[go: up one dir, main page]

WO1996040199A1 - Procedes d'inhibition de la phagocytose - Google Patents

Procedes d'inhibition de la phagocytose Download PDF

Info

Publication number
WO1996040199A1
WO1996040199A1 PCT/US1996/010494 US9610494W WO9640199A1 WO 1996040199 A1 WO1996040199 A1 WO 1996040199A1 US 9610494 W US9610494 W US 9610494W WO 9640199 A1 WO9640199 A1 WO 9640199A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
cells
syk
sequence
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1996/010494
Other languages
English (en)
Other versions
WO1996040199A9 (fr
Inventor
Alan D. Schreiber
Jong-Gu Park
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Pennsylvania Penn
Original Assignee
University of Pennsylvania Penn
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Pennsylvania Penn filed Critical University of Pennsylvania Penn
Priority to IL12233896A priority Critical patent/IL122338A0/xx
Priority to JP9502301A priority patent/JPH11507824A/ja
Priority to EP96923327A priority patent/EP0831875A4/fr
Priority to CA2223402A priority patent/CA2223402C/fr
Priority to AU63869/96A priority patent/AU723595B2/en
Publication of WO1996040199A1 publication Critical patent/WO1996040199A1/fr
Publication of WO1996040199A9 publication Critical patent/WO1996040199A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates, in general, to methods of treating diseases resulting from
  • the present invention relates to methods of modulating the clearance of antibody-coated cells, viruses, or soluble antigens by inhibiting
  • the invention also relates to the modulation of those immune reactions for which the reaction of antigen-antibody complexes with Fc
  • Certain immunological disorders are characterized by a disturbance in the expression of monocyte or macrophage Fc (IgG) receptors.
  • An increase in the number of Fc receptors can result from an increase in the level of Fc receptor mediators such as gamma interferon or infection or the release of bacterial products.
  • a decrease in the number of Fc receptors that can bind IgG can result not only from a reduction in the actual number of functional receptors but also from the saturation of Fc receptors by immune
  • autoimmune diseases such as systemic lupus erythematosus
  • levels of circulating immune complexes can be high and thus receptor
  • autoimmune diseases the body's mechanisms for distinguishing between itself and foreign invaders malfunction.
  • the body begins to make antibodies to certain parts of itself; these antibodies trigger the immune system which then destroys the tissue identified by the abnormal antibodies.
  • autoimmune hemolytic anemias represent a group of disorders in which individuals produce
  • erythrocytes to one or more of their own erythrocyte membrane antigens. Coating of erythrocytes by the abnormal antibodies is followed by their clearance from the circulation by splenic macrophages and subsequent destruction in the spleen.
  • Representative diseases in this class are immune hemolytic anemia, immune
  • autoimmune disease Another type of autoimmune disease is the type represented by systemic lupus erythematosus and
  • rheumatoid arthritis In these diseases, chronic inflammation is present in the joints, tendons, kidneys, lung, heart and other organs. In rheumatoid arthritis, for example, breakdown of joint cartilage into the synovial fluid of the joint is present in later stages of the disease. In systemic lupus
  • IgG-containing complexes in the circulation It is believed that recognition of these complexes in tissues by cells having Fc receptors initiates or increases tissue destruction by macrophages and possibly other cells such as polymorphonuclear leukocytes in these tissues. Reaction with these Fc receptors initiates a range of immune-associated reactions that may harm body tissues in proximity to these Fc receptor bearing cells.
  • IgG-containing immune complexes with macrophage Fc receptors are often treated with corticosteroids, or immunosuppressants. These treatments can have diverse and serious side effects.
  • the present invention offers alternative treatment approaches that can be used alone or in combination with more conventional drug
  • the present invention relates to a method of preventing the phagocytosis of immune complexes (eg IgG-containing immune complexes) and/or the release of intracellular biologically active products by cells interacting with immune complexes.
  • An example of the present method comprises introducing into phagocytic cells of the mammal that are in contact with the immune complexes (eg, IgG-containing immune complexes) an inhibitor of a kinase endogenous to the cells that activates an Fc receptor present at the membrane of the cells.
  • the present invention relates to a method of preventing the clearance of immune complexes (eg, IgG-containing immune complexes) from a mammal that comprises introducing into
  • hematopoietic cells eg phagocytic cells
  • the present invention relates to a method of inhibiting the binding of immune complexes (eg, IgG-containing immune complexes) present in a mammal to membrane-bound Fc receptors.
  • the method comprises introducing into the mammal a soluble Fc receptor that competes with the membrane-bound Fc receptor for binding to the immune complex.
  • the introduction is effected under conditions such that binding of the immune complex to the membrane-bound Fc receptor is inhibited.
  • the present invention relates to a method of inhibiting the phagocytic potential of a mammalian cell bearing an Fc receptor.
  • the method comprises introducing into the cell a construct comprising, in the 5'-3' direction of
  • transcribed strand of which comprises a sequence complementary to endogenous mRNA encoding the Fc receptor
  • a termination sequence (polyadenylation signal) functional in the cell.
  • the construct is introduced under conditions such that the
  • complementary strand is transcribed and binds to the endogenous mRNA thereby reducing expression of the Fc receptor and inhibiting the phagocytic potential of the cell.
  • Fc ⁇ RIIIA ⁇ wild type and mutants Shown above the schematic diagram of the ⁇ chain are signal sequence (S) , external peptides (E), transmembrane domain (TM), and cytoplasmic domain (CY).
