WO2003064662A1 - Molecules bispecifiques reticulant itim et itam pour le traitement des allergies - Google Patents

Molecules bispecifiques reticulant itim et itam pour le traitement des allergies Download PDF

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WO2003064662A1
WO2003064662A1 PCT/US2001/008671 US0108671W WO03064662A1 WO 2003064662 A1 WO2003064662 A1 WO 2003064662A1 US 0108671 W US0108671 W US 0108671W WO 03064662 A1 WO03064662 A1 WO 03064662A1
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determinant
targets
bispecific
bispecific molecule
itam
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PCT/US2001/008671
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David Thomas
Sunny Tam
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Tanox, Inc.
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Priority to PCT/US2001/008671 priority Critical patent/WO2003064662A1/fr
Priority to AU2001247543A priority patent/AU2001247543A1/en
Publication of WO2003064662A1 publication Critical patent/WO2003064662A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the immune system is our defense against a variety of antigens, including those associated with bacteria, viruses, and cancerous cells.
  • Allergic diseases including asthma, allergic rhinitis, and atopic dermatitis affect as much as 5 to 10% of the U.S. population. Asthma alone is estimated to affect more than 15 million Americans, with more than 5000 dying annually. More than 10% of children have allergic dermatitis at some point during their childhood (Leung, D., Mol. Gen. and Metab. 63:157-167 (1998).
  • the prevalence of allergic diseases and their associated morbidity has risen dramatically over the last 20 years. Effective therapies that lack deleterious side effects for the management of these diseases are lacking.
  • TCR T-cell antigen receptor
  • BCR B-cell antigen receptor
  • FCR Fc portion of different immunoglobulin subclasses
  • Activation or inhibition of the immune response occurs at the receptor site and involves two different motifs: the immunoreceptor tyrosine-based activation motif (ITAM) and the immunoreceptor tyrosine-based inhibition motif (ITIM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • IgE is a well known mediator of the immediate-type hypersensitivity allergic reactions including, e.g., allergic rhinitis ("hay fever"), extrinsic asthma, and food and drug allergies. IgE is secreted by, and expressed on the surface of B-cells or B-lymphocytes. Upon exposure of a mammal to an allergen (antigen), the B-cells specific for that particular antigen are "activated” and develop into IgE-secreting plasma cells. The allergen-specific IgE circulates through the bloodstream. In IgE-mediated allergic reactions, IgE binds through its Fc portion to Fc ⁇ RI receptors present on the surface of basophils, mast cells, and Langerhans cells.
  • IgE bound to the surface of these cells then contacts and binds an allergen, this causes a cross-linking of the bound IgE molecules and hence the underlying receptors, and triggers the release of pharmacologic mediators, such as histamine, serotonin, leukotrienes and the slow-reacting substance of anaphylaxis. These mediators cause the pathologic manifestations of allergic reactions. Many IgE-mediated allergic conditions or diseases could be inhibited or ameliorated if mast cell activation could be avoided. Cross- linking of an ITAM with an ITIM may cause such a negative regulation of mast cell activation.
  • the coligation in this International patent application was accomplished using an F(ab') 2 antibody fragment targeting the light chains of both the B23.1 antibody and rat IgE.
  • the mast cells were primed with mAb B23.1 and rat IgE, and then the F(ab') 2 fragment was added. Inhibition of exocytosis as compared with controls was evident. This inhibitory effect is believed to be caused by inhibition of the signal transduction cascade that otherwise leads to the release of such mast cell mediators.
  • the present invention relates to bispecfic molecules comprising a first determinant that targets an Immunoreceptor Tyrosine-Based Activation Module (ITAM) and a second determinant that targets an Immunoreceptor Tyrosine-Based Inhibition Module (ITIM) in order to inhibit mast-cell degranulation or inhibit cytokine release.
  • TAM Immunoreceptor Tyrosine-Based Activation Module
  • ITIM Immunoreceptor Tyrosine-Based Inhibition Module
  • embodiments of this invention include first a determinant that targets, e.g., IgE, allergen, or Fc ⁇ RI, as well as a second determinant that targets, e.g., HM18.
  • bispecific molecules of the present invention may include proteins (including peptides), antibodies, or fragments thereof.
