispecific antibody molecules of the present invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to the FcR like the FcyRIIB and another with a specificity to an activating receptor; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single- chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al, Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (vi) a Tanda
  • bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization.
  • Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies.
  • the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology.
  • a bispecific antibody is formed by "Fab-arm" or "half- molecule” exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions.
  • the resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.
  • bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is a antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is an antibody of the present invention and the second antibody has a different binding specificity, or vice versa.
  • the reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine.
  • Step d) may further comprise restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting.
  • the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety.
  • the method of the present invention is thus particularly suitable for inhibiting or preventing activation of the targeted cell.
  • the present invention is particularly suitable for inhibiting or preventing degranulation of mast cells, activation and proliferation of mast or B cells.
  • the method of the present invention is particularly suitable for inhibiting mast cell activation induced by the interaction between an antigen and an IgE bound to an activating receptors FCERI of the mast cell.
  • the term "inhibiting" in relation to receptor activity means decreasing cell activation compared to uninhibited cells. Inhibition can be assessed by at least a 5% , 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100 % decrease. Methods for assaying inhibition are well known in the art and are particularly described in the EXAMPLE.
  • the method of the present invention is thus particular suitable for therapeutic purposes.
  • the method of the present invention is particularly suitable for the treatment of cancer, autoimmune inflammatory diseases and allergies.
  • the further object of the present invention relates to a method of treating a disease selected from the group consisting of cancers, autoimmune inflammatory diseases and allergies in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a molecule which is capable of co-engaging the FcR like the FcyRIIB receptor with an activating receptor.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • the subject suffers from a myeloid proliferation disease.
  • Prototypic examples are Chronic Myeloid Leukemias and Mast cell proliferations such as mastocytosis.
  • the term "mastocytosis” as used herein, relates to systemic mastocytosis, or mastocytoma. Mastocytosis is a myeloproliferative disorder with limited treatment options. The pathogenesis of mastocytosis has been attributed to constitutive activation of the receptor tyrosine kinase KIT.
  • B-cell malignancy includes any type of leukemia or lymphoma of B cells.
  • B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, B-cell pro lymphocytic leukemia, B cell lymphomas (e.g.
  • Hodgkin's disease B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g. Waldenstrom's macroglobulinaemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphomas), leukemias (e.g. acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also termed B cell chronic lymphocytic leukemia BCLL), hairy cell leukemia and chronic myoblastic leukemia) and myelomas (e.g. multiple myeloma).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • BCLL B cell chronic lymphocytic leukemia
  • hairy cell leukemia and chronic myoblastic leukemia hairy cell leukemia and chronic myoblastic leukemia
  • myelomas e.g. multiple myeloma
  • Additional B cell malignancies include small lymphocytic lymphoma, B cell prolymphocyte leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.
  • MALT mucosa-associated lymphoid tissue
  • the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile- onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and a
  • the autoimmune inflammatory diseases is secondary to therapeutic treatment, in particular a treatment with an immune checkpoint inhibitor.
  • the term "immune checkpoint inhibitor” has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade.
  • Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins.
  • the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
  • the subject suffers from an allergic disorder.
  • allergic disorder refers to any disorder resulting from antigen activation of mast cells that results in an "allergic reaction” or state of hypersensitivity and influx of inflammatory and immune cells.
  • Those disorders include without limitation: systemic allergic reactions, systemic anaphylaxis or hypersensitivity responses, anaphylactic shock, drug allergies, and insect sting allergies; respiratory allergic diseases, such asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis and interstitial lung diseases (ILD) (e.g.
  • ILD interstitial lung diseases
  • idiopathic pulmonary fibrosis ILD associated with rheumatoid arthritis, or other autoimmune conditions
  • rhinitis hay fever, conjunctivitis, allergic rhinoconjunctivitis and vaginitis
  • skin and dermatological disorders including psoriasis and inflammatory dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiforms, linear IgA disease, acute and chronic urticaria and scleroderma
  • vasculitis e.g.
  • necrotizing, cutaneous, and hypersensitivity vasculitis necrotizing, cutaneous, and hypersensitivity vasculitis
  • spondyloarthropathies e.g., intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, enteritis, nontropical sprue and celiac disease).
  • the subject suffers from asthma.
  • asthma refers to an inflammatory disease of the respiratory airways that is characterized by airway obstruction, wheezing, and shortness of breath.
  • the subject suffers from any allergic disease in which mast cell and/or basophil activation plays a critical role.
  • the allergic disease may be asthma, skin allergy like eczema, urticaria, dermatographism, allergic rhinitis, drug allergy or food allergy.
  • the subject suffers from anaphylaxis.
  • anaphylaxis refers to a life threatening allergic reaction characterized by decreased blood pressure, respiratory failure with bronchoconstriction, and skin rash due to release of mediators from cells such as mast cells.
  • a “therapeutically effective amount” of the molecule of the present invention is meant a sufficient amount of the molecule to treat the disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the molecule of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific antibody employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibody employed; the duration of the treatment; drugs used in combination or coincidental with the specific antibody employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the molecule is administered to the subject in the form of a pharmaceutical composition.
  • the active agent is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • pharmaceutically acceptable excipients such as biodegradable polymers
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- so lid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the active agent can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • 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.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Trans-inhibition of mast cell activation.
  • A Concentration-dependent induction of trans-inhibition in mast cells. FcyRIHA " " BMMCs sensitized with mlgE anti-DNP and rlgE (doubly sensitized) were challenged with increasing concentrations of DNP-BSA alone or mixed with increasing concentrations of MAR IgG. The release of ⁇ -hexosaminidase was measured and plotted as a function of the concentration of antigen. Data are representative of three independent experiments.
  • B Trans-inhibition requires the Fc portion of IgG.
  • SHIP1 is involved in and necessary for trans-inhibition. Trans-inhibition requires SHIP1. Doubly sensitized SHIP 1 +/+ and SHIP ⁇ ⁇ ' ⁇ BMMCs were challenged as indicated with DNP-BSA and MAR IgG for 10 min. ⁇ -hexosaminidase release was measured and plotted as a function of the concentration of DNP-BSA (E). Data are representative of two independent experiments. Cell lysates were electrophoresed and Western blotted with the indicated antibodies (F). Data are representative of two independent experiments.
  • FIG. 3 SHIP1 is sufficient for trans-inhibition.
  • A Trans-inhibition can be induced equally well by wild-type human FcyRIIB, or by human FcyRIIB whose intracytoplasmic domain was replaced by the catalytic domain of human SHIP1. Doubly sensitized RBL-FcyRIIB/mh and RBL-FcyRIIB-hSHIPl transfectants were challenged with DNP-HSA alone or with DNP-HSA and MAR IgG. ⁇ -hexosaminidase release was measured and plotted as a function of the concentration of DNP-HSA. Data are representative of three independent experiments.
  • B Trans- inhibition abrogates antigen-induced PI(3,4,5)P 3 generation.
  • Wild-type (WT) and FcyRIIB ⁇ ⁇ BMMCs sensitized with mlgE anti-DNP were challenged with increasing concentrations of DNP-BSA or with increasing concentrations of DNP- BSA and immune complexes made with the rat anti-Kit mAb ACK2 and MAR IgG.
  • ⁇ - hexosaminidase release was measured and plotted as a function of the concentration of DNP-BSA.
  • Data are representative of three independent experiments performed with WT BMMCs. One experiment only included FcyRIIB ⁇ ⁇ BMMCS.
  • Trans-inhibition can inhibit the in vitro proliferation and the in vivo growth of mastocytoma cells transformed by a v-oncogene.
  • Trans-inhibition induced by co-aggregating FcyR with Kit affects the in vitro proliferation of MMC-1 cells.
  • A MMC-1 cells were loaded with CFSE, preincubated with 2.4G2 or without, and incubated with immune complexes made with the indicated concentrations of GST-SCF and rabbit IgG anti-GST antibodies over a 5-day period. Cell-associated CFSE fluorescence was measured and plotted as a function time.
  • Trans-inhibition induced by co-aggregating FcyR affects the in vitro proliferation of MMC-1 cells.
  • MMC-1 cells were incubated for 72h with immune complexes made of GST and the indicated concentrations of rabbit IgG-anti-GST antibodies. Cells were counted in triplicates, and mean ⁇ SD cell numbers were plotted as a function of the concentration of IgG anti-GST antibodies.
  • FIG. 6 Trans-inhibition in murine and human basophils.
  • D Trans-inhibition in human basophils. Trans-hibition operates in human basophils. Human blood basophils sensitized with rlgE were challenged with increasing concentrations of RAHE F(ab') 2 alone or increasing concentrations of RAHE F(ab') 2 and MAR IgG. CD203c on basophils was measured in duplicates and the mean increase in CD203c expression (AMFI) was plotted as a function of the concentration of RAHE F(ab') 2 . Data shown in (A) are representative of nine independent experiments. Histamine released in supernatants was measured in triplicates and mean values ⁇ SD were plotted.
  • BMMCs were obtained by culturing mouse bone marrow cells in IL-3 -containing medium as described in (9).
  • MMC-1 cells (32) were a kind gift of Dr.
  • PBMC peripheral blood mononuclear cells
  • the rat anti-mouse Kit mAb ACK-2 (47), the rat anti-mouse FcyRIIB/IIIA mAb 2.4G2 (48) and the mouse anti-FcyRIIB K9.361 were affinity-purified on Protein G-sepharose.
  • the rat anti-mouse FcyRIIIA mAb 275003 (49) was from R&D Systems (Lille, France).
  • DNP-BSA and TNP-BSA-Biotin were prepared as described.
  • Glutathion S-transferase (GST)-SCF was produced in E. Coli and affinity-purified on Glutathion-agarose.
  • DNP-HSA was purchased from Sigma- Aldrich.
  • Mouse IgG anti-rat Ig (MAR IgG) and corresponding F(ab') 2 fragments [MAR F(ab') 2 ], Fluorescein isothiocyanate (FITC)-conjugated Rat anti-mlgE antibodies and FITC-conjugated Mouse anti-rat Ig antibodies were from Jackson Immunoresearch; Rabbit IgG anti-human IgE (RAHE) from Dako-Cytomation; Phycoerythrine (PE)-conjugated anti-CD203c antibodies and Allophycocyanin (APC)-conjugated anti- human FcsRIa antibodies from Bio legend; Rabbit anti- GST antibodies, Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies, normal mouse IgG and mouse anti-pPLCy-l antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); Rabbit anti-Akt, pAkt, pSyk, pGab2, pNF- ⁇ , pp
  • K9.361 and RAHE F(ab') 2 were prepared by pepsin digestion. Mast cell activation. BMMCs were sensitized by an overnight incubation at 37°C with 0.1 ⁇ g/ml mlgE anti-DNP 2682-1 (50) and 3 ⁇ g/ml rlgE IR162 (51) and challenged for various periods of times with the indicated concentrations of ligands. ⁇ -hexosaminidase was quantitated in 10-min supernatants using an enzymatic assay (9). LTC-4 and MIPl-a were titrated in 30-min supernatants by ELISA (Neogen-corporation and R&D systems respectively). TNF-a was titrated in 3-h supernatants by an ELISA (R&D systems).
  • C57BL/6J mice (Charles River) were injected i.v. with supernatants from BMMCs sensitized with mlgE 2682-1 anti-DNP and rlgE IR162, harvested 10 min after challenge with indicated ligands.
  • C57BL/6J mice were injected i.v. with 4 x 10 6 BMMCs sensitized with mlgE 2682-1 anti-DNP and rlgE IR162 and incubated with MAR IgG for 10 min.
  • Mice were challenged by an i.v. injection of 500 ⁇ g DNP-BSA 15 min later. Rectal temperature was monitored using a Precision Digital Thermometer 4600 (YSI, Dayton, Ohio, USA).
  • BMMCs sensitized with mlgE 2682-1 anti-DNP and rlgE IR162 were loaded with 0.5 ⁇ Fluo-3-AM (Invitrogen, Carlsbad, CA) for lh at room temperature, and analyzed by flow cytometry (Becton Dickinson) before and after stimulation.
  • BMMCs sensitized with mlgE anti-DNP 2682-1 and rlgE IR162 were challenged with 10 ng/ml DNP-BSA, 15 ⁇ g/ml MAR IgG or both for 1 , 3 or 10 min, lysed in SDS at 95°C, fractionated by SDS-PAGE and Western blotted with indicated antibodies.
  • HRP was detected using an ECL kit (Amersham Biosciences, Buckinghamshire, UK).
  • BMMCs sensitized with mlgE anti-DNP 2682-1 and rlgE IR162 were labeled for 8 h with 1 mCi/ml [ 32 P]orthophosphate (Perkin Elmer) in phosphate-free DMEM (Invitrogen) before they were challenged for 10 min with DNP-HSA, MAR IgG or both. Lipids were immediately extracted, separated by thin-layer chromatography and analyzed by HPLC as described previously (52).
  • Proliferation assays 1) 3 H-thymidine incorporation. MMC-1 cells were preincubated with 10 ⁇ g/ml 2.4G2 or without for 15min at 37°C and seeded at lxl0 5 /ml with culture medium, with preformed immune complexes made of 5 ⁇ g/ml GST-SCF and 50 ⁇ g/ml Rabbit a-GST antibodies, or with immune complexes made of 10 ⁇ g/ml GST and 50 ⁇ g/ml Rabbit a-GST antibodies. 48h later, the same immune complexes were added, with 2.4G2 or without. 3 H-thymidine was added at 72h, and cells were further incubated for 18h before cell-associated radioactivity was measured.