  • S signal sequence
  • E external peptides
  • TM transmembrane domain
  • CY cytoplasmic domain
  • the expanded area shows an area of the nucleotide sequence of the y chain containing the conserved motif.
  • the murine y chain is shown.
  • the conserved amino acids of the gene family of the ⁇ and ⁇ chain genes are denoted by the underline.
  • the N-proximal tyrosine encoded by the TAC codon of the nucleotides 235-237 (Ra et al, J. Biol. Chem.
  • FIGS 2A and 2B show binding and phagocytosis of IgG-sensitized RBCs (EA) by transfected COS-1 cells. Binding of EA by transfected COS-1 cells (left panel: A, C, E and G). Phagocytosis of EA by transfected COS-1 cells (right panel; B, D, F, and H). (A) and (B): binding and phagocytosis of COS-1 cells
  • phosphorylated samples were run on a 12.5% reducing SDS-PAGE gel. The gel was treated with IN KOH to remove phosphoserine and threonine, dried and the autoradiogram was examined after 4 days.
  • lane 1 Sham transfectants with Fc ⁇ RIIIA- ⁇ and pSVL vector without ⁇ cDNA insert.
  • lanes 2 Fc ⁇ RIIIA ⁇ + wild type human y.
  • lane 3 Fc ⁇ RIIIA ⁇ + wild type mouse ⁇ .
  • lane 4 :
  • Fc ⁇ RIIIA ⁇ + DMA The phosphorylated ⁇ chains are denoted by an arrow (shown on the lower right side).
  • the arrow with an asterisk (shown on the upper right side) is a specific tyrosine phosphoprotein band at approximately 40 kDa.
  • transfectants were greatly decreased compared to WT transfectants.
  • Figure 5 shows selection of a target sequence (target III) for the stem-loop antisense ODN.
  • target III target sequence
  • the entire Syk mRNA sequence was scanned three times with a RNA secondary structure prediction program to find sequences free of secondary structures. Each scanning was performed 33 bases apart in a 99-nucleotide frame (denoted as frames A, B, and C) sequentially. The most open sequence in three staggered scannings was chosen as a target sequence.
  • the top rectangle with dots represents cDNA sequence of human Syk mRNA (Law et al, J. Biol . Chem . 269:12310 (1994)).
  • Target sites I, II and III correspond to nucleotides no. 159 to 173 (the area surrounding the translation initiation codon), no. 451 to 463 and no. 802 to 816, respectively.
  • Target III is shown as an example in this Figure.
  • Targets I and II were chosen in the same manner.
  • Putative secondary structures in the area of Syk mRNA containing the target III sequence are shown in the three staggered frames of 99 nts each, frame A, frame B, and frame C. Circled nucleotides in the three staggered frames are the common sequence of target III with minimum
  • the stem domain of the 7 nucleotide length is formed by complementary terminal sequence with nucleotide content of only G and C in the 5' and 3' termini.
  • the loop domain consists of three antisense sequences; the 5'-CTGTCAGCCATGCCG-3' sequence shown with squares is complementary to target I in Syk mRNA (see Figure 5), the 5'-GCTTCTTGAGGAG-3' sequence shown in triangles is complementary to target II, and the 5'-TGTCTTGTCTTTGTC-3' sequence shown with circles is complementary to target III which is also denoted with circles in Figure 5.
  • antisense sequences were tandemly joined in the 5' to 3' order for targets I, III, and II, respectively.
  • ⁇ S indicates the phosphorothioate modification
  • 5-prime terminus has one phosphorothioate modification and the 3-prime terminus has two.
  • FIG. 7 shows inhibition of Syk antisense ODNs on phagocytosis in monocytes.
  • Monocytes (1 x 10 5
  • Phagocytic index (PI) number of ingested RBCs/100 cells. Each bar represents the mean ⁇ SEM of three separate experiments.
  • monocytes (1 x 10 5 cells/ml) treated with complexes of 4 ⁇ g/ml of LIPOFECTAMINE and the ODNs (1.0 ⁇ M each of the linear control or the linear Syk antisense ODN, or 0.2 ⁇ M each of the stem-loop control or the stem-loop Syk antisense ODN) for 2 days, and cDNA was synthesized from total RNA with random hexanucleotide primers. PCR was performed with Syk cDNA as templates with two Syk primers (Syk-H and Syk-M). PCR products were analyzed by Southern hybridization and hybridized bands were visualized by chemiluminescent detection reagents.
  • Lane 1 cells treated with Syk antisense ODN: Lane 2, cells treated Syk sense ODN; lane 3, reagent control; Lane 4, no treatment; Lane 5, molecular weight markers.
  • the present invention relates, at least in part, to methods of modulating the clearance from a mammal (eg, from the circulation of a mammal) of
  • the invention provides methods of treating immunologic disorders, such as autoimmune diseases, characterized by
  • immune complexes eg, IgG-containing immune complexes
  • Fc receptors for example, those present on the surface of macrophages
  • the methods of the invention result in Fc receptor expression and/or function being altered so that phagocytosis of IgG antibody-coated cells is reduced.
  • liver and splenic macrophages designed so as to increase clearance of circulating immune complexes in the liver and spleen and thereby prevent their deposition in tissues such as the kidney and in the joints. This increase can be effected by stimulating liver and splenic macrophages using
  • the invention provides methods of inhibiting Fc receptor function by inhibiting the phosphorylation of Fc receptor components
  • circulation soluble Fc receptors that compete with the membrane bound receptor for immune complex (eg,
  • the invention also provides a method of inhibiting expression of Fc receptors by introducing into receptor-producing cells Fc receptor antisense constructs.