  • Antibody fragments may include, e.g., Fab, F(ab') 2 , and Fv.
  • compositions comprising the bispecific molecules of the invention together with a pharmaceutically acceptable carrier, excipient, stabilizer or other additive.
  • Another embodiment of the present invention relates to methods of treating IgE-mediated allergic diseases and other conditions associated with cell activation, wherein therapeutically effective amounts of the bispecific molecule or a pharmaceutical preparation thereof are administered.
  • Another embodiment of the present invention relates to gene therapy approaches using nucleotides encoding such bispecific molecules for expression in vivo.
  • the bispecific molecules may include bispecific antibodies or diabodies, having one determinant derived from an anti-ITAM antibody (including Fc ⁇ RI) and the other from an anti-ITIM antibody (including HM18).
  • bispecific molecules or antibodies can be used, one example being two single chain Fv antibodies linked together.
  • these bispecific molecules bind, directly or indirectly, to an ITAM (e.g., Fc ⁇ RI) and to an ITIM (e.g. HM18), which are both present on mast cell surfaces. This binding will likely result in the inhibition of cell activation. In the case where the bispecific molecule binds Fc ⁇ RI and HM18, this would inhibit exocytosis and release of the pharmacologic mediators of allergic diseases.
  • ITAM e.g., Fc ⁇ RI
  • ITIM e.g. HM18
  • bispecific molecules of the invention is formed by conjugating two single chain antibodies, one derived from an antibody specific for an ITAM (e.g., Fc ⁇ RI) and the other from an antibody specific for an ITIM (e.g., HM18).
  • ITAM e.g., Fc ⁇ RI
  • ITIM e.g., HM18
  • Another embodiment is a fusion protein including a monoclonal antibody to Fc ⁇ RI, or a fragment thereof, and an antibody to HM18, or a fragment thereof.
  • Figure 1 shows the percentage of inhibition of histamine release from mast cells using GAM (a goat anti-mouse antibody) and 7.3 (a monoclonal anti-human Fc ⁇ RII antibody)to cross-link Fc ⁇ RI and Fc ⁇ RII after IgE binds.
  • GAM a goat anti-mouse antibody
  • 7.3 a monoclonal anti-human Fc ⁇ RII antibody
  • Figure 2 shows the inhibition of ⁇ -hexosaminidase release from C57BL/6 mouse mast cells upon crosslinking of
  • Fc ⁇ RI and Fc ⁇ RII receptors by a polyclonal rabbit IgG anti-HSA.
  • Figure 3 shows the inhibition of ⁇ -hexosaminidase release from C57BL/6 mouse mast cells upon crosslinking of Fc ⁇ RI (which IgE bound to DNP is absorbed) and Fc ⁇ RII/RIII receptors by a bispecific 2.4G2-anti-DNP antibody.
  • Figure 4 shows the inhibition of passive cutaneous anaphylaxis in Balb/c mice upon crosslinking of Fc ⁇ RI and Fc ⁇ RII receptors by a bispecific 2.4G2-anti-DNP antibody. IgE was added at increasing doses, as shown.
  • Figure 5A shows HPLC analyses of the bispecific antibody (E.10.10.3 x AT.10). and 5B depicts the SDS-PAGE of the antibody electrophoresed on a reduced NuPage Bis-Tris gel (4-12% gradient gel).
  • Figures 6A-6E show FACS analysis of Daudi cells stained with the bispecific antibody (E10.10.3 X AT.10) at 5 ⁇ g/ml and 25 ⁇ g/ml, one of its parental antibody clones (AT.10), and an irrelevant control antibody.
  • FIG. 7 shows the inhibition of histamine release by bispecific antibody (bsAb) on human cultured mast cells.
  • Mast cells were first exposed to human IgE specific to the viral glycoprotein gp120, and then incubated with the bispecific antibody, E10.10.3, AT.10, or anti-CD81 (a leukocyte cell surface protein). These cells were then challenged with ovalbumin-conjugated gp120 peptide. Inhibition of mast cell degranulation ranged from 50-75%.