  • MMC-1 cells were labeled with CFSE for 10 min at room temperature, incubated with 2.4G2 or without, challenged with immune complexes made of GST-SCF and Rabbit a-GST for 10 min, and cultured at lxlO 5 cells/ml for the indicated periods of time. Fluorescence was analyzed by flow cytometry using a FACScalibur.
  • Tumor growth assessment 3 x 10 5 MMC-1 cells were incubated for 15 min with preformed GST-SCF-rabbit anti-GST, GST-rabbit anti-GST immune complexes or culture medium, and injected s.c. into Nude, Rag 7" or Rag 7 VFcRy 7" mice. Tumor volume was measured over 15 d.
  • Human basophil stimulation Human PBMC (lxlO 6 cells/ml in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 1% hepes buffer and 1% penicillin-streptomycin) were incubated overnight at 37°C with 3 ⁇ g/ml rlgE (53) IR162 or without.
  • Non-sensitized PBMC were challenged with Rabbit anti- human IgE (RAHE).
  • PBMC sensitized with rlgE were challenged with the indicated concentrations of RAHE and MAR.
  • CD203c up-regulation was monitored in Fc8RI + cells using a FACScalibur (BD-Biosciences) or a MACSQuant (Miltenyi-Biotec). Histamine was measured in 45 -min supernatants by ELISA (Neogen Corporation). For histamine release, PBMC sensitized with rlgE were challenged with 10 ⁇ g/ml RAHE F(ab') 2 alone or with 10 ⁇ g/ml RAHE F(ab') 2 and 30 ⁇ g/ml MAR IgG. Histamine released in supernatants was measured 45 min later by ELISA (Neogen Corporation).
  • FcyRIIB inhibit mast cell activation induced by FCERI molecules other than those with which FcyRIIB are co-engaged.
  • Bone marrow-derived mast cells from FcyRIIIA-deficient mice possess activating IgE receptors (FcsRI) and inhibitory IgG receptors (FcyRIIB). Such cells were used to exclude the possibility that activating IgG receptors (FcyRIIIA) could be co-engaged with FcyRIIB by IgG antibodies.
  • Cells were sensitized with a mouse IgE (mlgE) anti-dinitrophenyl (DNP) and a myeloma rat IgE (rlgE) of an unknown specificity, which could be engaged independently by specific ligands (data not shown).
  • MAR IgG also inhibited cell activation induced by the engagement of FcsRI -bound mlgE with antigen in doubly sensitized cells (Fig. 1 A).
  • FcyRIIB not only inhibited activation signals induced by FcsRI molecules with which they were co-aggregated (cis-inhibition), but also activation signals induced by other FcsRI molecules that were independently aggregated on the same cells (trans-inhibition).
  • Trans-inhibition was induced neither by MAR F(ab')2 that could engage FcsRI but not FcyRIIB (Fig. IB) nor by intact normal mouse IgG that could not engage any receptor (data not shown). Trans-inhibition was abrogated in FcyRIIB-deficient cells and, importantly, it was as efficient in wild-type cells as in FcyRIIIA-deficient cells (data not shown). Trans-inhibition therefore required that the Fc portion of MAR IgG bound to FcyRIIB, and the negative effect of FcyRIIB was dominant over the positive effect of FcyRIIIA that were co-engaged with FcyRIIB by MAR IgG in wild-type mast cells.
  • Trans-inhibition affected cell activation by other anti-DNP IgE antibodies (data not shown) or by IgE antibodies with another specificity (data not shown). It was not due to a decreased accessibility of IgE antibodies for antigen (data not shown). Trans- inhibition persisted if antigen challenge was delayed after the addition of MAR IgG (fig. S6), but it was abrogated after a few hours (data not shown).
  • Trans-inhibition was observed when assessing the release of granular mediators or the secretion of eicosanoids, chemokines, and cytokines (data not shown). It decreased the production of all the mediators that account for systemic anaphylaxis. Indeed, trans-inhibition abrogated the loss of body temperature in wild-type mice injected intravenously with supernatant collected 10 min after the challenge of doubly sensitized BMMCs with antigen (data not shown). Trans- inhibition also abrogated the loss of body temperature induced by an intravenous injection of antigen into mice injected previously with doubly sensitized BMMCs that were challenged with MAR IgG 10 min earlier (data not shown). Trans-inhibition can therefore dampen in vitro reactions and their systemic in vivo consequences.
  • Trans-inhibition involves and requires the lipid phosphatase SHIP1.
  • Trans-inhibition abrogated mediator release induced by antigen, but had no effect mediator release induced by PMA and ionomycin (data not shown), i.e. it acted upstream of the Ca2+ response. It indeed dampened antigen-induced Ca2+ mobilization (data not shown), as well as Akt, Erkl/2, JNK, p38 and NF- ⁇ phosphorylation but not Syk, LAT, Gab2 and PLCy-l phosphorylation (data not shown), i.e. distal, but not proximal signals that were rather enhanced (data not shown). SHIP1 was heavily phosphorylated (data not shown).
  • KIR2DL3 is a typical NK cell inhibitory receptor whose intracytoplamsic domain contains two ITIMs that have an affinity for the SH2 domain-containing tyrosine phosphatases 1 and 2 (SHP-1 and SHIP- 2), but not for SHIP1 or SHIP2 (22).
  • Trans-inhibition can be explained if PI(3,4,5)P3 can diffuse from activating signalosomes where this phospholipid was generated, as shown in immunological synapses (26), and meet SHIP1 where this enzyme was recruited. It can also be explained if SHIP1 can leave inhibitory signalosomes where it was recruited, as shown in B cells (27), and meet PI(3,4,5)P3 where it was generated. Two sets of data discriminated between these two possibilities.
  • chimeric molecules made of mouse (m) FcyRIIB whose intracytoplasmic domain was replaced by that of human (h) FcyRIIB (FcyRIIB/mh) or by the catalytic domain of human SHIP1 (FcyRIIB/hSHIPl) induced trans-inhibition of comparable magnitudes Fig. 3 A.
  • SHIP1 is therefore not only necessary but also sufficient for trans-inhibition, and trans-inhibition may not require the phosphatase to leave inhibitory signalosomes.
  • antigen-induced increase of intracellular PI(3,4,5)P3 concentration was prevented in both cis- and trans-inhibition (Fig. 3B).
  • SHIP1 therefore hydrolyzed not only PI(3,4,5)P3 generated by FcsRI that were co-ligated with FcyRIIB, but also PI(3,4,5)P3 generated by FcsRI that were not. Consequently, co-engaging FcyRIIB with a fraction of FcsRI can induce a global inhibition of PI(3,4,5)P3-dependent cell signaling.
  • Trans-inhibition can prevent the in vitro and the in vivo growth of transformed cells whose proliferation depends on a viral oncogene.
  • FcyRIIB can cis-inhibit proliferation signals when co-ligated with the Stem Cell Factor (SCF) Receptor-Tyrosine Kinase (RTK) Kit (30) and that inhibition depends on SHIP1 (31).
  • SCF Stem Cell Factor
  • RTK Receptor-Tyrosine Kinase
  • MMC-1 is a mouse mastocytoma tumor that originated in mice infected with the Abelson murine leukemia virus (32).
  • MMC-1 cells express the transforming oncogene v-Abl (33), activating (FcyRIIIA) and inhibitory (FcyRIIB) IgG receptors (FcyR) but no FcsRI, and Kit (fig. S9) the transmembrane and intracytoplasmic domains of which contained no mutation.
  • v-Abl which was constitutively phosphorylated in MMC-1 cells (data not shown), accounted for their proliferation as the latter was inhibited by the Abl inhibitor STI571 (34) at 100-fold lower concentrations than by the multi-RTK inhibitor SU11248 (35) (data not shown).
  • MMC-1 proliferation was also dose-dependently inhibited when FcyR were co-aggregated with Kit by GST-SCF-anti-GST immune complexes (Fig. 5 A), and inhibition was prevented by the blocking anti-FcyRIIB+IIIA mAb 2.4G2.
  • FcyRIIB trans-inhibited v-Abl-dependent proliferation signals when co-engaged with FcyRIIIA and Kit in MMC-1 cells.
  • trans-inhibition did not require Kit as it was similarly induced by GST-anti-GST immune complexes that could co-engage FcyRIIB with FcyRIIIA only (Fig. 5 B). This was observed both in vitro (Fig. 5 B) and in vivo (data not shown). Thus, trans-inhibition both decreased the in vitro proliferation and prevented the in vivo growth of v-oncogene-transformed tumor cells in immunodeficient mice, when FcyRIIB were co-aggregated with FcyRIIIA and Kit or with FcyRIIIA only, by IgG immune complexes.
  • Trans-inhibition operates and is long-lasting in human basophils.
  • MAR F(ab')2 and IgG induced similar IL-4 secretion in FcyRIIB-deficient BMB (data not shown). As previously observed (11), cis-inhibition therefore operates in mouse basophils.
  • Antigen- induced IL-4 secretion was comparable in wild-type and FcyRIIB-deficient BMB doubly sensitized with mlgE anti-DNP and rlgE. It was decreased by MAR IgG in wild-type BMB, but markedly increased in FcyRIIB-deficient BMB (data not shown). Trans-inhibition therefore operates in primary mouse basophils.
  • human basophils can be activated by rabbit anti- human IgE (RAHE) F(ab')2 that aggregate FcsRI -bound human IgE (Fig. 6 A) and, when sensitized with rlgE, by MAR F(ab')2 (data not shown). Basophil activation was much milder in response to MAR IgG that co-engage FcsRI with FcyR (data not shown). As previously observed (11), FcyR-dependent cis-inhibition therefore operates in human basophils.
  • MAR IgG also inhibited RAHE F(ab')2-induced basophil activation, whether assessed by CD203c up-regulation (Fig.
  • MAR F(ab')2 displayed a bell-shaped curve (Fig. 6 B). RAHE F(ab')2-induced basophil activation was abrogated by an excess of MAR F(ab')2 in rlgE-sensitized cells (Fig. 6 C). Trans-inhibition can therefore be induced in human basophils as in mouse mast cells, by over-aggregating FcsRI.
  • Trans-inhibition a novel regulatory mechanism by which antibodies can anergize mast cells and basophils when engaging Fc Receptors.
  • Trans-inhibition operated in normal primary mast cells and basophils, whether of murine or human origin, and human FcyRIIB trans-inhibited similarly as murine FcyRIIB. It dampened all IgE-induced mast cell secretory responses, including the release of all mediators that account for anaphylaxis. It also acted upon the growth factor-induced proliferation of normal mast cells and, importantly, the growth factor- independent autonomous proliferation of cells transformed by the oncogene v-Abl. It could also prevent the growth of tumors in immunodeficient mice injected with these cells. Trans-inhibition is therefore likely to affect a wide array of biological responses, whether physiological or pathological. It can possibly be exploited as a novel therapeutic tool, especially but not exclusively, in allergy.
  • Trans-inhibition and cis-inhibition have common characteristics. Both require a similar co- aggregation or over-aggregation of Fc receptors. Both use the lipid phosphatase SHIP1 which prevents the PH domain-mediated membrane translocation of critical signaling molecules by hydrolyzing PI(3,4,5)P3. Cis- and trans-inhibition, however, differ in their consequences. Unlike cis-inhibition that acts upon signals generated by the activating receptors that are either co- aggregated with inhibitory receptors or over-aggregated, trans-inhibition acts upon signals generated by activating receptors that are engaged independently. Trans-inhibition is a global phenomenon whereas cis-inhibition is a local phenomenon.
  • Trans-inhibition is a collateral effect that goes along with cis-inhibition and dramatically expands the regulatory properties of Fc Receptors. Another difference is that all ITIM-containing receptors can cis-inhibit activation signals, whereas FcyRIIB, but not other ITIM-containing receptors, can trans-inhibit.
  • Co-aggregation enables tyrosine phosphatases to dephosphorylate phospho-proteins in signalosomes generated by activating receptors.
  • FcyRIIB need to be co-aggregated with activating receptors in order to be phosphorylated and to recruit SHIP 1/2. These phosphatases can then hydrolyze PI(3,4,5)P3 generated by activating receptors, whether the latter were co-engaged with FcyRIIB or not.
  • Trans-inhibition is a property of lipid phosphatases, but not of protein phosphatases.
  • SHIP1 was indeed 1) involved, 2) necessary and 3) sufficient for trans-inhibition.
  • SHIP1 was heavily tyrosyl-phosphorylated in cells submitted to trans-inhibition, indicating that it was recruited in signalosomes where it became a substrate of Lyn (36).
  • PI(3,4,5)P3 can diffuse from activating signalosomes where it was generated, and reach SHIPl in inhibitory signalosomes.
  • PI(3,4,5)P3 is generated at the leading edge of immunological synapses where PI3K is activated. Increased amounts of PI(3,4,5)P3 were however observed not only within immunological synapses, but also outside (26).
  • SHIPl can leave inhibitory signalosomes where it was recruited and reach PI(3,4,5)P3 elsewhere.
  • PLC-yl When recruited to the plasma membrane, PLC-yl is embedded in a cluster of molecules (40) including the cytosolic adapters SLP76, Grb2, and GADS that cooperatively bind to the transmembrane adapter LAT1 (41). Under these conditions, reduced PI(3,4,5)P3 levels may be sufficient to reduce the influx of extracellular Ca2+, but not to prevent PLC-yl from being recruited and activated.