  • the invention also provides methods of degrading Fc receptor RNA using, for example, ribozymes.
  • the present invention relates to a method of preventing ingestion (eg phagocytosis) of immune complexes (eg IgG-coated cells) by inhibiting phosphorylation of core sequences within the
  • cytoplasmic domains of the ⁇ and ⁇ chains of FcRIIIA is required for phagocytic signal transduction (the numbers following the letter X denote the number of amino acids at that position; X can be any amino acid but X2 within a Y-X2-L is preferably the amino acids present in a Y-X2-L sequence of the cytoplasmic domain of Fc ⁇ RIIA or the ⁇ chain of Fc ⁇ RIII). It appears that the second Y-X2-L of these core sequences (motifs) is particularly important for phagocytosis.
  • the present invention contemplates the introduction into target cells of an inhibitor of the kinase (s) responsible for phosphorylation.
  • the kinase responsible for phosphorylation
  • inhibitor is a peptide that includes a sequence similar to, if not identical to, at least a functional portion of a tyrosine-containing motif (note, for example, the underlined portions of the motifs set forth above) and thus serves as a competitive inhibitor of the
  • the inhibitor can take the form of an Fc receptor devoid of the extracellular domain or devoid of the extracellular and transmembrane domains.
  • the inhibitor can be
  • a mimetic of the active peptide can be used having a structural conformation similar to the binding site of the active peptide.
  • the sequences of the ⁇ chain of FceRI necessary for mediator release eg, histamine, cytokines and leukotrienes
  • the peptide inhibitor of the invention, or mimetic thereof can be introduced into target cells directly, for example, using liposomes.
  • a DNA sequence encoding the peptide inhibitor can be introduced using gene therapy protocols so that the peptide is produced intracellularly.
  • the inhibitor or inhibitor encoding sequence can be administered to the cells of the lung, including macrophages, in the form of an aerosol.
  • the inhibitor or inhibitor encoding sequence can be present in the aerosol as a particle (e.g. liposome, or non-infectious bacteria, for example, Listeria, in the case of the encoding sequence) that is phagocytosed by the particle.
  • pulmonary macrophages Phagocytosis results in the introduction into the macrophages of the inhibitor or inhibitor encoding sequence.
  • Viral vectors can also be used to introduce the peptide inhibitor encoding sequence of the invention into cells of the pulmonary tree.
  • the vectors can be introduced as an aerosol and can take the form of a replication defective herpes or adenoviral vector. Retroviral vectors can also be used. (See, generally, Bajocchi et al, Nat. Genet.
  • Blood monocytes can be transformed (infected) ex vivo with the peptide inhibitor encoding sequence of the invention and then reintroduced into the patient so that the inhibitor is produced in vivo .
  • phosphorylation involves the use of ribozymes that recognize RNA sequences specifying Fc receptor
  • ribozyme can be effected using a carrier such as a liposome coated with IgG so as to direct insertion to Fc ⁇ receptor bearing cells.
  • a carrier such as a liposome coated with IgG so as to direct insertion to Fc ⁇ receptor bearing cells.
  • IgE-coated liposomes can be used to direct the ribozyme to mast cells or basophiles, or other cells bearing the IgE receptor FceRI with its associated ⁇ subunit.
  • the ⁇ subunit of the IgE receptor is responsible for transmitting the signal inducing the release of intracellular mediators by Fee receptor bearing cells such as mast cells.
  • the destruction of the ⁇ chain RNA is predicted to inhibit the release of these bioactive products.
  • ribozymes administered as described would bind to a few selected sequences (eg, RNA splicing and 5' untranslated
  • RNA sequences for which they were specific, for example, in Fc ⁇ RIIA RNA or Fc ⁇ RIIIA ⁇ chain RNA) and the enzymatic activity associated with the ribozyme would result in digestion and thus removal of the RNA specifying functional sequences of the receptor necessary for phagocytic signal transduction.
  • cytokines and leukotrienes can be eliminated using this strategy.
  • phosphorylation involves the use of a ribozyme or an antisense construct that targets Syk encoding sequences (see Example V).
  • the Syk gene product but not the gene product of ZAP-70 of the Syk kinase family, has been shown to stimulate Fc ⁇ RI and Fc ⁇ RIIIA phagocytosis mediated by both the ⁇ and ⁇ chains.
  • ZAP-70 in the presence of certain Src related tyrosine kinases can stimulate Fc ⁇ RI and Fc ⁇ RIIIA phagocytosis.
  • This chimera acts like ZAP-70 in that it does not increase Fc ⁇ receptor mediated signaling (COS-1 cell transfectants and phagocytic signaling are one readout). Further chimeras of Syk and ZAP-70 have been produced. A Syk mutant has been constructed in which the SH 2 domain has been replaced with the SH2 domain of ZAP-70. This chimera acts like Syk kinase. Similarly, a ZAP-70 mutant has been constructed in which the SH2 domain has been replaced with the SH2 domain of Syk kinase. This chimera acts like ZAP-70. A Syk mutant has been constructed in which the interval region between the second SH2 domain and the catalytic
  • Chimeras were produced using overlap PCR using wild type Syk and ZAP-70.