  • Figure 8 shows the inhibition of histamine release by bispecific antibody on human cultured basophils. Basophils cultured from human cord blood were exposed to human IgE expressed from clone SE44 and then incubated with varying concentrations of bispecific antibody. When challenged with antigen, a greater than 60% inhibition of histamine release was seen at concentrations as low as 0.1 ⁇ g/ml of bispecific antibody.
  • Figure 9 shows the inhibition of histamine release from exogenous IgE by bispecific antibody on human circulating basophils.
  • the leukocyte-enriched fraction of a human buffy coat sample which included circulating basophils, was treated with human IgE (clone SE44, 10 ⁇ g/ml) and then incubated with bispecific antibody at concentrations ranging from 0.03 ⁇ g/ml to 100 ⁇ g/ml.
  • parental antibodies E10.10.3 and AT.10 were also used, as well as anti-DC81.
  • the sample was then challenged with gp120. A biphasic inhibitory pattern was seen, higher concentrations exhibiting a less inhibitory effect than the lower concentrations, and at the lowest concentration (0.03 ⁇ g/ml) there is not enough crosslinking to give the same percentage inhibition.
  • Figure 10 shows the inhibition of TNF- ⁇ release by bispecific antibody on human cultured mast cells.
  • Cultured mast cells were first treated with IgE (clone SE44, 10 ⁇ g/ml) and then incubated with bispecific antibody at a concentration of 0.1 ⁇ g/ml or 1.0 ⁇ g/ml or without bispecific antibody.
  • the cells were then challenged with ovalbumin-conjugated gp120 for 2 and 24 hours before TNF- ⁇ release was measured. It appears to take 24 hours for TNF- ⁇ transcription to take place thus demonstrating inhibition of TNF- ⁇ release.
  • Bispecific molecules are intended to include molecules having at least two determinant regions having specificity for a specific ligand.
  • bispecific molecules can be antibodies including, e.g., monoclonal antibodies, including full-length, polyclonal antibodies, chimeric antibodies, humanized antibodies, deimmunized antibodies, single chain antibodies, and antibody fragments (e.g., Fab, F(ab') 2 , Fv, and other fragments which retain the antigen binding function of the parent antibody), provided that the antibody exhibits the desired biological activity.
  • the chains of the antibody molecule may be joined by a synthetic linker enabling them to be made as a single protein chain by recombinant methods.
  • “Humanized” forms of antibodies are antibodies (e.g., murine) comprised of chimeric immunoglobulins, immunoglobulin chains, or fragments thereof, which contain minimal sequences derived from non- human immunoglobulin.
  • a “humanized” antibody only the complementarity determining regions (CDRs), which are responsible for antigen binding and specificity, are non-human derived and have an amino acid sequence corresponding to the non-human antibody, and substantially all of the remaining portions of the molecule (except, in some cases, small portions of the framework regions within the variable region) are human derived and have an amino acid sequence corresponding to a human antibody. See, e.g., L.
  • “Diabodies” are antibody fragments with two antigen binding sites, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) in the same polypeptide chain. The domains pair with complementary domains due to a linker that is too short to allow pairing between the two domains of the same chain.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • Such “diabodies” are described in U.S. Patent No. 5,534,254 (Creative Biomolecules, Inc.) In addition to diabodies, it is possible to construct other bispecific molecules capable of coligating HM18 and Fc ⁇ RI, and suitable for treatment of IgE-mediated allergic diseases in accordance with the teachings of the invention.
  • bispecific molecules can be small molecules, antibody homologues or analogues, nucleotides or other molecules capable of coligating HM18 and Fc ⁇ RI.
  • Such bispecific molecules can be isolated from libraries or otherwise by screening against HM18 and Fc ⁇ RI, and conducting any further in vitro functional assays, as necessary, including the cellular assays described below.
  • Human antibodies of the present invention can be made by several different methods, including the use of human immunoglobulin expression libraries (Stratagene Corp., La Jolla, California; Cambridge Antibody Technology Ltd., London, England) to produce fragments of human antibodies (VH, V , FV, Fd, Fab, or (Fab') 2 ) and use of these fragments to construct whole human antibodies by fusion of the appropriate portion thereto, using techniques similar to those for producing chimeric antibodies. Human antibodies can also be produced in transgenic mice with a human immunoglobulin genome.