  • Trans-inhibition was demonstrated by co-engaging FcyRIIB with FcsRI sensitized with two non-crossreacting IgE on mouse mast cells, mouse basophils and human basophils. This condition mimics the situation in allergic patients, especially patients subjected to immunotherapy, and possibly in non-allergic individuals. Under this condition, trans-inhibition was 1) induced by FcyRIIB, 2) when these inhibitory receptors were co-ligated with FcsRI, and 3) assessed on biological responses triggered by FcsRI. However, neither the induction nor the effects of trans- inhibition were restricted to these receptors.
  • Trans-inhibition could be induced when FcyRIIB were co-engaged with receptors other than FcsRI. It was indeed observed when co-engaging FcyRIIB with a growth factor receptor such as Kit. It was probably also induced by co-engaging FcyRIIB with FcyRIIIA in MMC-1. Possibly pertaining to trans-inhibition, the co-aggregation of FcyRIIB with BCR inhibited chemotaxis in murine lymphoma A20 B cells (42). These findings suggest that trans-inhibition may be induced whenever FcyRIIB are co-aggregated with activating receptors that use a src family kinase capable of phosphorylating the FcyRIIB ITIM.
  • Trans-inhibition could be induced by receptors other than FcyRIIB, including activating immunoreceptors. It could indeed be induced by FcsRI, independently of FcyRIIB, in mouse mast cells and human basophils challenged with a supra-optimal concentration of antigen, which induces a massive recruitment of SHIP1 by FcsRI (29). This finding indicates that the capability to trans-inhibit is restricted neither to FcyRIIB nor to IgG antibodies. It suggests that trans- inhibition may be induced by any receptor, whether inhibitory or activating, capable of recruiting enough SHIP1 into signalosomes, whether during co-regulation or during auto-regulation (fig. SI 6). Trans-inhibition is neither a property of antibodies nor a property of Fc Receptors that are engaged by antibodies. It is a property of the lipid phosphatase SHIP1.
  • Trans-inhibition could act upon signals generated by receptors other than FcsRI or even other than immunoreceptors, including proliferation signals generated by a transforming oncogene. It indeed decreased not only the SCF-induced Kit-dependent proliferation of normal mast cell, but also the constitutive v-Abl-dependent proliferation of mastocytoma cells. A variety of biological responses can therefore be trans-inhibited by FcyRIIB. This finding suggests that trans-inhibition may affect biological responses induced by any receptor that activates PI3K and uses PI(3,4,5)P3 to recruit PH domain-containing signaling molecules.
  • G Protein-coupled receptors might be such receptors as FcyRIIB-dependent inhibition of CXCL12-induced chemotaxis in A20 cells also depended on SHIP1 (42). Numerous receptors that control the activation and the proliferation of hematopoietic cells use PI(3,4,5)P3 and are under the control of SHIP1, as shown by the pleiotropic effects of SHIP1 deletion (43). Many among these receptors are expressed by mast cells (44). The biological significance of trans-inhibition was further enhanced by the discovery that most SH2 domains bind to plasma membrane lipids with a high affinity and, many as specifically as PH domains (45).
  • trans-inhibition is a novel SHIP- 1 -dependent regulatory mechanism that can prevent Fc Receptor-expressing cells from responding to a variety of extracellular or intracellular activation and proliferation signals that depend on PI(3,4,5)P3.
  • Fc Receptor-dependent trans- inhibition endows antibodies with previously unsuspected regulatory properties and new biological significance. It provides theoretical grounds for novel therapeutic approaches.
  • Ligands capable of co-engaging FcyRIIB with receptors that use PI3K without activating mast cells might be engineered and used for an allergen-nonspecific "universal" desensitization in allergic patients.
  • Peritoneal cell-derived mast cells an in vitro model of mature serosal-type mouse mast cells. J Immunol 178, 6465-6475 (2007).
  • RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells. Immunity 12, 347-358 (2000).

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Abstract

The present invention relates to methods of inhibiting biological responses induced by a receptor that uses PI3K to signal in a cell. The inventors found that the inhibitory low-affinity IgG receptors FcγRIIB that are highly expressed in mouse mast cells and human basophils, not only cis-inhibit signals triggered by activating receptors with which they are co-engaged, but also trans-inhibit signals triggered by receptors engaged independently. In particular, the present invention relates to a method of inhibiting biological responses induced by a receptor that uses PI3K to signal in a cell that coexpresses receptors for the Fc portion of antibodies (FcR) that recruit the lipid phosphatase SHIP1 and/or SHIP2.

Description

METHODS OF INHIBITING BIOLOGICAL RESPONSES INDUCED BY A
RECEPTOR THAT USES PI3K TO SIGNAL IN A CELL
FIELD OF THE INVENTION:
The present invention relates to methods of inhibiting biological responses induced by a receptor that uses PI3K to signal in a cell.
BACKGROUND OF THE INVENTION:
High-affinity IgE receptors (FcERI) are thought to initiate allergic manifestations when they are aggregated on mast cells and/or basophils. FcERI aggregation occurs when a plurivalent allergen binds to receptor-bound allergen-specific IgE antibodies. FcERI oligomerization is sufficient to activate basophils and mast cells in vitro, as demonstrated using bivalent haptens (1), IgE dimers (2), or anti-receptor antibodies (3). Mast cells and basophils of an individual, however, carry, each, many receptor-bound polyclonal IgE directed against the various epitopes of the different proteins of an allergen. Besides, allergic patients who are polysensitized have IgE antibodies against more than one allergen. Distinct large FcERI aggregates, made each by polyclonal IgE against one allergen, can therefore form on mast cells and basophils in vivo. Finally, one million-fold more IgG than IgE (mg/ml vs ng/ml) circulate in the bloodstream, and allergens are likely to form complexes with polyclonal IgG antibodies of several specificities before they reach FcERI -bound IgE. Rather than being aggregated by allergens, mast cell and basophil FCERI are therefore co-aggregated with low-affinity IgG receptors expressed on the same cells. These comprise activating [FcyRIIIA in mice (4,5), FcyRIIA in humans (6)] and inhibitory [FcyRIIB in both species (5, 7)] receptors. Co-aggregation further increases when high amounts of specific IgG are induced in patients undergoing allergen immunotherapy (8). FcyRIIB were demonstrated to inhibit FcERI-dependent mast cell (9) and basophil (7) activation. They were recently found to be critical for the efficacy of allergen immunotherapy (10, 11).
The mechanism by which FcyRIIB inhibit IgE-induced mast cell and basophil activation is well known. Inhibition requires that FcyRIIB be co-aggregated with FCERI (9). Co-aggregation enables the FcERI-associated Src kinase Lyn to phosphorylate both Immunoreceptor Tyrosine- based Activation Motifs (ITAMs) in FCERI, and the Immunoreceptor Tyrosine-based Inhibition Motif (ITIM) in FcyRIIB (12). When tyrosyl-phosphorylated, FcyRIIB recruit the Src-homology 2 (SH2) domain-containing inositol 5-phosphatase SHIP1 (13-15) into FCERI signalosomes where it hydrolyzes phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] (15), generated by phosphoinositide 3-kinase (PI3K), thereby preventing the recruitment of Pleckstrin Homology (PH) domain-containing molecules that are critical for cell activation (16,17).
SUMMARY OF THE INVENTION:
The present invention relates to methods of inhibiting biological responses induced by a receptor that uses PI3K to signal. In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
The inventors found that the inhibitory low-affinity IgG receptors FcyPJIB that are highly expressed in mouse mast cells and human basophils, not only cis-inhibit signals triggered by activating receptors with which they are co-engaged, but also trans-inhibit signals triggered by receptors engaged independently. Trans-inhibition affected high-affinity IgE receptor (FCERI)- dependent mouse mast cell, mouse basophil and human basophil activation, as well as growth factor receptor (Kit)-dependent normal mouse mast cell proliferation, and even the constitutive in vitro proliferation and the in vivo growth of oncogene (v-Abl)-transformed mastocytoma cells. Trans- inhibition was induced by receptors that recruit SHIPl, whether inhibitory such as FcyRIIB, or activating such as FcERI. It was indeed due to the lipid phosphatase SHIPl which, by hydrolyzing PI(3,4,5)P3, induced a global unresponsiveness that affected biological responses triggered by receptors that use phosphoinositide 3-kinase (PI3K) to signal. Trans-inhibition can therefore control numerous processes, in physiology and pathology. It provides mechanistic grounds for unanticipated new therapeutic approaches, especially of allergic diseases.
Accordingly, the first aspect of the present invention relates to a method of inhibiting biological responses induced by a receptor that uses PI3K to signal in a cell that coexpresses receptors for the Fc portion of antibodies (FcRs) that recruit the lipid phosphatase SHIPl and/or SHIP2.
In one embodiment, the receptors for the Fc portion of antibodies (FcRs) may be the inhibitory low-affinity IgG receptors FcyRIIB.
As used herein, the term "receptor for the Fc portion of antibodies" or "FcR" denote a protein found on the surface of certain cells - including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells - that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc (Fragment, crystallizable) region. Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. Some viruses such as flaviviruses use Fc receptors to help them infect cells, by a mechanism known as antibody-dependent enhancement of infection. There are several different types of Fc receptors, which are classified based on the type of antibody that they recognize. For example, those that bind the most common class of antibody, IgG, are called Fc-gamma receptors (FcyR), those that bind IgA are called Fc-alpha receptors (FcaR) and those that bind IgE are called Fc-epsilon receptors (FcsR).
Accordingly, the receptors for the Fc portion of antibodies may be a Fc-gamma receptor like FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD 16), FcyRIIIB (CD 16b), a Fc-alpha receptor like FcaRI (CD89) or Fc-epsilon receptors like FcsRI or FcsRII (CD23).
As used herein, the term "FcyRIIB" has its general meaning in the art and refers to receptors for the Fc portion of IgG encoded by FCGR2B gene (Gene ID: 2113). The term is also known as CD32; FCG2; CD32B; FCGR2; IGFR2. The receptor has a low affinity for monomeric IgG but it binds IgG immune complexes with a high avidity. The encoded protein is involved in the endocytosie of soluble immune complexes, the phagocytosis of particulate immune complexes, the regulation of cell activation induced by ITAM-containing receptors (an example of which is the regulation of antibody production initiated by B cells), the regulation of cell proliferation induced by Receptor Tyrosine Kinase Receptors. An exemplary human amino acid sequence is represented by the NCBI reference sequence NP 001002273.1, NP 001002274.1, NP_001002275.1, NP_001177757.1 or NP 003992.3.
As used herein the term "PI3K" has its general meaning in the art and refers to a phosphoinositide 3-kinase or variant thereof, which is capable of phosphorylating the inositol ring of PI in the D-3 position. The term "PI3K variant" is intended to include proteins substantially homologous to a native PI3K, i.e., proteins having one or more naturally or non-naturally occurring amino acid deletions, insertions, or substitutions {e.g., PI3K derivatives, homologs, and fragments), as compared to the amino acid sequence of a native PI3K. Examples of PI3K include, but are not limited to pl l0-a, ρ110-β, ρ110-γ, ρ110-δ, PI3K-C2a, PI3K-C2P, PI3K-C2y and Vps34. PI3Ks are classified into at least four classes. Class I includes pi 10-a, pi 10-β, pi 10-γ, and pi 10-δ. Class I PI3Ks are responsible for the production of phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), and phosphatidylinositol (3,4,5)- trisphosphate (PI(3,4,5)P3). The PI3K is activated by G protein-coupled receptors and tyrosine kinase receptors. Class II includes PI3K-C2a, PI3K-C2p, and PBK-C2y. Class III includes Vps34. Class IV includes mTOR, ATM, ATR, and DNA-PK. In some embodiments, the PI3K is a Class I kinase. In some embodiments, the PI3K is selected among pi 10-a, pi 10-β, pi 10-γ, and pi 10-δ. In some embodiments, the expression "receptor that uses PI3K to signal" refers to any receptor that activate a PI3K and in particular a class I PI3K.