  • interval sequence of Syk kinase as being responsible for signal transduction events, including those involved in phagocytosis, makes possible a screen that can be used to test compounds (eg peptides or mimetics) for their ability to
  • a test compound can be contacted with a polypeptide comprising the Syk interval region, or portion thereof of at least 3, 5 or 7 amino acids or larger portions, for example, of at least 20, 50 or 100 amino acids (eg a chimera comprising the ZAP-70 SH2 and kinase domains and the Syk interval sequence), and a polypeptide comprising the ZAP-70 interval region (eg a chimera comprising the Syk SH2 and kinase domains and the ZAP-70 interval sequence).
  • Compounds that bind the former polypeptide but not the latter are putative selective inhibitors of signaling events mediated by Syk interval sequences (including phagocytosis and mediator release from mast cells and other Fee receptor bearing cells).
  • Such compounds can also be tested by introducing into Syk- deficient, potentially phagocytic cells (eg Fc ⁇
  • COS cells bearing an Fc ⁇ receptor including COS cells bearing an Fc ⁇ receptor
  • a construct encoding a polypeptide comprising the Syk interval sequence eg a construct encoding the chimera described above, contacting that cell with the test compound and assaying for the ability of the cell to carry out phagocytosis;
  • phagocytosis being a readout for signaling by Syk kinase.
  • Compounds that inhibit phagocytosis can be expected to inhibit other signaling events mediated by Syk interval region sequences.
  • Compounds that inhibit the phagocytic potential of the cells expressing the Syk interval region can then be tested for stability, toxicity, etc in accordance with standard protocols.
  • This approach can also be used to screen for compounds (eg peptides or mimetics) that inhibit mast cell, or other Fee receptor bearing cell, mediator release (eg histamine release).
  • compounds eg peptides or mimetics
  • mediator release eg histamine release
  • Peptides and mimetics identified using the above- described screen, or otherwise identified can be formulated as pharmaceutical compositions and
  • Syk interval sequence eg between the second SH2 domain and the catalytic
  • kinase domain (kinase) domain) (eg in purified or isolated form) or portion thereof of at least 5 or 6 amino acids, or mimetics thereof, are within the scope of the invention and can be formulated and used as described above.
  • the present invention also contemplates the use of Syk antisense constructs to inhibit mediator (eg histamine) release from cells bearing an Fee receptor, such as mast cells (see
  • Example VI Inhibition of histamine (a mast cell mediator) release, for example, is of therapeutic importance in the treatment of asthma.
  • Preferred targets of Syk antisense constructs are described below (see also Examples V and VI).
  • the constructs can be administered systemically or directly to the lung (eg aerosol administration). Delivery can be effected using the techniques described herein (including liposome formulations). Optimum dosing will depend on the patient, and the construct and mode of
  • the present invention relates to a method of inhibiting the interaction between immune complexes (eg, IgG-containing immune complexes) and membrane-associated Fc receptors and thereby suppressing the clearance of such complexes by phagocytosis (alternatively, the signalling through the Fc receptor resulting in the release of intracellular mediators).
  • the method involves introducing into the circulation a soluble form of the Fc receptor that competes with the membrane bound form for immune complex binding. Transcripts of certain soluble forms have been identified in cells of megakaryocytic and monocyte/myeloid lineages (Rappaport et al, Exp.
  • transcripts lack sequences coding for the transmembrane receptor region but retain sequences coding for the cytoplasmic domain.
  • the present invention contemplates the production and use of soluble Fc receptors that include an extracellular domain alone or in combination with a cytoplasmic domain. Suitable receptors are capable of competing with membrane bound Fc receptors for binding of
  • Soluble receptors of the invention can take the form of Fc ⁇ RI, Fc ⁇ RII or Fc ⁇ RIII extracellular domains alone or binding portions thereof (alternatively, a soluble receptor of FceRI can be employed taking the form of an extracellular domain alone or binding portion thereof). As noted above, cytoplasmic domains, or portions thereof, can also be present.
  • Fc ⁇ RIIA Fc ⁇ RIIA, respectively, and where ⁇ and ⁇ correspond to the ⁇ and ⁇ chains of Fc ⁇ RIII, the first designation indicating the source of the extracellular domain and the second the source of the cytoplasmic domain: I:I, I, IIA, IIA:IIA, I: IIA, ⁇ : ⁇ , ⁇ , ⁇ .IIA, I: ⁇ .
  • Soluble receptors depending on their nature, can be prepared chemically or recombinantly (Horton et al, Biotechniques 8:528 (1990)).
  • the soluble receptors can be administered systemically or to the lung as
  • the present invention relates to a method of inhibiting Fc receptor
  • double-stranded DNA double-stranded DNA, the transcribed strand of which includes a sequence complementary to the endogenous mRNA of the Fc receptor the expression of which is to be inhibited, and iii) a termination sequence
  • This embodiment of the invention makes it possible to regulate the expression of a specific Fc receptor in cells producing multiple receptor classes. This specificity can be achieved by selecting for inclusion in the DNA segment ( (ii) above) sequences unique to the mRNA of the endogenous Fc receptor.
  • the invention also relates to antisense constructs that target Syk kinase encoding sequences.
  • constructs (ii) above is a segment of double-stranded DNA, the transcribed strand of which includes a sequence complementary to endogeneous mRNA of Syk kinase.
  • Factors that affect the efficacy of antisense oligonucleotides include stability of the antisense oligonucleotides, their delivery into the cell
  • Syk antisense oligonucleotides of the present invention are modified in three steps to address these issues.
  • the stem has complementary terminal sequences, for example, with only Gs and Cs.
  • the loop domain has, for example, three antisense sequences targeting different sites of Syk mRNA.