  • mice are available from Abgenix, Inc., Fremont, California, and Medarex, Inc., Annandale, New Jersey.
  • Fab can be constructed and expressed by similar means (M.J. Evans et al., J. Immunol. Meth., 184: 123-138 1995).
  • Single chain antibodies (“ScFv”) and the method of their construction are described in U.S. Patent No. 4,946,778.
  • the antibodies of the present invention are either wholly or partially human and are less immunogenic than wholly murine or non- human-derived antibodies, as are the fragments and single chain antibodies. All these molecules (or derivatives thereof) are therefore less likely to evoke an immune or allergic response. Consequently, they are better suited for in vivo administration in humans than wholly non-human antibodies, especially when repeated or long-term administration is necessary, as may be needed for treatment with the bispecific antibodies of the invention.
  • 5,534,254 (Creative Biomolecules, Inc.) describes several different embodiments of bispecific antibodies, including linking single chain Fv with peptide couplers, including Ser- Cys, (Gly) -Cys, (His) 6 -(Gly) -Cys, chelating agents, and chemical or disulfide couplings including bismaleimidohexane and bismaleimidocaproyl.
  • Another embodiment is a dimer having single chain FvLi and FvH 2 linked and FvHi linked with FvL 2. All such linkers and couplings can be used with the bispecific antibodies of the invention.
  • the bispecific molecules of the invention are administered as a pharmaceutical composition at a dosage effective to inhibit mast cell exocytosis.
  • the effective dosage can be readily determined in routine human clinical trials or by extrapolation from animal models.
  • the pharmaceutical composition is administered by injection, intravenously, subcutaneously or intraperitoneally. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes. Preferred routes of administration include oral, dermal, transdermal, and injection.
  • compositions Before administration to patients, formulants and excipients, well known in the art, are preferably added to the pharmaceutical composition. These substances may be co-administered with the bispecific molecule of the claimed invention, including solvents, dispersion media, delay agents, emulsions, and the like.
  • additives may include buffering agents, stabilizing agents, preservatives, non-ionic detergents, antioxidants, and other miscellaneous additives (Remington's Pharmaceutical Sciences, 16 th ed., A. Osol. (1980).
  • pharmaceutical compositions can be chemically modified by covalent conjugation to a polymer to increase their circulating half-life. Polymers, and methods to attach them to peptides, are shown in U.S. Patent Nos. 4,766,106; 4,179,337;
  • Monoclonal antibodies against mouse gp49 (the mouse counterpart of HM18) is described in Katz et al., 1989, J. Immunol. 142:919-926.
  • Monoclonal antibodies against human HM18 would be made using analogous methods and techniques, with the human HM18 polypeptide described in International Patent Application WO 98/09638 as the immunogen.
  • Monoclonal antibodies against Fc ⁇ RI can be made using similar techniques, using a recombinant peptide representative of the sequence of the receptor. These peptides can be linked to a carrier, for example, keyhole limpet hemocyanin, to increase the immunogenicity and the production of antibodies to the immunogen.
  • the antibodies would be screened against mast cells bearing HM18 and Fc ⁇ RI or against recombinant versions of these receptors. Similar techniques could be used to generate bispecific antibodies capable of indirectly cross-linking HM18 and Fc ⁇ RI, e.g., bispecific antibodies with one arm binding to IgE or an allergen, and the other arm binding to HM18.
  • bispecific antibodies against other ITAM/ITIM receptor pairs could be made using similar techniques, with an appropriate ITAM and ITIM used as immunogens for the mice.
  • the antibody candidates would be screened against the receptor pair, or, if appropriate, the indirect linker(s).
  • Other bispecific molecules including peptides, DNA, RNA, other organic molecules or homologues or analogues thereof, can also be made against any ITAM/ITIM receptor pair, or, an indirect linker(s) thereto.
  • Activating receptors include BCR, Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RI, Fc ⁇ RIIIA and TCR.
  • ITAM receptors are carried on a variety of human cells and could therefore be regulated by interaction with the appropriate ITIM receptor.
  • the bispecific molecule candidates can then be screened for binding to the designated ITAM/ITIM receptor pair, or the indirect linker(s), using conventional procedures.