In some embodiments, the receptor that uses PI3k to signal is a G protein-coupled receptor. As used herein, the term "G protein-coupled receptor" or "GPCR" has its general meaning in the art and refers to a large protein family of receptors, that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. The term is also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptor, and G protein-linked receptors (GPLR). GPCRs can be grouped into 6 classes based on sequence homology and functional similarity: Class A (or 1) (Rhodopsin-like), Class B (or 2) (Secretin receptor family), Class C (or 3) (Metabotropic glutamate/pheromone), Class D (or 4) (Fungal mating pheromone receptors), Class E (or 5) (Cyclic AMP receptors) and Class F (or 6) (Frizzled/Smoothened). Examples of GPCRs include but are not limited to Chemokine (C-C motif) receptor 1 (CCRl, CKRl); Chemokine (C-C motif) receptor 2 (CCR2, CKR2); Chemokine (C-C motif) receptor 3 (CCR3, CKR3); Chemokine (C-C motif) receptor 4 (CCR4, CKR4); Chemokine (C-C motif) receptor 5 (CCR5, CKR5); Chemokine (C-C motif) receptor 8 (CCR8, CKR8); Chemokine (C-C motif) receptor-like 2 (CCRL2, CKRX); chemokine (C motif) receptor 1 (XCR1, CXC1) InterPro: IPR005393; chemokine (C-X3-C motif) receptor 1 (CX3CR1, C3X1) InterPro: IPR005387; GPR137B (GPR137B, TM7SF1); Chemokine receptor InterPro: IPR000355; Chemokine (C-C motif) receptor-like 1 (CCRLl CCRL1, CCR11); Chemokine (C-C motif) receptor 6 (CCR6, CKR6); Chemokine (C-C motif) receptor 7 (CCR7, CKR7); Chemokine (C-C motif) receptor 9 (CCR9, CKR9); Chemokine (C-C motif) receptor 10 (CCR10, CKRA); CXC chemokine receptors InterPro: IPR001053; Chemokine (C-X-C motif) receptor 3 (CXCR3); Chemokine (C-X-C motif) receptor 4 (CXCR4, Fusin); Chemokine (C-X-C motif) receptor 5 (CXCR5); Chemokine (C-X-C motif) receptor 6 (CXCR6, BONZO); Chemokine (C-X-C motif) receptor 7 (CXCR7, RDC1) InterPro: IPR001416; Interleukin-8 InterPro: IPR000174 (IL8R) ; IL8R-a (IL8RA, CXCR1); IL8R-P (IL8RB, CXCR2) ; Adrenomedullin receptor (GPR182) ; Duffy blood group, chemokine receptor (DARC, DUFF); G Protein-coupled Receptor 30 (GPER, CML2, GPCR estrogen receptor); Angiotensin II receptor InterPro: IPR000248; Angiotensin II receptor, type 1 (AGTR1, AG2S); Angiotensin II receptor, type 2 (AGTR2, AG22); Apelin receptor (AGTRLl, APJ) InterPro: IPR003904; Bradykinin receptor InterPro: IPR000496; Bradykinin receptor Bl (BDKRB1, BRB1); Bradykinin receptor B2 (BDKRB2, BRB2); GPR15 (GPR15, GPRF); GPR25 (GPR25); Opioid receptor InterPro: IPR001418; delta Opioid receptor (OPRD1, OPRD); kappa Opioid receptor (OPRK1, OPRK); mu Opioid receptor (OPRM1, OPRM); Nociceptin receptor (OPRLl, OPRX); Somatostatin receptor InterPro: IPR000586; Somatostatin receptor 1 (SSTR1, SSR1); Somatostatin receptor 2 (SSTR2, SSR2); Somatostatin receptor 3 (SSTR3, SSR3); Somatostatin receptor 4 (SSTR4, SSR4); Somatostatin receptor 5 (SSTR5, SSR5); GPCR neuropeptide receptor InterPro: IPR009150; Neuropeptides B/W receptor 1 (NPBWR1, GPR7); Neuropeptides B/W receptor 2 (NPBWR2, GPR8); GPR1 orphan receptor (GPR1) InterPro: IPR002275; Galanin receptor InterPro: IPR000405; Galanin receptor 1 (GALR1, GALR); Galanin receptor 2 (GALR2, GALS); Galanin receptor 3 (GALR3, GALT); Cysteinyl leukotriene receptor InterPro: IPR004071; Cysteinyl leukotriene receptor 1 (CYSLTR1) Cysteinyl leukotriene receptor 2 (CYSLTR2) Leukotriene B4 receptor InterPro: IPR003981; Leukotriene B4 receptor (LTB4R, P2Y7); Leukotriene B4 receptor 2 (LTB4R2); Relaxin receptor InterPro: IPR008112; Relaxin/insulin-like family peptide receptor 1 (RXFP1, LGR7); Relaxin/insulin-like family peptide receptor 2 (RXFP2, GPR106); Relaxin/insulin-like family peptide receptor 3 (RXFP3, SALPR); Relaxin/insulin-like family peptide receptor 4 (RXFP4, GPR100/GPR142); KiSSl-derived peptide receptor (GPR54) (KISS1R) InterPro: IPR008103; Melanin-concentrating hormone receptor 1 (MCHR1, GPRO) InterPro: IPR008361; Urotensin-II receptor (UTS2R, UR2R) InterPro: IPR000670; Cholecystokinin receptor InterPro: IPR009126; Cholecystokinin A receptor (CCKAR, CCKR); Cholecystokinin B receptor (CCKBR, GASR); Neuropeptide FF receptor InterPro: IPR005395; Neuropeptide FF receptor 1 (NPFFR1, FF1R); Neuropeptide FF receptor 2 (NPFFR2, FF2R); Orexin receptor InterPro: IPR000204; Hypocretin (orexin) receptor 1 (HCRTR1, OX1R); Hypocretin (orexin) receptor 2 (HCRTR2, OX2R); Vasopressin receptor InterPro: IPR001817; Arginine vasopressin receptor 1A (AVPRIA, VIAR); Arginine vasopressin receptor IB (AVPR1B, V1BR); Arginine vasopressin receptor 2 (AVPR2, V2R); Gonadotrophin releasing hormone receptor (GNRHR, GRHR) InterPro: IPR001658; Pyroglutamylated RF amide peptide receptor (QRFPR, GPR103); GPR22 (GPR22, GPRM); GPR176 (GPR176, GPR); Bombesin receptor InterPro: IPR001556; Bombesin-like receptor 3 (BRS3); Neuromedin B receptor (NMBR); Gastrin-re leasing peptide receptor (GRPR); Endothelin receptor InterPro: IPR000499; Endothelin receptor type A (EDNRA, ET1R); Endothelin receptor type B (EDNRB, ETBR); GPR37 (GPR37, ETBR-LP2) InterPro: IPR003909; Neuromedin U receptor InterPro: IPR005390; Neuromedin U receptor 1 (NMUR1); Neuromedin U receptor 2 (NMUR2); Neurotensin receptor InterPro: IPR003984; Neurotensin receptor 1 (NTSRl, NTRl); Neurotensin receptor 2 (NTSR2, NTR2); Thyrotropin-releasing hormone receptor (TRHR, TRFR) InterPro: IPR009144; Growth hormone secretagogue receptor (GHSR) InterPro: IPR003905; GPR39 (GPR39); Motilin receptor (MLNR, GPR38) ;Anaphylatoxin receptors InterPro: IPR002234; C3a receptor (C3AR1, C3AR); C5a receptor (C5AR1, C5AR); Chemokine-like receptor 1 (CMKLR1, CML1) InterPro: IPR002258; Formyl peptide receptor InterPro: IPR000826; Formyl peptide receptor 1 (FPR1, FMLR); Formyl peptide receptor-like 1 (FPRL1, FML2); Formyl peptide receptor-like 2 (FPRL2, FML1); MAS1 oncogene InterPro: IPR000820; MAS1 (MAS1, MAS); MAS1L (MAS1L, MRG); GPR1 (GPR1); GPR32 (GPR32, GPRW); GPR44 (GPR44); GPR77 (GPR77, C5L2); Melatonin receptor InterPro: IPR000025; Melatonin receptor 1A (MTNR1A, ML1A); Melatonin receptor IB (MTNR1B, ML IB); Neurokinin receptor InterPro: IPR001681; Tachykinin receptor 1 (TACR1, NK1R); Tachykinin receptor 2 (TACR2, NK2R) ;Tachykinin receptor 3 (TACR3, NK3R); Neuropeptide Y receptor InterPro: IPR000611; Neuropeptide Y receptor Yl (NPYIR, NYIR); Neuropeptide Y receptor Y2 (NPY2R, NY2R); Pancreatic polypeptide receptor 1 (PPYR1 , NY4R); Neuropeptide Y receptor Y5 (NPY5R, NY5R); Prolactin- releasing peptide receptor (PRLHR, GPRA) InterPro: IPR001402; Prokineticin receptor 1 (PROKR1, GPR73); Prokineticin receptor 2 (PROKR2, PKR2); GPR19 (GPR19, GPRJ); GPR50 (GPR50, ML1X); GPR75 (GPR75); GPR83 (GPR83, GPR72); Glycoprotein hormone receptor InterPro: IPR002131; FSH-receptor (FSHR); Luteinizing hormone/choriogonado tropin receptor (LHCGR, LSHR); Thyrotropin receptor (TSHR); Leucine-rich repeat-containing G protein- coupled receptor 4 (LGR4, GPR48); Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, GPR49); Leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6); GPR40- related receptor InterPro: IPR013312; Free fatty acid receptor 1 (FFARl, GPR40); Free fatty acid receptor 2 (FFAR2, GPR43); Free fatty acid receptor 3 (FFAR3, GPR41); GPR42 (GPR42, FFAR1L); P2 purinoceptor InterPro: IPR002286; Purinergic receptor P2Y1 (P2RY1); Purinergic receptor P2Y2 (P2RY2); Purinergic receptor P2Y4 (P2RY4); Purinergic receptor P2Y6 (P2RY6); Purinergic receptor P2Y8 (P2RY8); Purinergic receptor P2Y11 (P2RY11); Hydroxycarboxylic acid receptor 1 (HCAR1 , GPR81); Hydroxycarboxylic acid receptor 2, Niacin receptor 1 (HCAR2, GPR109A); Hydroxycarboxylic acid receptor 3, Niacin receptor 2 (HCAR3, GPR109B, HM74); GPR31 (GPR31, GPRV); GPR82 (GPR82); Oxoglutarate (alpha-ketoglutarate) receptor 1 (OXGR1, GPR80); Succinate receptor 1 (SUCNR1, GPR91); P2 purinoceptor InterPro: IPR002286; Purinergic receptor P2Y12 (P2RY12); Purinergic receptor P2Y13 (P2RY13, GPR86) InterPro: IPR008109; Purinergic receptor P2Y14 (P2RY14, UDP-glucose receptor, KI01) InterPro: IPR005466; GPR34 (GPR34); GPR87 (GPR87); GPR171 (GPR171, H963); Platelet- activating factor receptor (PTAFR, PAFR) InterPro: IPR002282; Cannabinoid receptor InterPro: IPR002230; Cannabinoid receptor 1 (brain) (CNR1, CB1R); Cannabinoid receptor 2 (macrophage) (CNR2, CB2R); Lysophosphatidic acid receptor InterPro: IPR004065; Lysophosphatidic acid receptor 1 (LPAR1); Lysophosphatidic acid receptor 2 (LPAR2); Lysophosphatidic acid receptor 3 (LPAR3); Sphingosine 1-phosphate receptor InterPro: IPR004061; Sphingosine 1-phosphate receptor 1 (S1PR1); Sphingosine 1-phosphate receptor 2 (S1PR2); Sphingosine 1 -phosphate receptor 3 (S1PR3); Sphingosine 1 -phosphate receptor 4 (S1PR4); Sphingosine 1 -phosphate receptor 5 (S1PR5); Melanocortin/ACTH receptor InterPro: IPR001671; Melanocortin 1 receptor (MC1R, MSHR); Melanocortin 3 receptor (MC3R); Melanocortin 4 receptor (MC4R); Melanocortin 5 receptor (MC5R); ACTH receptor (MC2R), ACTR); GPR3 (GPR3); GPR6 (GPR6); GPR12 (GPR12, GPRC); Eicosanoid receptor InterPro: IPR008365; Prostaglandin D2 receptor (PTGDR, PD2R); Prostaglandin El receptor (PTGER1, PE21); Prostaglandin E2 receptor (PTGER2, PE22): Prostaglandin E3 receptor (PTGER3, PE23); Prostaglandin E4 receptor (PTGER4, PE24); Prostaglandin F receptor (TGFR, PF2R); Prostaglandin 12 (prostacyclin) receptor (PTGIR, PI2R); Thromboxane A2 receptor (TBXA2R, TA2R); Lysophosphatidic acid receptor InterPro: IPR004065; Lysophosphatidic acid receptor 4 (LPAR4); Lysophosphatidic acid receptor 5 (LPAR5); Lysophosphatidic acid receptor 6 (LPAR6) InterPro: IPR002188; P2 purinoceptor InterPro: IPR002286; Purinergic receptor P2Y10 (P2RY10, P2Y10), Protease-activated receptor InterPro: IPR003912; Coagulation factor II (thrombin) receptor-like 1 (F2RL1, PAR2); Coagulation factor II (thrombin) receptor-like 2 (F2RL2, PAR3); Coagulation factor II (thrombin) receptor- like 3 (F2RL3, PAR4); Epstein-Barr virus induced gene 2 (lymphocyte-specific G protein-coupled receptor) (GPR183); Proton-sensing G protein-coupled receptors; GPR4 (GPR4) InterPro: IPR002276; GPR65 (GPR65) InterPro: IPR005464; GPR68 (GPR68) InterPro: IPR005389; GPR132 (GPR132, G2A) InterPro: IPR005388; GPR17 (GPR17, GPRH); GPR18 (GPR18, GPRI); GPR20 (GPR20, GPRK); GPR35 (GPR35); PR55 (GPR55); Coagulation factor II receptor (F2R, THRR) ; Opsins InterPro: IPR001760; Rhodopsin (RHO, OPSD); Opsin 1 (cone pigments), short-wave-sensitive (color blindness, tritan) (OPN 1 SW, OPSB) (blue-sensitive opsin); Opsin 1 (cone pigments), medium-wave-sensitive (color blindness, deutan) (OPNIMW, OPSG) (green-sensitive opsin); Opsin 1 (cone pigments), long-wave-sensitive (color blindness, protan) (OPN1LW, OPSR) (red-sensitive opsin); Opsin 3, Panopsin (OPN3); Opsin 4, Melanopsin (OPN4); Opsin 5 (OPN5, GPR136); Retinal G protein coupled receptor (RGR); Retinal pigment epithelium-derived rhodopsin homolog (RRH, OPSX) (visual pigment-like receptor opsin) InterPro : IPR001793 ; 5 -Hydro xytryptamine (5 -HT) receptor InterPro : IPR002231 ; 5-HT2A (HTR2A, 5H2A); 5-HT2B (HTR2B, 5H2B); 5-HT2C (HTR2C, 5H2C); 5-HT6 (HTR6, 5H6) InterPro: IPR002232; Adrenergic receptor InterPro: IPR002233; AlphalA (ADRA1A, A1AA); AlphalB (ADRA1B, A1AB); AlphalD (ADRA1D, A1AD); Alpha2A (ADRA2A, A2AA); Alpha2B (ADRA2B, A2AB); Alpha2C (ADRA2C, A2AC); Betal (ADRB1, B1AR); Beta2 (ADRB2, B2AR); Beta3 (ADRB3, B3AR); Dopamine receptor InterPro: IPR000929; Dl (DRD1, DADR); D2 (DRD2, D2DR); D3 (DRD3, D3DR); D4 (DRD4, D4DR4); D5 (DRD5, DBDR); Trace amine receptor InterPro: IPR009132; TAAR1 (TAAR1, TAR1); TAAR2 (TAAR2, GPR58); TAAR3 (TAAR3, GPR57);TAAR5 (TAAR5, PNR); TAAR6 (TAAR6, TAR4); TAAR8 (TAAR8, GPR102); TAAR9 (TAAR9, TAR3); Histamine H2 receptor (HRH2, HH2R) InterPro: IPR000503; Histamine HI receptor (HRHl , HHIR) InterPro: IPR000921 ; Histamine H3 receptor (HRH3) InterPro: IPR003980; Histamine H4 receptor (HRH4) InterPro: IPR008102; Adenosine receptor InterPro: IPR001634; Al (ADORA1 , AA1R); A2a (ADORA2A, AA2A); A2b (ADORA2B, AA2B); A3 (ADORA3, AA3R); Muscarinic acetylcholine receptor InterPro: IPR000995; Ml (CHRM1 , ACM1); M2 (CHRM2, ACM2); M3 (CHRM3, ACM3); M4 (CHRM4, ACM4); M5 (CHRM5, ACM5); GPR21 (GPR21 , GPRL); GPR27 (GPR27); GPR45 (GPR45, PSP24); GPR52 (GPR52); GPR61 (GPR61); GPR62 (GPR62); GPR63 (GPR63); GPR78 (GPR78); GPR84 (GPR84); GPR85 (GPR85); GPR88 (GPR88); GPR101 (GPR101); GPR161 (GPR161 , RE2); GPR173 (GPR173, SREB3); 5-Hydroxytryptamine (5-HT) receptor InterPro: IPR002231 ; 5-HT1A (HTR1A, 5H1A); 5-HT1B (HTR1B, 5H1B); 5-HT1D (HTR1D, 5H1D); 5- HT1E (HTR1E, 5H1E); 5-HT1F (HTR1F, 5H1F); 5-HT4 (HTR4) InterPro: IPR001520; 5-HT5A (HTR5A, 5H5A); and 5-HT7 (HTR7, 5H7) InterPro: IPR001069.