  • mRNA forms secondary structures by intramoleculear
  • Syk mRNA secondary structures may inhibit access of antisense oligonucleotides to target sequences.
  • the entire Syk mRNA sequence was scanned with an RNA secondary-structure predication program.
  • Syk mRNA was scanned in three staggered frames, and the most "open" sequences with minimum secondary structures were chosen.
  • Stem-loop antisense oligonucleotide reduced the phogocytic signal more dramatically than a mixture of three linear antisense oligonucleotides.
  • the higher efficacy of the stem-loop Syk antisense oligonucleotide may be due to better stability from nuclease digestion.
  • antisense oligonucleotides were also complexed, for example, with cationic liposomes, to improve delivery to the cells.
  • the stablility of the stem-loop Syk antisense oligonucleotides markedly improved when complexed with liposomes.
  • a stem-loop antisense oligonucleotide directed at, for example, the Fc ⁇ RIIIA ⁇ subunit mRNA has also be used.
  • Liposomes can be delivered to the reticuloendothelial system, for which monocytes/macrophages are a major residential cell population.
  • the complex of liposome- stem-loop Syk antisense oligonucleotide is advantageous for use as a therapeutic agent (s) for immunologic disorders requiring down-regulation of Fc ⁇ receptor- mediated function in monocytes/macrophages.
  • Syk kinase is also associated with FceRI and with the B-cell antigen receptor.
  • oligonucleotide is also useful for investigating intracellular signaling events through these receptors and for developing therapeutic agents to modulate the signals mediated by these receptors.
  • the sequence complementary to the endogenous mRNA target is at least 15 nucleotides in length, preferably, at least 30 and, most preferably, at least 50.
  • the sequence is typically less than 5000 nucleotides in length, preferably less than 2000, and most preferably less than 1000.
  • the sequence can be complementary to a translated or untranslated region of the target mRNA (see, for example, McKenzie et al, Molec. Immunol. 29:1165 (1992), Matsuda et al, Mol.
  • Administration of the antisense construct for example, to the lung and to the spleen, can be carried out as described above using both in vivo and ex vivo
  • antisense transcript itself can be introduced directly into the target cells using methods known in the art, including those described above (see also Example V - there, linear and stem-loop Syk antisense oligonucleotides (ODNs) modified with
  • phosphorothioate show partial resistance to serum nucleases.
  • antisense ODNs with phosphorothioate modifications at 5' and 3' termini are even more stable.
  • the present invention also relates to a method of effecting inhibition by introducing into a cell having phagocytic potential Fc ⁇ RIIB (eg Fc ⁇ RIIB2), which is capable of inhibiting the function of Fc ⁇ receptors, including Fc ⁇ RIIA (Hunter et al, FASEB J. June 1996, New La, LA).
  • Fc ⁇ RIIB phagocytic potential Fc ⁇ RIIB
  • Introduction of Fc ⁇ RIIB can be effected by transfecting/infecting a target cell with a construct comprising a sequence encoding
  • Fc ⁇ RIIB or portion thereof that effects the inhibition (Brooks et al, J. Exp. Med. 170:1369 (1989); Indik et al, Blood 83:2072 (1994)).
  • Suitable constructs can be selected by one skilled in the art.
  • Recombinant soluble Fc ⁇ RIII proteins can be produced using expression vectors as described below.
  • the soluble protein can correspond to Fc ⁇ RIII with the transmembrane domain removed.
  • the constructs can be introduced into mammalian cells under conditions such that expression of the receptor encoding sequence occurs.
  • the recombinant proteins thus produced are isolated both from the cell lysates and from the supernatants.
  • Transfection of adherent cells or cells in suspension Transfection of adherent cells, eg, CHO cells or COS cells, or an appropriate suspension cell system will be performed. Permanent transfectants expressing soluble forms of Fc ⁇ receptor will be established by
  • Transfected cells will be allowed to grow 48 hours and selected in media containing Geneticin at 2 mg/ml (Gibco BRL, Gaithersburg, Maryland) or other selection drug. After approximately twelve weeks, positive colonies will be isolated and expanded for further characterization of the clones. The isolated clones will be examined by enzyme-linked immunoassay (ELISA) using ELISA plates (Dynatech, Alexandria, Virginia) to select a transfectant cell line expression the highest quantity of the soluble receptor. Mass culture of adherent transfectants will be achieved by employing the hollow-fiber tissue culture system.
  • ELISA enzyme-linked immunoassay
  • soluble Fc ⁇ RIII proteins are assessed both in vi tro and in vivo .
  • the effect of soluble Fc receptors on IgG-immune complex binding to cellular membrane-bound receptors depends on several factors including the local concentrations of the ligand and soluble receptor, the surface density of the membrane-bound receptor, the valence of the ligand and the relative affinities of the two receptor forms for ligand.
  • the limiting factors in the interaction of soluble Fc ⁇ RIII receptors with ligand and cellular membranes can be deciphered using available model systems.
  • the in vi tro assay systems rely on the competition of soluble receptors with cell membrane receptors for labeled IgG ligand and IgG-coated erythrocytes (EA). Fc ⁇ receptor-negative cells are transfected with transmembrane Fc ⁇ RIII molecules that retain the
  • the pSVL eucaryotic expression vector (Pharmacia LKB, Piscataway, NJ) was employed for expression of Fc ⁇ RIIIA in COS-1 cells.
  • huFc ⁇ RIIIA ⁇ cDNA was cloned into the Xbal and BamHI cloning sites of pSVL.