  • Gene constructs that direct the expression in vivo of the diabodies or bispecific proteins can be administered by an appropriate vector system, including a retrovirus, an adenovirus, a parvovirus or any other vector permitting cellular transfer of the gene constructs, or by incorporation of the gene construct into liposomes with or without the viral vector.
  • nucleotides can be used to transfect cells ex vivo, using known methods including electroporation, calcium phosphate transfection, microinjection, or incorporation of the gene constructs into liposomes followed by transfection. The cells are then introduced into the patient for expression in vivo.
  • Use of gene therapy for antibody expression is well known in the art. See, e.g., International Patent Application No. WO 98/31808.
  • bispecific molecules of the claimed invention may also be subjected to further modifications, depending on the intended use of the molecule. Such modifications can include, further alteration of the amino acid sequence, fusion with heterologous proteins, or other covalent modifications.
  • Example I Generation of single chain antibody fragments from monoclonal antibodies to HM18 and to Fc ⁇ RI.
  • the first contains a Hindlll and Sfil restriction site for cloning purposes followed by a degenerated sequence annealing to the 5' V H region.
  • the second contains a degenerate sequence for the 3' part of the V H region followed by a sequence encoding a ((Gly) 4 Ser) 3 linker and the 5' part of the V regions.
  • the third is a degenerated primer having homology with the 5' part of the V L region, while the last primer contains a Notl restriction site and anneals to the 3' part of the V region.
  • Bispecific bivalent molecules can be generated by shortening the flexible linker sequence in the anti-HM18 ScFv and in the anti- Fc ⁇ RI ScFv, from fifteen residues to five (Gly 4 Ser) and by cross-pairing the variable heavy and light chain domains from the two single chain Fv fragments with the different antigen recognition.
  • the construction is preferably performed in three steps.
  • the light chain variable fragments are exchanged in the ScFv constructs from an anti-HM18 ScFv and an anti-Fc ⁇ RI ScFv by using restriction enzyme sites located in the 5'-end and just outside the 3'-part of the light chain variable gene.
  • the 15-residue linker of the chimeric construct V H -a HM18/15AA-linker/V L -a- Fc ⁇ RI is replaced by the 5 residue linker (Gly 4 Ser) by using sites located in the 3'-part of V H and the 5'-part of V L .
  • a chimeric cassette is combined in the vector pUC119- fabsol (a pUC119 derivative similar to pUC119His6mycXba (Low et al., J. Mol. Biol. 260:359 (1996)), but with all Apall-sites in the vector backbone deleted by in vitro mutagenesis) containing a bi-cistronic expression cassette.
  • a diabody-producing clone containing both ScFv- cassettes is identified and used for expression of the recombinant diabody molecule.
  • Example III Cellular Assays to Screen for Non-Activating High Affinity Antibodies against IgE or Fc ⁇ RI Receptor and HM18 Antibodies
  • Human Fc ⁇ RI receptor (Invitrogen Inc. or Beth Israel Hospital) and human HM18 receptor (Brigham & Women Hospital) can be co- transfected into the rat mast cell line, RBL-2H3 (ATCC collection). Briefly, transfected cells are resuspended in RPMI 1640 medium supplemented with 10% FCS at 1 X 10 6 cells/ml and incubated at 37°C for 1 hour with 2 ⁇ Ci/ml [ 3 H] serotonin (Amersham Corp.). The cells are washed and reincubated for another hour at 37°C and transferred to 96-well microculture plates at 2 X 10 5 cells/well.
  • Cells are then treated with individual bispecific hybridoma supernatant or carrier coupled diabody specific for human Fc ⁇ RI and HM18 receptors and left for 1 hour.
  • human IgE and IgE crosslinking reagent for 30 minutes, the attached cells are washed and warmed for 15 minutes at 37°C. Reactions are terminated by adding 50 ⁇ l of ice- cold medium and by placing plates on ice. 50 ⁇ l of supernatants are mixed with 1 ml of emuisifier safe scintillation liquid and counted in a Beckman counter. The percentage of serotonin release is calculated using as 100% the cpm obtained from the same number of pulsed cells lysed in 0.5% SDS and NP-40. The diabodies that can inhibit maximum serotonin release would be the choice for further characterization.