In some embodiments, the receptor that uses PI3K to signal is a Receptor Tyrosine Kinase
(RTK). As used herein, the term "RTK" refers to a transmembrane receptor that contains a tyrosine kinase in its intracytoplasmic domain n. The RTKs have been divided into a number of classes as follows: RTK class I (EGF receptor family); II (insulin receptor family); III (PDGR receptor family); IV (FGF receptor family); V (VEGF receptor family); VI (HGF receptor family); VII (Trk receptor family); VIII (AXL receptor family); IX (AXL receptor family); X (LTK receptor family); XI (TIE receptor family); XII (ROR receptor family); XIII (DDR receptor family); XV (KLG receptor family); XVI (RYK receptor family); arid XVII (MuSK receptor family). The RTKs that depend upon cytosolic receptors include integrins, interferon receptors, interleukin receptors, GP130 associated proteins, etc. Examples of RTKs include but are not limited to EPOR, GHR, CFSR, PRLR, MPL; IFN Family: IFNAR1 , 2, IFNGR1 ,2; yC Family: IL2RA, B, G, IL4R, IL2RG (Type 1 receptor), IL4R-IL13RAl(Type II receptor), IL7R, IL2RG, IL9R, IL15RA, IL2RB, IL10RA, B, IL12RB 1 , 2, IL13RA1; IL3 Family: IL3RA, CSF2RA, B, IL5RA, GP130 Family: IL6R, IL6ST, IL1 1RA, LIFR, OSMR, IL6GT, CNTFR, IL6ST, and LIFR. Examples of such receptors includes epidermal growth factor receptor (EGFR), insulin receptor, platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR), and nerve growth factor receptor (NGFR). In some embodiments, the RTK is a member of the EGFR family such as EGFR or erbB-1 , erbB-2, erbB-3, or erbB-4. More preferably, the RTK is EGFR, which is a 170 kDa membrane-spanning glycoprotein that binds to, for example, EGF, TNF-a, amphiregulin, heparin-binding EGF (HB-EGF), betacellulin, epiregulin, and NRG2-a. Also preferably, the RTK is HER2, a proto-oncogene that encodes a transmembrane receptor protein of 185 kDa. The RTK may also be a member of the VEGF receptor (VEGFR) family, which includes VEGFR-1, VEGFR-2, VEGFR-3, neuropilin-1 and neuropilin-2. Ligands that bind to VEGFR-1 and VEGFR- 2 include isoforms of VEGF (VEGF121, VEGF 145, VEGF 165, VEGF 189 and VEGF206). In some embodiments, the RTK is a member of t the type III family of receptor tyrosine kinase. As used herein the term "type III family of receptor tyrosine kinases" or "type III RTKs" is intended to include receptor tyrosine kinases which typically contain five immunoglobulin like domains, or Ig-like domains, in their ectodomains. Examples of type III RTKs include, but are not limited to PDGF receptors, the M-CSF receptor, the FGF receptor, the Flt3-receptor (also known as Flk2) and the KIT receptor. In a preferred embodiment of the invention, the type III RTK is KIT (also known in the art as the SCF receptor). KIT, like other type III RTKs is composed of a glycosylated extracellular ligand binding domain (ectodomain) that is connected to a cytoplasmic region by means of a single transmembrane (TM) domain (reviewed in Schlessinger (2000) Cell 103: 211- 225). Another hallmark of the type III RTKs, e.g., KIT, is a cytoplasmic protein tyrosine kinase (PTK) domain with a large kinase-insert region. At least two splice isoforms of the KIT receptor are known to exist, the shorter making use of an in- frame splice site. All isoforms of KIT, and the other above described RTKs, are encompassed by the present invention. The terms "Kit", "KIT" and "KIT receptor", as used herein, include the type III transmembrane receptor tryosine kinase (RTK) that plays crucial roles in mediating diverse cellular processes including cell differentiation, proliferation and cell survival, among other activities, upon binding by any KIT ligand, e.g., Stem Cell Factor (SCF) (reviewed in Schlessinger (2000) Cell 103: 211-225). KIT is also known as the SCF receptor. Like other members of the type III subfamily of RTKs, KIT is composed of an extracellular domain that includes five Ig-like domains (designated D1-D5), a single transmembrane domain, a juxtamembrane region , a tyrosine kinase domain split by a kinase insert and a C-terminal tail. In one embodiment of the invention, the KIT is human KIT. The term "KIT" is also intended to include recombinant human KIT (rhKIT), which can be prepared by standard recombinant expression methods.
In one embodiment, the receptor that uses PI3K to signal is an immunoreceptor. Immunoreceptors comprise, among others, B Cell receptors (BCR), T Cell Receptors (TCR), Fc Receptors (FcR).
In some embodiments, the cell is a B cell. The term "B cell" has its general meaning in the art. B cells are lymphocytes that play a large role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by T cells). In some embodiments, the cell is a mast cell or a basophil. As used herein, the term "mast cell" refers to a granulocyte that contains granules with histamine and heparin. In some embodiments, the term "mast cell" refers to a mastocyte. In some embodiments, the term "mast cell" refers to a basophil. In some embodiments, the term "mast cell" refers to an inactivated mast cell. In some embodiments the term "mast cell" refers to an activated mast cell. In some embodiments, the term "mast cell" refers to a mast cell residing in the bone marrow, in the systemic circulatory system, and/or in organ tissues. In some embodiments, the organ tissue is the lung, the skin, the heart, the brain, the eye, the gastrointestinal tract, the thymus, the spleen, the ear, the nose or combinations thereof. As used herein, the term "basophil" refers to a basophil granulocyte. In some embodiments, the term "basophil" refers to a human basophil progenitor. In some embodiments, the term "basophil" refers to a basophil lineage-committed progenitor. In some embodiments, the term "basophil" refers to a human common myeloid progenitor (hCMP). In some embodiments, the term "basophil" refers to any combination of a basophil granulocyte, a human basophil progenitor, a basophil lineage-committed progenitor, and a human common myeloid progenitor (hCMP). In some embodiments, the term "basophil" refers to a basophil residing in the bone marrow, in the systemic circulatory system, and/or in organ tissues. In some embodiments, the organ tissue is the lung, the skin, the heart, the brain, the eye, the gastrointestinal tract, the thymus, the spleen, the ear, the nose or combinations thereof.
In some embodiments, the cell is a malignant cell. In some embodiments, the malignant cell expresses FcRs. These cells are primarily, but not exclusively, cells of the myeloid lineage. Malignant cells can also be a transformed B cell. Non-hematopietic cells can also express FcRs. One example is malignant melanoma cells that express FcRIIB.
The inventors demonstrated that trans-inhibition is a consequence of SHIPl/2-mediated cis-inhibition.
The inventors demonstrated that SHIP 1/2 is recruited by receptors that are phosphorylated by a Src family tyrosine kinase and that SHIP 1/2 inhibits signals generated by receptors that use PI3K, by hydro lyzing PIP3. SHIP 1/2 can therefore inhibit PIP3 -dependent activation/proliferation signals that depend on all receptors which use PI3K and which are co-expressed with receptors capable of recruiting SHIP 1/2.
Trans-inhibition therefore comprises two phases:
• an induction phase leading to the recruitment of SHIP 1/2
• an effector phase leading to the extinction of PIP3 -dependent signals
SHIP 1/2 can be recruited either: • by activating receptors themselves that use it as an auto-regulation mechanism. These can be ITAM-containing immunoreceptors such as FcRs, BCRs, TCRs, activating NK receptors, etc., RTKs including most growth factor receptors such as KIT, Insulin receptors, EGFR, VEGFR, hormone receptor etc...
• by inhibitory receptors that are co engaged with activating receptors. Prototypic SHIPl/2-recruiting inhibitory receptors are FcyRIIB. FcyRIIB need to be co- aggregated with activating receptors that use a Src kinase that phosphorylates the FcyRIIB ITIM. FcyRIIB can therefore recruit SHIP 1/2 when co-engaged with a variety of activating receptors.
As a consequence, trans-inhibition can be induced by all ligands that co-engage activating receptor or inhibitory receptor like FcyRIIB with activating receptors that use a Src kinase to signal. Such a co-engagement can be induced by bi-specific ligands that can bind to FcyRIIB and to activating receptors. Because FcyRIIB are low-affinity IgG receptors, they can be engaged very efficiently by the Fc portion of antibodies that recognize activating receptors expressed on the same membrane. Antibodies are therefore appropriate tools to induce trans-inhibition.
Thus, in another aspect, the invention relates to a molecule which is capable of co-engaging a FcR like the FcyRIIB with an activating receptor that use a Src kinase to signal.
According to the invention, an activating receptor that use a Src kinase to signal may be an immunoreceptor such as activating FcRs, BCRs, TCRs, RTKs including most growth factor receptors such as KIT, Insulin receptors, - EGFR or VEGFR.
According to the invention, activating FcRs may be FcyRI (CD64), FcyRIIA (CD32a), FcyRIIIA (CD 16), FcyRIIIB (CD 16b), a Fc-alpha receptor like FcaRI (CD89) or Fc-epsilon receptors like FcsRI or FcsRII (CD23).
In one embodiment, the molecule which is capable of co-engaging a FcR like the FcyRIIB with an activating receptor may be a antibody or a plurispecific molecule like a bispecific antibody.
In some embodiments, the molecule which is capable of co-engaging the FcR like the FcyRIIB with an activating receptor is a plurispecific antibody comprising at least one binding site that specifically binds to the FcR like the FcyRIIB receptor, and at least one binding site that specifically binds to the activating receptor.
Exemplary formats for the plurispecific antibody molecules of the present invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to the FcR like the FcyRIIB and another with a specificity to an activating receptor; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single- chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al, Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (viii) a so called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivaient bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab- arm; and (x) a diabody. Another exemplary format for bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies. In some embodiments, the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in WO2008119353 and WO 2011 131746 (both by Genmab A/S). In one exemplary method, described in WO 2008119353, a bispecific antibody is formed by "Fab-arm" or "half- molecule" exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences. In another exemplary method, described in WO 2011131746, bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is a antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is an antibody of the present invention and the second antibody has a different binding specificity, or vice versa. The reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. Step d) may further comprise restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting. Preferably, the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety.
The method of the present invention is thus particularly suitable for inhibiting or preventing activation of the targeted cell. In particular, the present invention is particularly suitable for inhibiting or preventing degranulation of mast cells, activation and proliferation of mast or B cells. In particular, the method of the present invention is particularly suitable for inhibiting mast cell activation induced by the interaction between an antigen and an IgE bound to an activating receptors FCERI of the mast cell. As used herein, the term "inhibiting" in relation to receptor activity means decreasing cell activation compared to uninhibited cells. Inhibition can be assessed by at least a 5% , 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100 % decrease. Methods for assaying inhibition are well known in the art and are particularly described in the EXAMPLE.