  • muFc ⁇ RIIIA ⁇ cDNA was cloned into Xhol and BamHI cloning sites.
  • TCR/Fc ⁇ RIIIA ⁇ was cloned into the Xbal and BamHI cloning sites of pSVL.
  • Transfected cells were harvested with staining buffer (PBS containing 0.02 % sodium azide and 0.1% BSA) using transfer pipettes. Cells were
  • EA IgG-sensi tized RBCs
  • EA IgG-sensitized RBCs
  • Inhibitors of phosphatases and proteases (ImM EGTA, 1 mM Na orthovanadate, 1 mM PMSF, 10 ⁇ g/ml aprotinin, 50 ⁇ g/ml leupeptin, and 100 ⁇ g/ml soybean trypsin inhibitor) were added fresh to lysis buffer. After 15 minutes of lysis on ice, cell lysates were centrifuged for 30 minutes at 4°C to clarify. The Fc ⁇ RIIIA- ⁇ chain was immunoprecipitated with anti-human ⁇ antiserum (provided by Jean-Pierre Kinet, NIAID-N1H, Rockville, MD) and Protein A-sepharose CL4B (Signa, St.
  • COS-1 cells plated on glass coverslips were incubated with 2 ⁇ M Fura-2/AM (Calbiochem. San Diego, CA) for 30 minutes, washed twice and the coverslips then
  • Fc ⁇ RIIIA receptors were crosslinked either with biotinylated anti-Fc ⁇ RIII followed by the addition of streptavidin or with anti-Fc ⁇ RIII mAB 3G8 whole IgG.
  • 10 ⁇ M epinephrine was added to crosslink epinephrine receptors expressed on COS cells.
  • Calcium imaging was performed using a 4Ox fluorescence objective on a Nikon Diaphot microscope with the image-1 AT quantitative fluorescence system (Universal Imaging, West Chester, PA).
  • 340/380 ratio images were calculated on a pixel by pixel basis and the average 340/380 ratio within each cell determined at each time point. 340/380 ratios were converted to [Ca2+ ] i based on solution
  • FMI fluorescence intensity
  • cotransfected cells stained with anti-Fc ⁇ RIII mAB increased by 15 fold compared to cells stained with an IgG isotype control or compared to mock-transfected cells stained with anti-Fc ⁇ RIII mAB (Table 1).
  • the transfectants were examined for their ability to bind and phagocytose IgG sensitized RBCs (EA).
  • Transfection efriciency was determined by now cytometry.
  • the mean fluorescence intensity (MFI) is shown for one or 3 separate experiments with similar results.
  • IntcmaMzed RBCs were microscopically scored (1000x). Results are expressed as the mean ⁇ SEM for phagocytosis and binding (resetting) of EA. At least 3 separate experiments were performed for each clone. For each experiment. 1500 ceils were counted at 5 randomly selected sites. • Mean Fluorescence Intensity. $pI (Phagocytic bdex): number of RBCs internalized per 100 COS-1 cells
  • N-proximal (clones MIA and M1B) or C-proximal (clones M2A and M2B) tyrosines were individually replaced by phenylalanine.
  • both tyrosines were replaced by
  • results summarized in Table 2 are as follows : M1 ⁇ mutants showed more than 99% reduction in phagocytic activity as shown by phagocytic index (PI) ( ⁇ 1 % of transfectants with ingested EA and minimal ingested EA per phagocytosing cell) (p(0.02); M2 and DM ⁇ mutants demonstrated essentially no phagocytosis (1 among 5000 cells examined) (Table 2, Fig. 2).
  • PI phagocytic index
  • phagocytosis in a dose dependent manner, with 50% inhibition at 25 ⁇ M and complete inhibition at 200-400 ⁇ M (p(0.01) (Table 3).
  • tyr 23 did not affect the binding of EA. Inhibition of phagocytosis was not associated with reduction in viability, since transfectants pretreated with tyr 23 (400 ⁇ M) followed by washing had phagocytic activity partially (3 hr wash, Table 3) or completely (overnight wash, data not shown) restored.
  • Tyrosine Residues of the ⁇ Subunit are Phosphorylated In Vi tro :
  • the in vi tro kinase assay demonstrated a distinct band of approximately 40 kDa present in all lanes except the sham transfectants. This band may represent an associated phosphoprotein coprecipitating with ⁇ .
  • Fc ⁇ RIII in its native state on pulmonary macrophage or cultured monocytes (M) was examined in order to study the physiologically relevant protein tyrosine kinases (PTK) and phosphotyrosine containing substrates during macrophage signal transduction.
  • PTK protein tyrosine kinases
  • Fab antibody Western blot analysis revealed a characteristic pattern of
  • Phosphotyrosine patterns were indistinguishable in fresh macrophage and cultured monocytes, validating the latter as a useful in vitro model.
  • P62 a protein associated with p120 ras GAP, although not GAP itself, was identified by specific immunoprecipitation as one of these phosphotyrosine substrates.
  • a second substrate was found to be p95 , a hematopoietic oncogene product which is also tyrosine phosphorylated after TCR, slg and FceR1 activation.
  • the kinase PTK72/Syk heretofore identified only in B cell slg and mast cell FceRI signaling, was also a major phosphotyrosine substrate after macrophage
  • Syk has also been found to be present in immunoprecipitates of the ⁇ chain Fc ⁇ RIIIA suggesting that Syk is associated with phosphorylated y chain.
  • ODN antisense oligonucleotides
  • a linear antisense ODN was used to target the area surrounding the translation initiation codon.