  • a secondary confirmatory human cord blood derived mast cell culture could be used.
  • cultured human mast cells can be raised from commercially available CD34+ purified human umbilical cord blood mononuclear cells through the addition of 80 ng/ml of rhSCF, 50 ng/ml rhlL-6 and 5 ng/ml rhlL-10 for 6 to 8 weeks.
  • the confirmed mast cell population after FACS sorting will be plated into 96-well microculture plates. Cells are then treated with selected diabody supernatants for 1 hour.
  • human IgE and IgE crosslinking reagent for example, human IgE and IgE crosslinking reagent for
  • the histamine release into the media is measured with the ELISA detection system from Immunotech, Inc.
  • the diabodies that can inhibit maximum histamine release would be the choice for further characterization.
  • Example IV Reduction of Mast Cell Degranulation by Cross- Linking of the ITIM and ITAM receptors
  • GAM goat anti-mouse antibody
  • An isotype matched mouse IgG Fab' 2 was used as a control, showing the importance of crosslinking the inhibitory receptor Fc ⁇ RII to the IgE receptor (see Figure 1). The experiment is described briefly below.
  • CD34+ cells were isolated from human cord blood using Dynal CD34 coated beads. The isolated cells were cultured in the presence of 80 ng/ml of recombinant human stem cell factor, 50 ng/ml of recombinant human interleukin 6 and 5 ng/ml of recombinant human interleukin 10 for about 8 weeks. For each group, 1 X 10 5 human mast cells were treated with mouse IgE (10 ⁇ g/ml), mouse IgE + mouse anti- human Fc ⁇ RII antibody, clone 7.3 (1 or 10 ⁇ g/ml), or isotype matched mouse IgG Fab' 2 (10 ⁇ g/ml) for the first hour of incubation at 37°C.
  • the cells were washed twice with PBS before applying the goat anti-mouse Fab' 2 (GAM, 10 ⁇ g/ml) to the selected groups for another hour of incubation at 37°C.
  • the histamine release into the supernatant from each treatment group was tested with the human histamine ELISA kit from Immunotech, Inc.
  • Figure 1 shows histamine release inhibition of human cord blood mast cells upon crosslinking of Fc ⁇ RI and Fc ⁇ RII receptors. The percentage of inhibition was the comparison of the amount of histamine release (in nM) between the crosslinked groups versus the positive control group (IgE+GAM) in lane 2 in Figure 1.
  • mice mast cells (2 x 10 5 cells/group) were incubated with IgE anti-DNP (1 ⁇ g/ml) for 45 minutes at 37°C.
  • DNP-HSA (5 ng/ml) was incubated with rabbit IgG anti-HSA (at various concentrations, ranging from 0.01 to 100 ⁇ g/ml) for 15 minutes at 37°C.
  • mice were incubated with the IgG complexed antigen for another 45 minutes at 37°C.
  • the ⁇ -hexosaminidase activity released into the supernatant was measured by the hydrolysis of pNpp.
  • Figure 2 shows the Inhibition of ⁇ -hexosaminidase release from C57BL/6 mouse mast cells upon crosslinking of Fc ⁇ RI and Fc ⁇ RII receptors by a rabbit IgG anti-HSA.
  • the percent of inhibition was the comparison of the amount of ⁇ -hexosaminidase release (in nM) between the crosslinked groups (lanes 2 through 6) versus the positive control group (IgE+DNP-HSA, lane 1).
  • Example VI Inhibition with Another Bispecific Antibody
  • DNP di-nitrophenyl moiety
  • clone 2.4G2 mouse Fc ⁇ RII/lll receptors
  • mice mast cells (2 x 10 5 cells/group) were incubated with IgE anti-DNP (1 ⁇ g/ml) both with and without 2.4G2-anti-DNP (at various concentrations, ranging from 0.1 to 100 ⁇ g/ml) for 45 minutes at 37°C. After washing with Tyrode's buffer, cells were incubated with the antigen, DNP-HSA (5 ng/ml) for another 45 minutes at 37°C. The ⁇ -hexosaminidase activity was measured by the hydrolysis of pNpp.