The method of the present invention is thus particular suitable for therapeutic purposes. In particular, the method of the present invention is particularly suitable for the treatment of cancer, autoimmune inflammatory diseases and allergies.
Accordingly the further object of the present invention relates to a method of treating a disease selected from the group consisting of cancers, autoimmune inflammatory diseases and allergies in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a molecule which is capable of co-engaging the FcR like the FcyRIIB receptor with an activating receptor.
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
In some embodiments, the subject suffers from a myeloid proliferation disease. Prototypic examples are Chronic Myeloid Leukemias and Mast cell proliferations such as mastocytosis. The term "mastocytosis" as used herein, relates to systemic mastocytosis, or mastocytoma. Mastocytosis is a myeloproliferative disorder with limited treatment options. The pathogenesis of mastocytosis has been attributed to constitutive activation of the receptor tyrosine kinase KIT.
In some embodiments, the subject suffers from a B-cell malignancy. As used herein, the term "B-cell malignancy" includes any type of leukemia or lymphoma of B cells. B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, B-cell pro lymphocytic leukemia, B cell lymphomas (e.g. various forms of Hodgkin's disease, B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g. Waldenstrom's macroglobulinaemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphomas), leukemias (e.g. acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also termed B cell chronic lymphocytic leukemia BCLL), hairy cell leukemia and chronic myoblastic leukemia) and myelomas (e.g. multiple myeloma). Additional B cell malignancies include small lymphocytic lymphoma, B cell prolymphocyte leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.
In some embodiments, the autoimmune inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile- onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic scleroderma, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, Rasmussen's encephalitis, limbic and/or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes such as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospho lipid antibody syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens- Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus, autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP, Guillain- Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non- cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis, optionally benign monoclonal gammopathy or monoclonal garnmopathy of undetermined significance, MGUS, peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post- vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthamopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired splenic atrophy, infertility due to antispermatozoan antobodies, non- malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as Lesihmania, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T- lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen- antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Lo filer's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, ischemic re-perfusion disorder, reduction in blood pressure response, vascular dysfunction, antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, reperfusion injury of myocardial or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.
In some embodiments, the autoimmune inflammatory diseases is secondary to therapeutic treatment, in particular a treatment with an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
In some embodiments, the subject suffers from an allergic disorder. As used herein, "allergic disorder" refers to any disorder resulting from antigen activation of mast cells that results in an "allergic reaction" or state of hypersensitivity and influx of inflammatory and immune cells. Those disorders include without limitation: systemic allergic reactions, systemic anaphylaxis or hypersensitivity responses, anaphylactic shock, drug allergies, and insect sting allergies; respiratory allergic diseases, such asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis and interstitial lung diseases (ILD) (e.g. idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, or other autoimmune conditions); rhinitis, hay fever, conjunctivitis, allergic rhinoconjunctivitis and vaginitis; skin and dermatological disorders, including psoriasis and inflammatory dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiforms, linear IgA disease, acute and chronic urticaria and scleroderma; vasculitis (e.g. necrotizing, cutaneous, and hypersensitivity vasculitis); spondyloarthropathies; and intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, enteritis, nontropical sprue and celiac disease).
In some embodiments, the subject suffers from asthma. As used herein, the term "asthma" refers to an inflammatory disease of the respiratory airways that is characterized by airway obstruction, wheezing, and shortness of breath.
In some embodiments, the subject suffers from any allergic disease in which mast cell and/or basophil activation plays a critical role.
In one embodiment, the allergic disease may be asthma, skin allergy like eczema, urticaria, dermatographism, allergic rhinitis, drug allergy or food allergy.
In some embodiments, the subject suffers from anaphylaxis. As used herein, the term "anaphylaxis" refers to a life threatening allergic reaction characterized by decreased blood pressure, respiratory failure with bronchoconstriction, and skin rash due to release of mediators from cells such as mast cells.
By a "therapeutically effective amount" of the molecule of the present invention is meant a sufficient amount of the molecule to treat the disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the molecule of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific antibody employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibody employed; the duration of the treatment; drugs used in combination or coincidental with the specific antibody employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
According to the invention, the molecule is administered to the subject in the form of a pharmaceutical composition. Typically, the active agent is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- so lid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The active agent can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. 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. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media, which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1: Trans-inhibition of mast cell activation. (A) Concentration-dependent induction of trans-inhibition in mast cells. FcyRIHA" " BMMCs sensitized with mlgE anti-DNP and rlgE (doubly sensitized) were challenged with increasing concentrations of DNP-BSA alone or mixed with increasing concentrations of MAR IgG. The release of β-hexosaminidase was measured and plotted as a function of the concentration of antigen. Data are representative of three independent experiments. (B) Trans-inhibition requires the Fc portion of IgG. Doubly sensitized FcyRIHA"7" BMMCs were challenged with increasing concentrations of DNP-BSA alone, mixed with MAR F(ab')2, or mixed with MAR IgG. β-hexosaminidase release was measured and plotted as described in (B). Data are representative of four independent experiments.
Figure 2: SHIP1 is involved in and necessary for trans-inhibition. Trans-inhibition requires SHIP1. Doubly sensitized SHIP 1+/+ and SHIP \~'~ BMMCs were challenged as indicated with DNP-BSA and MAR IgG for 10 min. β-hexosaminidase release was measured and plotted as a function of the concentration of DNP-BSA (E). Data are representative of two independent experiments. Cell lysates were electrophoresed and Western blotted with the indicated antibodies (F). Data are representative of two independent experiments.
Figure 3: SHIP1 is sufficient for trans-inhibition. (A) Trans-inhibition can be induced equally well by wild-type human FcyRIIB, or by human FcyRIIB whose intracytoplasmic domain was replaced by the catalytic domain of human SHIP1. Doubly sensitized RBL-FcyRIIB/mh and RBL-FcyRIIB-hSHIPl transfectants were challenged with DNP-HSA alone or with DNP-HSA and MAR IgG. β-hexosaminidase release was measured and plotted as a function of the concentration of DNP-HSA. Data are representative of three independent experiments. (B) Trans- inhibition abrogates antigen-induced PI(3,4,5)P3 generation. Doubly sensitized wild-type BMMCs were labeled with 32Pi and challenged with culture medium, DNP-HSA, MAR IgG or DNP-HSA and MAR IgG for 10 min. Phosphoinositides were measured in lipid extracts from duplicates and mean values ± SD were plotted in the histogram. Data are representative of two independent experiments. Figure 4: Trans-inhibition can be induced by several receptors and can affect several cellular responses. Wild-type (WT) and FcyRIIB ~ ~ BMMCs sensitized with mlgE anti-DNP were challenged with increasing concentrations of DNP-BSA or with increasing concentrations of DNP- BSA and immune complexes made with the rat anti-Kit mAb ACK2 and MAR IgG. β- hexosaminidase release was measured and plotted as a function of the concentration of DNP-BSA. Data are representative of three independent experiments performed with WT BMMCs. One experiment only included FcyRIIB ~ ~ BMMCS.
Figure 5: Trans-inhibition can inhibit the in vitro proliferation and the in vivo growth of mastocytoma cells transformed by a v-oncogene. Trans-inhibition induced by co-aggregating FcyR with Kit affects the in vitro proliferation of MMC-1 cells. (A) MMC-1 cells were loaded with CFSE, preincubated with 2.4G2 or without, and incubated with immune complexes made with the indicated concentrations of GST-SCF and rabbit IgG anti-GST antibodies over a 5-day period. Cell-associated CFSE fluorescence was measured and plotted as a function time. (B) Trans-inhibition induced by co-aggregating FcyR affects the in vitro proliferation of MMC-1 cells. MMC-1 cells were incubated for 72h with immune complexes made of GST and the indicated concentrations of rabbit IgG-anti-GST antibodies. Cells were counted in triplicates, and mean ± SD cell numbers were plotted as a function of the concentration of IgG anti-GST antibodies.
Figure 6: Trans-inhibition in murine and human basophils. (D) Trans-inhibition in human basophils. Trans-hibition operates in human basophils. Human blood basophils sensitized with rlgE were challenged with increasing concentrations of RAHE F(ab')2 alone or increasing concentrations of RAHE F(ab')2 and MAR IgG. CD203c on basophils was measured in duplicates and the mean increase in CD203c expression (AMFI) was plotted as a function of the concentration of RAHE F(ab')2. Data shown in (A) are representative of nine independent experiments. Histamine released in supernatants was measured in triplicates and mean values ± SD were plotted. (B) Trans-inhibition can be induced by over-aggregating human FcsRI. Inhibition of human basophil activation induced by over-aggregating FcsRI. Human basophils sensitized with rlgE were challenged with increasing concentrations of MAR F(ab')2. CD203c on basophils was measured in duplicates and the mean increase in CD203c expression (AMFI) was plotted as a function of the concentration of MAR F(ab')2. Data are representative of three independent experiments. P values were calculated using an unpaired Student's t test: * P < 0.05, ** P < 0.01.
EXAMPLE:
Material & Methods Cells. 1) BMMCs were obtained by culturing mouse bone marrow cells in IL-3 -containing medium as described in (9). RBL cells expressing murine FcyRIIB 1 or a chimeric receptor made of mouse FcyRIIB the intracytoplasmic domain of which was replaced by that of KIR2DL3 (RBL- FcyRIIB-KIRL) (22) by that of human FcyRIIB 1 (RBL-FcyRIIB/mh) (46) or by the catalytic domain of human SHIP1 (RBL-FcyRIIB-hSHIPl) (31) were described previously. 2) MMC-1 cells (32) were a kind gift of Dr. Reuben Siraganian (NIH, Bethesda, MD). 3) Mouse basophils were obtained by culturing non-adherent mouse bone marrow cells in IL-3 -containing medium for 8 days. Basophils were enriched by magnetic depletion of Kit+ cells (Miltenyi). The purity of basophils thus obtained was generally 90%. 4) Human basophils were examined in peripheral blood mononuclear cells (PBMC) isolated by Ficoll-Hypaque density gradient centrifugation from blood samples obtained from the Etablissement Francais du Sang (EFS, Paris, France) in accordance with a convention between Institut Pasteur and EFS. This study was approved by the Comite de Protection des Personnes (CPP, He de France) and the Ministere de l'Education Nationale de la Recherche et de Technologie (Declaration collective 2008-68). All donors provided written informed consent for the collection of samples and subsequent analysis.
Reagents. The rat anti-mouse Kit mAb ACK-2 (47), the rat anti-mouse FcyRIIB/IIIA mAb 2.4G2 (48) and the mouse anti-FcyRIIB K9.361 were affinity-purified on Protein G-sepharose. The rat anti-mouse FcyRIIIA mAb 275003 (49) was from R&D Systems (Lille, France). DNP-BSA and TNP-BSA-Biotin were prepared as described. Glutathion S-transferase (GST)-SCF was produced in E. Coli and affinity-purified on Glutathion-agarose. DNP-HSA was purchased from Sigma- Aldrich. Mouse IgG anti-rat Ig (MAR IgG) and corresponding F(ab')2 fragments [MAR F(ab')2], Fluorescein isothiocyanate (FITC)-conjugated Rat anti-mlgE antibodies and FITC-conjugated Mouse anti-rat Ig antibodies were from Jackson Immunoresearch; Rabbit IgG anti-human IgE (RAHE) from Dako-Cytomation; Phycoerythrine (PE)-conjugated anti-CD203c antibodies and Allophycocyanin (APC)-conjugated anti- human FcsRIa antibodies from Bio legend; Rabbit anti- GST antibodies, Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies, normal mouse IgG and mouse anti-pPLCy-l antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); Rabbit anti-Akt, pAkt, pSyk, pGab2, pNF-κΒ, pp38 MAPK, pp44/42 MAPK and pJNK antibodies from Cell Signaling Technology (Beverly, MA); rabbit anti-pLAT antibodies from Upstate Biotechnology (Lake Placid, NY); rabbit anti-pSHIPl from Stemcell Technologies (Grenoble, France). K9.361 and RAHE F(ab')2 were prepared by pepsin digestion. Mast cell activation. BMMCs were sensitized by an overnight incubation at 37°C with 0.1 μg/ml mlgE anti-DNP 2682-1 (50) and 3 μg/ml rlgE IR162 (51) and challenged for various periods of times with the indicated concentrations of ligands. β-hexosaminidase was quantitated in 10-min supernatants using an enzymatic assay (9). LTC-4 and MIPl-a were titrated in 30-min supernatants by ELISA (Neogen-corporation and R&D systems respectively). TNF-a was titrated in 3-h supernatants by an ELISA (R&D systems).
Systemic Anaphylaxis. 6-8-week old C57BL/6J mice (Charles River) were injected i.v. with supernatants from BMMCs sensitized with mlgE 2682-1 anti-DNP and rlgE IR162, harvested 10 min after challenge with indicated ligands. Alternatively, C57BL/6J mice were injected i.v. with 4 x 106 BMMCs sensitized with mlgE 2682-1 anti-DNP and rlgE IR162 and incubated with MAR IgG for 10 min. Mice were challenged by an i.v. injection of 500 μg DNP-BSA 15 min later. Rectal temperature was monitored using a Precision Digital Thermometer 4600 (YSI, Dayton, Ohio, USA).