  • the other was designed to have a stem-loop structure, which can hybridize to three different sites of human Syk mRNA.
  • These Syk antisense ODNs were employed to investigate the role of the Syk tyrosine kinase in the Fey receptor mediated phagocytic signal in cultured monocytes.
  • Antisense or scrambled control ODNs were modified to be protected from nucleases.
  • One phosphodiester backbone at the 5-prime terminus and two at the 3 -prime terminus were modified with phosphorothioate.
  • Prediction of secondary structures of Syk mRNA (Law et al, J. Biol. Chem. 269:12310 (1994)) and ODNs were carried out with the MacDNASIS program (Hitachi Software, San Bruno, CA) on a Macintosh computer.
  • Linear 17 mer Syk antisense ODN having the sequence of 5'-CGCTGTCAGCCATGCCG-3', targets the area surrounding the translation initiation codon of Syk mRNA.
  • Stem-loop Syk antisense ODN is a 57 mer containing sequences complementary to three different target sites, target I (the area of the translation initiation, nucleotide no. 159 to 173), target II (451 to 463), and target III (802 to 816) of Syk mRNA (Law et al, J. Biol . Chem . 269:12310 (1994)) (Figure 5).
  • the stem-loop Syk antisense ODN forms a stem and loop structure by itself and was designed to contain minimal intramolecular secondary structures in the loop domain ( Figure 6).
  • the sequence of the stem-loop Syk antisense ODN is
  • Linear 17 mer control ODN has a random sequence of 5'-GCCCAAGATGATTCCAG-3'.
  • Stem-loop 61 mer control ODN has a random sequence of
  • linear and stem-loop control ODNs were also modified at both the 5-prime and 3-prime termini by phosphorothioates.
  • heparinized blood was centrifuged on Ficoll-Hypaque (Lymphocyte Separation Medium; Organon Teknika, Durham, NC) and interface cells were washed twice in PBS.
  • Ficoll-Hypaque Lymphocyte Separation Medium; Organon Teknika, Durham, NC
  • Mononuclear cells were resuspended in complete medium containing RPMI 1640 (GIBCO BRL, Life Technologies, Inc. Gaithersburg, MD) with 10% heat-inactivated FCS and 2 mM L-glutamine. Cells were allowed to adhere at 37°C onto tissue culture flasks precoated with FCS.
  • non-adherent cells were removed with extensive washing in HBSS .
  • Cells were harvested by vigorous agitation. The yield of monocytes ranged from 2 - 6 x 10 7 cells/500 ml of blood. Monocytes were routinely more than 98% viable judged by trypan blue exclusion. Isolated monocytes were maintained in RPMI 1640 supplemented with L-glutamine (2 mM) and 10% heat-inactivated FCS at 37°C with 5% CO 2 .
  • Oligodeoxynucleotides treatment of cells 1 x 10 5 Monocytes were incubated with ODN-liposome complexes containing 2 ⁇ g/ml of LIPOFECTAMINE and 0.5 ⁇ M of the linear control, 0.5 ⁇ M of the linear Syk antisense ODN or 0.1 ⁇ M of the stem-loop control, 0.1 ⁇ M of the stem-loop Syk antisense ODN in 0.3 ml of RPMI 1640 medium without FCS in a 24-well plate (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) at 37°C for 4 h. Medium was added to a 1 ml final volume with RPMI 1640 containing 10% FCS and cells were then incubated at 37°C for 2 days. The same volume of ODN-liposome mixture was also added to each well on Day 2,
  • LIPOFECTAMINE and 1.0 ⁇ M of the linear control, 1.0 ⁇ M of the linear Syk antisense ODN or 0.2 ⁇ M of the stem-loop control, or 0.2 ⁇ M of the stem-loop Syk antisense ODN for 2 days.
  • IgG-sensitized red blood cells 1 x 10 9 Sheep red blood cells (RBCs)/ml (Rockland Inc., Gilbertville, PA) were sensitized with an equal volume of the highest subagglutinating concentration of rabbit anti-sheep RBC antibody (Cappel Laboratories, West Chester, PA) at 37°C for 30 min.
  • the IgG-sensitized RBCs were washed twice and resuspended in PBS to a final concentration of 1 x 10 9 RBCs/ml as described previously (Schreiber et al, J. Clin. Invest. 56:1189 (1975)).
  • EA Phagocytosis of IgG-sensitized RBCs
  • F(ab')2 IgG (Tago, Burlingame, CA) at 4°C for 30 min. Cells were then washed and fixed with 1%
  • FACStar Becton Dickinson, Mountain View, CA
  • mean fluorescence intensity data and contour plots were generated using Consort 30 software. For all samples, 10,000 events were recorded on a logarithmic
  • Syk-H primer 5'-GGTGTGTGCCCTCCGGCC-3' corresponding to nucleotide No. 122 to 139 of Syk mRNA (Law et al, J. Biol. Chem. 269:12310 (1994)
  • Syk-M primer 5'-CTGCAGGTTCCATGT-3' (nucleotide No. 550 to 564).
  • the transferred membrane was hybridized with biotinylated internal probe (Syk-pS: 5'-GGGAGTGGTAGTGGCAGAGG-3', nucleotide No. 408 to 427) in 6 x SSPE and 50% formamide. After washing the membrane in 0.1 x SSC at 50°C, the hybridized bands were visualized by chemiluminescent detection reagent (PROTOGENETM Nucleic Acid Detection System, GIBCO BRL, Life Technologies, Inc. Gaithersburg, MD).