  • Figure 3 shows the inhibition of ⁇ -hexosaminidase release from
  • the ITAM/ITIM concept has been well documented with cellular models in the literature and demonstrated with human cultured mast cells, as described in Examples I to III.
  • the Segal's bispecific molecule was applied in a passive cutaneous anaphylaxis (PCA) animal model.
  • a bispecific chemically conjugated antibody having antigen binding regions from a mouse anti-human IgE (clone E.10.10.3) and a mouse anti-human Fc ⁇ RII (clone AT.10) was constructed using the chemical linker, o-phenylenedimaleimide, to conjugate the antigen binding regions.
  • the bispecific antibody [E.10.10.3 x AT.10 F(ab) 2 ] was analyzed using fluorescent antibody cell sorting (FACS) via cellular staining of a Burkitt lymphoma cell line, Daudi ( Figure 6A).
  • An irrelevant control antibody F(ab) 2 did not show positive staining ( Figure 6C).
  • the bispecific antibody was tested in an ELISA assay to confirm binding to human IgE coated microtiter plates (data not shown).
  • the bispecific antibody was then tested in cellular assays using human cultured mast cells and human cultured basophils to demonstrate the inhibition of histamine release.
  • Human CD34 + cells were cultured for 8 to10 weeks in the presence of 80 ng/ml stem cell factor (SCF), 50 ng/ml IL-6, and 5 ng/ml IL-10.
  • SCF stem cell factor
  • the cells were first exposed to human IgE specific for gp120 and the bispecific antibody before exposure to ovalbumin-conjugated gp120 peptide.
  • the bispecific antibody was assayed at concentrations of 0.3, 1, 3, 10, 30, and 100 ⁇ g/ml, as shown in figure 7.
  • Inhibition of histamine release was also measured in basophils from human blood PBMC using the bispecific antibody (see Figure 9).
  • Human buffy coat was ficoll-separated and the leukocyte-enriched fraction was collected.
  • Mixed leukocytes which included circulating basophils (1 x 10 5 cells per treatment) were first exposed to human IgE (clone SE44, 10 ⁇ g/ml) for 1 hour at 37°C, then washed and incubated with either the bispecific antibody at the indicated concentration or one of the parental antibodies, E10.10.3 (10 ⁇ g/ml) or AT10 (10 ⁇ g/ml), for 20 minutes at 37°C.
  • the cells were then exposed to ovalbumin- conjugated gp120 peptide for 1 hour at 37°C before histamine release was measured in the supernatant.
  • a known amount of exogenous human IgE (clone SE44, 10 ⁇ g/ml) was used to bind the "unoccupied" IgE receptors on the cell surface and then exposed to ovalbumin- conjugated gp120 peptide, in the presence of the bispecific antibody.
  • a greater than 60% inhibition was observed at concentrations as low as 0.03 ⁇ g/ml (See Figure 9, lane 10).
  • the control parental antibody, AT.10 alone did not show any inhibitory effect ( Figure 9, lane 14).
  • a biphasic response with the bispecific antibody was observed.
  • This response may be due to a stimulatory moiety of the bispecific antibody exerting its activatory effect at high concentrations (e.g. 100 ⁇ g/ml bispecific Ab, lanes 3-5), leading to less observable inhibition of histamine release. While at low concentration (e.g. 0.03 ⁇ g/ml bispecific antibody, lane 10), there is not enough ITAM/ITIM crosslinking to inhibit histamine release as significantly as seen in Figure 9, lanes 6-9.
  • the bispecific molecule was then tested in cellular assays using human cultured mast cells for the inhibition of cytokine release, such as TNF- ⁇ .
  • Human CD34 + cells were cultured for 8 to10 weeks in the presence of 80 ng/ml stem cell factor (SCF), 50 ng/ml IL-6 and 5 ng/ml IL-10 (Ochi et al., 1999).
  • SCF stem cell factor
  • IL-6 ng/ml IL-6
  • 5 ng/ml IL-10 8 ng/ml IL-10
  • Nine week old cultured mast cells (5 x 10 4 cells per treatment) were first treated with human IgE (clone SE44, 10 ⁇ g/ml) for 1 hour at 37°C, then washed and incubated with or without bispecific Ab at the indicated concentration for 20 minutes at 37°C.