Calcium mobilization. BMMCs sensitized with mlgE 2682-1 anti-DNP and rlgE IR162 were loaded with 0.5 μΜ Fluo-3-AM (Invitrogen, Carlsbad, CA) for lh at room temperature, and analyzed by flow cytometry (Becton Dickinson) before and after stimulation.
Western blot analysis. BMMCs sensitized with mlgE anti-DNP 2682-1 and rlgE IR162 were challenged with 10 ng/ml DNP-BSA, 15 μg/ml MAR IgG or both for 1 , 3 or 10 min, lysed in SDS at 95°C, fractionated by SDS-PAGE and Western blotted with indicated antibodies. HRP was detected using an ECL kit (Amersham Biosciences, Buckinghamshire, UK).
PI(3,4,5)P3 measurement. BMMCs sensitized with mlgE anti-DNP 2682-1 and rlgE IR162 were labeled for 8 h with 1 mCi/ml [32P]orthophosphate (Perkin Elmer) in phosphate-free DMEM (Invitrogen) before they were challenged for 10 min with DNP-HSA, MAR IgG or both. Lipids were immediately extracted, separated by thin-layer chromatography and analyzed by HPLC as described previously (52).
Proliferation assays. 1) 3H-thymidine incorporation. MMC-1 cells were preincubated with 10μg/ml 2.4G2 or without for 15min at 37°C and seeded at lxl05/ml with culture medium, with preformed immune complexes made of 5 μg/ml GST-SCF and 50 μg/ml Rabbit a-GST antibodies, or with immune complexes made of 10 μg/ml GST and 50 μg/ml Rabbit a-GST antibodies. 48h later, the same immune complexes were added, with 2.4G2 or without. 3H-thymidine was added at 72h, and cells were further incubated for 18h before cell-associated radioactivity was measured. 2) CFSE dilution assay. MMC-1 cells were labeled with CFSE for 10 min at room temperature, incubated with 2.4G2 or without, challenged with immune complexes made of GST-SCF and Rabbit a-GST for 10 min, and cultured at lxlO5 cells/ml for the indicated periods of time. Fluorescence was analyzed by flow cytometry using a FACScalibur.
Tumor growth assessment. 3 x 105 MMC-1 cells were incubated for 15 min with preformed GST-SCF-rabbit anti-GST, GST-rabbit anti-GST immune complexes or culture medium, and injected s.c. into Nude, Rag7" or Rag7 VFcRy7" mice. Tumor volume was measured over 15 d.
Human basophil stimulation. Human PBMC (lxlO6 cells/ml in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 1% hepes buffer and 1% penicillin-streptomycin) were incubated overnight at 37°C with 3 μg/ml rlgE (53) IR162 or without. Non-sensitized PBMC were challenged with Rabbit anti- human IgE (RAHE). PBMC sensitized with rlgE were challenged with the indicated concentrations of RAHE and MAR. After 20 min, cells were stained with PE-conjugated anti-CD203c and APC-conjugated anti-FcsRIa. CD203c up-regulation (AMFI) was monitored in Fc8RI+ cells using a FACScalibur (BD-Biosciences) or a MACSQuant (Miltenyi-Biotec). Histamine was measured in 45 -min supernatants by ELISA (Neogen Corporation). For histamine release, PBMC sensitized with rlgE were challenged with 10μg/ml RAHE F(ab')2 alone or with 10μg/ml RAHE F(ab')2 and 30μg/ml MAR IgG. Histamine released in supernatants was measured 45 min later by ELISA (Neogen Corporation).
Statistical analysis. Statistical analyses were performed with the GraphPad Prism 5 software (GraphPad Software). The statistical significance of differences between two groups was calculated with a two-tailed, unpaired Student's t test. The results are provided as P values, where P < 0.05 is considered statistically significant.
Results
FcyRIIB inhibit mast cell activation induced by FCERI molecules other than those with which FcyRIIB are co-engaged.
Bone marrow-derived mast cells (BMMCs) from FcyRIIIA-deficient mice possess activating IgE receptors (FcsRI) and inhibitory IgG receptors (FcyRIIB). Such cells were used to exclude the possibility that activating IgG receptors (FcyRIIIA) could be co-engaged with FcyRIIB by IgG antibodies. Cells were sensitized with a mouse IgE (mlgE) anti-dinitrophenyl (DNP) and a myeloma rat IgE (rlgE) of an unknown specificity, which could be engaged independently by specific ligands (data not shown). When doubly sensitized, cells were activated by either ligand (data not shown) that engaged one FcsRI -bound IgE only (data not shown). As expected, mouse anti-rat Ig (MAR) F(ab')2 activated BMMCs sensitized with rlgE, whereas intact MAR IgG did not (data not shown). When binding to receptor-bound rlgE, MAR F(ab')2 indeed aggregate FcsRI, and they activate cells, whereas MAR IgG co-aggregate FcsRI through their Fab portions and FcyRIIB through their Fc portion, and they inhibit cell activation. Surprisingly, MAR IgG also inhibited cell activation induced by the engagement of FcsRI -bound mlgE with antigen in doubly sensitized cells (Fig. 1 A). Thus, FcyRIIB not only inhibited activation signals induced by FcsRI molecules with which they were co-aggregated (cis-inhibition), but also activation signals induced by other FcsRI molecules that were independently aggregated on the same cells (trans-inhibition).
Trans-inhibition was induced neither by MAR F(ab')2 that could engage FcsRI but not FcyRIIB (Fig. IB) nor by intact normal mouse IgG that could not engage any receptor (data not shown). Trans-inhibition was abrogated in FcyRIIB-deficient cells and, importantly, it was as efficient in wild-type cells as in FcyRIIIA-deficient cells (data not shown). Trans-inhibition therefore required that the Fc portion of MAR IgG bound to FcyRIIB, and the negative effect of FcyRIIB was dominant over the positive effect of FcyRIIIA that were co-engaged with FcyRIIB by MAR IgG in wild-type mast cells. Trans-inhibition affected cell activation by other anti-DNP IgE antibodies (data not shown) or by IgE antibodies with another specificity (data not shown). It was not due to a decreased accessibility of IgE antibodies for antigen (data not shown). Trans- inhibition persisted if antigen challenge was delayed after the addition of MAR IgG (fig. S6), but it was abrogated after a few hours (data not shown).
Trans-inhibition was observed when assessing the release of granular mediators or the secretion of eicosanoids, chemokines, and cytokines (data not shown). It decreased the production of all the mediators that account for systemic anaphylaxis. Indeed, trans-inhibition abrogated the loss of body temperature in wild-type mice injected intravenously with supernatant collected 10 min after the challenge of doubly sensitized BMMCs with antigen (data not shown). Trans- inhibition also abrogated the loss of body temperature induced by an intravenous injection of antigen into mice injected previously with doubly sensitized BMMCs that were challenged with MAR IgG 10 min earlier (data not shown). Trans-inhibition can therefore dampen in vitro reactions and their systemic in vivo consequences.
Trans-inhibition involves and requires the lipid phosphatase SHIP1.
Trans-inhibition abrogated mediator release induced by antigen, but had no effect mediator release induced by PMA and ionomycin (data not shown), i.e. it acted upstream of the Ca2+ response. It indeed dampened antigen-induced Ca2+ mobilization (data not shown), as well as Akt, Erkl/2, JNK, p38 and NF-κΒ phosphorylation but not Syk, LAT, Gab2 and PLCy-l phosphorylation (data not shown), i.e. distal, but not proximal signals that were rather enhanced (data not shown). SHIP1 was heavily phosphorylated (data not shown). Trans-inhibition was abrogated in BMMCs from SHIP 1 -deficient mice, while mediator release was dramatically enhanced (Fig. 2), as well as Akt, Erkl/2, JNK, p38 and NF-κΒ phosphorylation (data not shown).
Trans-inhibition was also induced by FcyRIIB in RBL-2H3 transfectants, but not by FcyRIIB with a KIR2DL3 intracytoplasmic domain (FcyRIIB-KIRL) (data not shown). KIR2DL3 is a typical NK cell inhibitory receptor whose intracytoplamsic domain contains two ITIMs that have an affinity for the SH2 domain-containing tyrosine phosphatases 1 and 2 (SHP-1 and SHIP- 2), but not for SHIP1 or SHIP2 (22). We previously used these two chimeric molecules to demonstrate that both could similarly cis-inhibit FcsRI signaling in mast cells (data not shown) (23) although FcyRIIB and KIR2DL3 recruit SHIP1 and SHP-1, respectively (24). Trans- inhibition therefore involves, depends on and requires the lipid phosphatase SHIP1. It is not due to the mere addition of inhibitory signals of any nature. Trans-inhibition is a property that FcyRIIB do not share with the numerous other ITIM-containing inhibitory receptors which recruit SHP-1 but not SHIP1 (25).
Trans-inhibition can be explained if PI(3,4,5)P3 can diffuse from activating signalosomes where this phospholipid was generated, as shown in immunological synapses (26), and meet SHIP1 where this enzyme was recruited. It can also be explained if SHIP1 can leave inhibitory signalosomes where it was recruited, as shown in B cells (27), and meet PI(3,4,5)P3 where it was generated. Two sets of data discriminated between these two possibilities. First, chimeric molecules made of mouse (m) FcyRIIB whose intracytoplasmic domain was replaced by that of human (h) FcyRIIB (FcyRIIB/mh) or by the catalytic domain of human SHIP1 (FcyRIIB/hSHIPl) induced trans-inhibition of comparable magnitudes (Fig. 3 A). SHIP1 is therefore not only necessary but also sufficient for trans-inhibition, and trans-inhibition may not require the phosphatase to leave inhibitory signalosomes. Second, antigen-induced increase of intracellular PI(3,4,5)P3 concentration was prevented in both cis- and trans-inhibition (Fig. 3B). SHIP1 therefore hydrolyzed not only PI(3,4,5)P3 generated by FcsRI that were co-ligated with FcyRIIB, but also PI(3,4,5)P3 generated by FcsRI that were not. Consequently, co-engaging FcyRIIB with a fraction of FcsRI can induce a global inhibition of PI(3,4,5)P3-dependent cell signaling.
Mast cell activation is also inhibited when FcsRI are over-aggregated by an excess of extracellular ligand (28). Indeed, the over-aggregation of FcsRI generates SHIP 1 -dependent inhibition signals (29) that are dominant over activation signals. Further supporting a global effect of SHIP 1, we found that an excess of antigen inhibited not only cell activation induced by specific mlgE, but also cell activation induced by an optimal concentration of MAR F(ab')2 in doubly sensitized cells (data not shown). Trans-inhibition can therefore be induced either by co- aggregating FCKRI with FcyRIIB (co -regulation) or by over-aggregating FcsRI (auto-regulation).
Trans-inhibition can prevent the in vitro and the in vivo growth of transformed cells whose proliferation depends on a viral oncogene.
We reported previously that FcyRIIB can cis-inhibit proliferation signals when co-ligated with the Stem Cell Factor (SCF) Receptor-Tyrosine Kinase (RTK) Kit (30) and that inhibition depends on SHIP1 (31). We show here that antigen- induced FcsRI-dependent mast cell activation was also inhibited when co-engaging FcyRIIB with Kit (Fig. 4A). Conversely, SCF-induced mast cell proliferation was inhibited when co-aggregating FcsRI with FcyRIIB (Fig. 4B). Trans- inhibition can therefore act upon growth factor-induced RTK-dependent cell proliferation, i.e. a process other than a secretory response, and be induced by co-aggregating FcyRIIB with RTK i.e. with receptors other than ITAM-containing receptors.
MMC-1 is a mouse mastocytoma tumor that originated in mice infected with the Abelson murine leukemia virus (32). MMC-1 cells express the transforming oncogene v-Abl (33), activating (FcyRIIIA) and inhibitory (FcyRIIB) IgG receptors (FcyR) but no FcsRI, and Kit (fig. S9) the transmembrane and intracytoplasmic domains of which contained no mutation. v-Abl, which was constitutively phosphorylated in MMC-1 cells (data not shown), accounted for their proliferation as the latter was inhibited by the Abl inhibitor STI571 (34) at 100-fold lower concentrations than by the multi-RTK inhibitor SU11248 (35) (data not shown). MMC-1 proliferation was also dose-dependently inhibited when FcyR were co-aggregated with Kit by GST-SCF-anti-GST immune complexes (Fig. 5 A), and inhibition was prevented by the blocking anti-FcyRIIB+IIIA mAb 2.4G2. Thus, FcyRIIB trans-inhibited v-Abl-dependent proliferation signals when co-engaged with FcyRIIIA and Kit in MMC-1 cells. This trans-inhibition was observed in vitro, by assessing thymidine incorporation (data not shown), cell numbers (not shown) or CSFE dilution (Fig. 5 A). It was also observed in vivo, as MMC-1 tumor growth was prevented if cells were incubated for 15 min with GST-SCF-anti-GST immune complexes before they were injected into Nu/Nu or Rag-/- mice which are unable to reject allogeneic tumors. Tumor growth was also markedly reduced in Rag-/- FcRy-/- mice that lack activating Fc Receptors, excluding tumor rejection by antibody-dependent cell-mediated cytotoxicity (data not shown). Noticeably, trans-inhibition did not require Kit as it was similarly induced by GST-anti-GST immune complexes that could co-engage FcyRIIB with FcyRIIIA only (Fig. 5 B). This was observed both in vitro (Fig. 5 B) and in vivo (data not shown). Thus, trans-inhibition both decreased the in vitro proliferation and prevented the in vivo growth of v-oncogene-transformed tumor cells in immunodeficient mice, when FcyRIIB were co-aggregated with FcyRIIIA and Kit or with FcyRIIIA only, by IgG immune complexes.