  • the cells were then exposed to ovalbumin-conjugated gp120 peptide for 2 and 24 hours at 37°C before TNF- ⁇ release was measured in the supernatant. At two hours there is very little TNF- ⁇ present in the supernatant of any of the samples. Results suggest that it takes 24 hours for TNF- ⁇ to be secreted into the supernatant.
  • the bispecific antibody was tested at 1 and 0.1 ⁇ g/ml. Inhibition of TNF- ⁇ release is most pronounced at 1 ⁇ g/ml after 24 hours (see Figure 10). This demonstrates that in addition to histamine release, the ITAM/ITIM crosslinking also can inhibit cytokine release.
  • the examples and terms and expressions used above are exemplary only and not limiting, and the invention is defined only in the claims that follow, and includes all equivalents of the subject matter of the claims.

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Abstract

Molécules bispécifiques capables de réticuler les récepteurs ITAM (motif d'activation à base de tyrosine) et ITIM (motif d'inhibition à base de tyrosine) sur une cellule afin d'inhiber l'activation cellulaire, et approches dans le domaine de la thérapie génique basées sur des nucléotides codant ces molécules bispécifiques en vue de l'expression in vivo. Un exemple de paire de récepteurs ITAM/ITIM est représenté par FcεRI et HM18, et un autre par FcεRI et FcηRII. La réticulation de ces récepteurs avec une molécule bispécifique selon la présente invention semble conduire à l'inhibition de la libération de médiateurs allergiques et à l'amélioration des symptômes de maladies allergiques. D'autres maladies peuvent également être améliorées par la réticulation des paires de récepteurs ITIM/ITAM.
PCT/US2001/008671 2001-03-16 2001-03-16 Molecules bispecifiques reticulant itim et itam pour le traitement des allergies WO2003064662A1 (fr)

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PCT/US2001/008671 WO2003064662A1 (fr) 2001-03-16 2001-03-16 Molecules bispecifiques reticulant itim et itam pour le traitement des allergies
AU2001247543A AU2001247543A1 (en) 2001-03-16 2001-03-16 Bispecific molecules cross-linking itim and itam for therapy of allergies

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Cited By (4)

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WO2005095460A2 (fr) 2004-03-30 2005-10-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Complexes bi-specifiques pour le ciblage de cellules impliquees dans des reactions de type allergique, compositions et utilisations associees
US8198427B1 (en) 2002-11-14 2012-06-12 Dharmacon, Inc. SiRNA targeting catenin, beta-1 (CTNNB1)
US8236309B2 (en) 2001-10-12 2012-08-07 Schering Corporation Use of bispecific antibodies to regulate immune responses
WO2014188423A1 (fr) 2013-05-21 2014-11-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Traitement de pathologies associées aux mastocytes

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8236309B2 (en) 2001-10-12 2012-08-07 Schering Corporation Use of bispecific antibodies to regulate immune responses
US8198427B1 (en) 2002-11-14 2012-06-12 Dharmacon, Inc. SiRNA targeting catenin, beta-1 (CTNNB1)
WO2005095460A2 (fr) 2004-03-30 2005-10-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Complexes bi-specifiques pour le ciblage de cellules impliquees dans des reactions de type allergique, compositions et utilisations associees
WO2005095460A3 (fr) * 2004-03-30 2006-03-30 Yissum Res Dev Co Complexes bi-specifiques pour le ciblage de cellules impliquees dans des reactions de type allergique, compositions et utilisations associees
JP2007537157A (ja) * 2004-03-30 2007-12-20 イッサム リサーチ ディベロップメント カンパニー オブ ザ ヘブリュ ユニバーシティ オブ エルサレム アレルギー性反応に関与する細胞を標的化するための二重特異性抗体、ならびにその組成物および使用
JP4939397B2 (ja) * 2004-03-30 2012-05-23 イッサム リサーチ ディベロップメント カンパニー オブ ザ ヘブリュ ユニバーシティ オブ エルサレム アレルギー性反応に関与する細胞を標的化するための二重特異性抗体、ならびにその組成物および使用
WO2014188423A1 (fr) 2013-05-21 2014-11-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Traitement de pathologies associées aux mastocytes

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