Trans-inhibition operates and is long-lasting in human basophils.
Wild-type mouse bone marrow-derived basophils (BMB) sensitized with rlgE secreted IL- 4 upon challenge with MAR F(ab')2, which aggregate FcsRI-bound rlgE but, as expected (11), not with MAR IgG, which co-aggregate FcsRI-bound rlgE with basophil FcyRIIB and IIIA. MAR F(ab')2 and IgG, however, induced similar IL-4 secretion in FcyRIIB-deficient BMB (data not shown). As previously observed (11), cis-inhibition therefore operates in mouse basophils. Antigen- induced IL-4 secretion was comparable in wild-type and FcyRIIB-deficient BMB doubly sensitized with mlgE anti-DNP and rlgE. It was decreased by MAR IgG in wild-type BMB, but markedly increased in FcyRIIB-deficient BMB (data not shown). Trans-inhibition therefore operates in primary mouse basophils.
Likewise, human basophils can be activated by rabbit anti- human IgE (RAHE) F(ab')2 that aggregate FcsRI -bound human IgE (Fig. 6 A) and, when sensitized with rlgE, by MAR F(ab')2 (data not shown). Basophil activation was much milder in response to MAR IgG that co-engage FcsRI with FcyR (data not shown). As previously observed (11), FcyR-dependent cis-inhibition therefore operates in human basophils. MAR IgG also inhibited RAHE F(ab')2-induced basophil activation, whether assessed by CD203c up-regulation (Fig. 6 A) or histamine release (data not shown), in cells sensitized with rlgE. Conversely, RAHE IgG inhibited MAR F(ab')2-induced basophil activation (data not shown). Trans-inhibition therefore operates in primary human peripheral blood basophils. It persisted if RAHE F(ab')2 was added to rlgE-sensitized basophils after MAR IgG (data not shown), up to 72h (data not shown). Inhibition was not due to cell death as basophils still responded to PMA+ionomycin (data not shown). Trans-inhibition is therefore long-lasting in human basophils.
Finally, responses of rlgE-sensitized human basophils to increasing concentrations of
MAR F(ab')2 displayed a bell-shaped curve (Fig. 6 B). RAHE F(ab')2-induced basophil activation was abrogated by an excess of MAR F(ab')2 in rlgE-sensitized cells (Fig. 6 C). Trans-inhibition can therefore be induced in human basophils as in mouse mast cells, by over-aggregating FcsRI.
Discussion:
We describe here trans-inhibition, a novel regulatory mechanism by which antibodies can anergize mast cells and basophils when engaging Fc Receptors. Trans-inhibition operated in normal primary mast cells and basophils, whether of murine or human origin, and human FcyRIIB trans-inhibited similarly as murine FcyRIIB. It dampened all IgE-induced mast cell secretory responses, including the release of all mediators that account for anaphylaxis. It also acted upon the growth factor-induced proliferation of normal mast cells and, importantly, the growth factor- independent autonomous proliferation of cells transformed by the oncogene v-Abl. It could also prevent the growth of tumors in immunodeficient mice injected with these cells. Trans-inhibition is therefore likely to affect a wide array of biological responses, whether physiological or pathological. It can possibly be exploited as a novel therapeutic tool, especially but not exclusively, in allergy.
Trans-inhibition and cis-inhibition have common characteristics. Both require a similar co- aggregation or over-aggregation of Fc receptors. Both use the lipid phosphatase SHIP1 which prevents the PH domain-mediated membrane translocation of critical signaling molecules by hydrolyzing PI(3,4,5)P3. Cis- and trans-inhibition, however, differ in their consequences. Unlike cis-inhibition that acts upon signals generated by the activating receptors that are either co- aggregated with inhibitory receptors or over-aggregated, trans-inhibition acts upon signals generated by activating receptors that are engaged independently. Trans-inhibition is a global phenomenon whereas cis-inhibition is a local phenomenon. Trans-inhibition is a collateral effect that goes along with cis-inhibition and dramatically expands the regulatory properties of Fc Receptors. Another difference is that all ITIM-containing receptors can cis-inhibit activation signals, whereas FcyRIIB, but not other ITIM-containing receptors, can trans-inhibit.
These two differences can be explained as follows. The overwhelming majority of ITIM- containing receptors such as KIRLs, recruit the tyrosine phosphatases SHP-1 and SHP-2. Few ITIM-containing receptors, among which FcyRIIB, recruit the inositol phosphatases SHIP1 and SHIP2. Both types of inhibitory receptors require to be co-engaged with activating receptors to inhibit, but for different reasons: co-aggregation brings tyrosine phosphatases close to their substrates in the first case, whereas it brings tyrosine kinases close to their substrates in the second case. Aggregation is sufficient for KIRLs to be phosphorylated and to recruit SHP-1 and SHP-2. Co-aggregation enables tyrosine phosphatases to dephosphorylate phospho-proteins in signalosomes generated by activating receptors. FcyRIIB need to be co-aggregated with activating receptors in order to be phosphorylated and to recruit SHIP 1/2. These phosphatases can then hydrolyze PI(3,4,5)P3 generated by activating receptors, whether the latter were co-engaged with FcyRIIB or not. Trans-inhibition is a property of lipid phosphatases, but not of protein phosphatases.
SHIP1 was indeed 1) involved, 2) necessary and 3) sufficient for trans-inhibition. First, SHIP1 was heavily tyrosyl-phosphorylated in cells submitted to trans-inhibition, indicating that it was recruited in signalosomes where it became a substrate of Lyn (36). Second, trans-inhibition was abrogated in SHIP 1 -deficient mast cells. Third, trans-inhibition could be induced by FcyRIIB whose intracytoplasmic domain was replaced by the catalytic domain of SHIPl . Unlike activating signalosomes subjected to cis-inhibition, activating signalosomes subjected to trans-inhibition are distinct from inhibitory signalosomes where SHIPl is recruited. Two conditions can enable this enzyme to meet its substrate. PI(3,4,5)P3 can diffuse from activating signalosomes where it was generated, and reach SHIPl in inhibitory signalosomes. In T cells, PI(3,4,5)P3 is generated at the leading edge of immunological synapses where PI3K is activated. Increased amounts of PI(3,4,5)P3 were however observed not only within immunological synapses, but also outside (26). Alternatively, SHIPl can leave inhibitory signalosomes where it was recruited and reach PI(3,4,5)P3 elsewhere. When phosphorylated upon BCR engagement by antigen (27) or co- engagement with FcyRIIB by IgG antibodies (37) in B cells, SHIPl and Dok-1 can form bidentate complexes. These complexes can leave receptor aggregates, and Dok-1 can target SHIPl to other PI(3,4,5)P3-enriched membrane areas via its PH domain. Our finding that FcyRIIB containing the catalytic domain of SHIPl as a surrogate intracytoplasmic domain trans-inhibited similarly as wild-type FcyRIIB has two implications. It suggests that the tyrosine/proline-rich C-terminal domain of SHIP 1 is not mandatory for trans-inhibition and, therefore, that the adapters Dok- 1 (37), Grb2 and Grap (38) are dispensable for trans-inhibition, once SHIPl has been recruited by FcyRIIB. It also suggests that SHIPl may not need to leave the inhibitory signalosome in order to trans-inhibit. This assumption is supported by the observation that the co-engagement of FcyRIIB with a fraction of FcsRI led to a global decrease in cellular PI(3,4,5)P3 that was not restored by an additional engagement of FcsRI. This observation explains that trans-inhibition was as efficient in wild-type as in FcyRIIIA-deficient BMMCs. FcyRIIB-dependent negative regulation is indeed dominant over FcyRIIIA- or FcyRIIA-dependent cell activation in mouse BMMCs (9) and in human basophils (11), respectively. Incidentally, the recruitment of not every PH-domain- containing molecule is equally sensitive to the hydrolysis of PI(3,4,5)P3 by SHIPl . As observed previously (39), SHIPl reduced Ca2+ responses but did not detectably decrease the phosphorylation of PLC-yl . When recruited to the plasma membrane, PLC-yl is embedded in a cluster of molecules (40) including the cytosolic adapters SLP76, Grb2, and GADS that cooperatively bind to the transmembrane adapter LAT1 (41). Under these conditions, reduced PI(3,4,5)P3 levels may be sufficient to reduce the influx of extracellular Ca2+, but not to prevent PLC-yl from being recruited and activated.
Trans-inhibition was demonstrated by co-engaging FcyRIIB with FcsRI sensitized with two non-crossreacting IgE on mouse mast cells, mouse basophils and human basophils. This condition mimics the situation in allergic patients, especially patients subjected to immunotherapy, and possibly in non-allergic individuals. Under this condition, trans-inhibition was 1) induced by FcyRIIB, 2) when these inhibitory receptors were co-ligated with FcsRI, and 3) assessed on biological responses triggered by FcsRI. However, neither the induction nor the effects of trans- inhibition were restricted to these receptors.
Trans-inhibition could be induced when FcyRIIB were co-engaged with receptors other than FcsRI. It was indeed observed when co-engaging FcyRIIB with a growth factor receptor such as Kit. It was probably also induced by co-engaging FcyRIIB with FcyRIIIA in MMC-1. Possibly pertaining to trans-inhibition, the co-aggregation of FcyRIIB with BCR inhibited chemotaxis in murine lymphoma A20 B cells (42). These findings suggest that trans-inhibition may be induced whenever FcyRIIB are co-aggregated with activating receptors that use a src family kinase capable of phosphorylating the FcyRIIB ITIM.
Trans-inhibition could be induced by receptors other than FcyRIIB, including activating immunoreceptors. It could indeed be induced by FcsRI, independently of FcyRIIB, in mouse mast cells and human basophils challenged with a supra-optimal concentration of antigen, which induces a massive recruitment of SHIP1 by FcsRI (29). This finding indicates that the capability to trans-inhibit is restricted neither to FcyRIIB nor to IgG antibodies. It suggests that trans- inhibition may be induced by any receptor, whether inhibitory or activating, capable of recruiting enough SHIP1 into signalosomes, whether during co-regulation or during auto-regulation (fig. SI 6). Trans-inhibition is neither a property of antibodies nor a property of Fc Receptors that are engaged by antibodies. It is a property of the lipid phosphatase SHIP1.
Trans-inhibition could act upon signals generated by receptors other than FcsRI or even other than immunoreceptors, including proliferation signals generated by a transforming oncogene. It indeed decreased not only the SCF-induced Kit-dependent proliferation of normal mast cell, but also the constitutive v-Abl-dependent proliferation of mastocytoma cells. A variety of biological responses can therefore be trans-inhibited by FcyRIIB. This finding suggests that trans-inhibition may affect biological responses induced by any receptor that activates PI3K and uses PI(3,4,5)P3 to recruit PH domain-containing signaling molecules. G Protein-coupled receptors might be such receptors as FcyRIIB-dependent inhibition of CXCL12-induced chemotaxis in A20 cells also depended on SHIP1 (42). Numerous receptors that control the activation and the proliferation of hematopoietic cells use PI(3,4,5)P3 and are under the control of SHIP1, as shown by the pleiotropic effects of SHIP1 deletion (43). Many among these receptors are expressed by mast cells (44). The biological significance of trans-inhibition was further enhanced by the discovery that most SH2 domains bind to plasma membrane lipids with a high affinity and, many as specifically as PH domains (45). Interactions between PI(4,5)P2 and the C- terminal SH2 domain of ZAP70 enables this Syk family cytosolic kinase to associate with the plasma membrane before TCR engagement and to be further recruited by PI3K-induced PI(3,4,5)P3 upon TCR ligation. Such sequential interactions with membrane lipids facilitate the binding of ZAP70 to phosphorylated TCRD ITAMS and the subsequent T cell activation. Many SH2 domain mutations involved in diseases are located in the lipid-binding sites of SH2 domains.
In conclusion, trans-inhibition is a novel SHIP- 1 -dependent regulatory mechanism that can prevent Fc Receptor-expressing cells from responding to a variety of extracellular or intracellular activation and proliferation signals that depend on PI(3,4,5)P3. Fc Receptor-dependent trans- inhibition endows antibodies with previously unsuspected regulatory properties and new biological significance. It provides theoretical grounds for novel therapeutic approaches. Ligands capable of co-engaging FcyRIIB with receptors that use PI3K without activating mast cells, might be engineered and used for an allergen-nonspecific "universal" desensitization in allergic patients.
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Claims

CLAIMS:
1. A method of inhibiting biological responses induced by a receptor that uses PI3K to signal in a cell that coexpresses receptors for the Fc portion of antibodies (FcR) that recruit the lipid phosphatase SHIP1 and/or SHIP2.
2. The method of inhibiting according to claim 1 wherein the FcR is FcyRIIB.
3. A molecule which is capable of co-engaging a FcR like the FcyRIIB with an activating receptor that uses a Src kinase to signal.
4. The molecule according to claim 3 wherein the activating receptor that uses a Src kinase to signal may be an immunoreceptor such as activating FcRs, BCRs, TCRs or RTKs including most growth factor receptors such as KIT, Insulin receptors, EGFR or VEGFR.
5. The molecule according to claims 3 or 4 wherein this molecule is an antibody.
PCT/EP2017/070997 2016-08-22 2017-08-21 Methods of inhibiting biological responses induced by a receptor that uses pi3k to signal in a cell WO2018036947A1 (en)

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