  • ODN (1 ⁇ M) exhibited a reduced level of phagocytosis. Phagocytosis reduced by 49%, shown as the phagocytic index (PI, from 220 ⁇ 8.8 to 113 ⁇ 12.3). Monocytes incubated with the stem-loop Syk antisense ODN (0.2 ⁇ M) exhibited an even greater reduction in phagocytosis by 89% (PI from 220 ⁇ 8.8 to 24 ⁇ 4.2) ( Figure 7). Both scrambled control ODNs, the linaer (1 ⁇ M) or stem-loop (0.2 ⁇ M) ODN, did not significantly affect Fc ⁇ RIIA mediated phagocytosis.
  • RBL-2H3 cells histamine containing rat mast cells
  • RBL-2H3 cells histamine containing rat mast cells
  • minimal essential medium supplemented with 17% fetal bovine serum, 100 U of penicillin and 100 ⁇ g of streptomycin per ml and 4mM glutamine at 37°C in 5% CO 2 .
  • Cells were seeded onto 1.6 cm plates or 24 well plates at a concentration of 1 x 10 5 cells per well for 24h before assay.
  • antisense ODNs To protect from nuclease digestion, antisense and sense control ODNS were modified by adding one phosphorothioate at the 5- prime terminus and two at the 3-prime terminus of the phosphodiester backbone. Three linear Syk antisense ODNs were designed: Target- I linear Syk antisense ODN (5'ATTGCCCGCCATGTCT3', nucleotides 319 to 333 including the translating initiation codon of Syk mRNA),
  • Target-II (5'GATTTGATTCTTGAG3', nucleotides 1175 to 1189)
  • Target-III (5'ATTTGGTAGTATCCCT3', nucleotides 1465 to 1479).
  • Stem-loop Syk antisense ODN is a 60 mer comprising sequences complementary to the three target sites (Fig. 9) (see also Example V above).
  • ODN treatment of cells 5 x 10 4 or 1 x 10 5 RBL-2H3 cells were seeded in each well of a 24 well plate 24hr before lipofection details. ODN-liposome complexes were added twice, once on day 2 and once on day 3.
  • DOTAP a "lipofectamine”
  • ODN a "lipofectamine”
  • Rat-5 Syk 5'-TTTGGCAACATCACCCGG-3' (nucleotides 368 to 386 and Rat-3 Syk primer: 5'-ACTTATGATGGCTTGCTC-3 '
  • Histamine release assay The histamine release assay was performed by cross-linking the rat RBL-2H3 cell IgE receptor FceRI as described below, and measuring histamine release using an enzyme immunoassay kit
  • RBL-2H3 cells containing 1X10 5 RBL-2H3 cells per well in 1.0 ml EMEM were incubated overnight at 37°C. The cells were washed once with PBS and incubated on ice with 1.0 ml PAGCM (a standard histamine release buffer) and 100 ⁇ l of FceRI antibody for 30 min. Following one gentle wash with PBS, the RBL-2H3 cells were incubated as follows: 1.0 ml of PAGCM buffer alone (negative control), 1.0 ml of PAGCM buffer containing 10 ⁇ l of calcium ionophore (50 ⁇ g/ml stock) (positive control), or 1.0 ml of PAGCM containing 10 ⁇ l of goat anti-mouse antibody (1 mg/ml) for 30 min. at 37°C. The PAGCM buffer containing histamine was removed from the cells and assayed by enzyme immunoassay. One hundred ⁇ l standards were included to produce a standard curve.
  • RBL-2H3 cells treated with stem-loop Syk antisense ODNs released 74% less histamine compared to control cells treated with sense DNA.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Transplantation (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention se rapporte, en général, à des procédés de traitement de maladies dues à des interactions entre des complexes immuns et des récepteurs Fc. La présente invention se rapporte notamment à des procédés permettant de moduler l'élimination de la circulation de cellules recouvertes d'anticorps, qui consistent à inhiber la phagocytose, et à des procédés de modulation de l'interaction des complexes immuns avec les récepteurs Fc des tissus. L'invention se rapporte en outre à des procédés de modulation de l'activation des processus immunologiques induits par l'activation des récepteurs Fc due à l'interaction anticorps-antigènes/récepteurs.
PCT/US1996/010494 1995-06-07 1996-06-07 Procedes d'inhibition de la phagocytose Ceased WO1996040199A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
IL12233896A IL122338A0 (en) 1995-06-07 1996-06-07 Methods of inhibiting phagocytosis
JP9502301A JPH11507824A (ja) 1995-06-07 1996-06-07 食作用を阻害する方法
EP96923327A EP0831875A4 (fr) 1995-06-07 1996-06-07 Procedes d'inhibition de la phagocytose
CA2223402A CA2223402C (fr) 1995-06-07 1996-06-07 Procedes d'inhibition de la phagocytose
AU63869/96A AU723595B2 (en) 1995-06-07 1996-06-07 Methods of inhibiting phagocytosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US48353095A 1995-06-07 1995-06-07
US08/483,530 1995-06-07

Publications (2)

Publication Number Publication Date
WO1996040199A1 true WO1996040199A1 (fr) 1996-12-19
WO1996040199A9 WO1996040199A9 (fr) 1997-04-03

Family

ID=23920431

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/010494 Ceased WO1996040199A1 (fr) 1995-06-07 1996-06-07 Procedes d'inhibition de la phagocytose

Country Status (6)

Country Link
EP (1) EP0831875A4 (fr)
JP (1) JPH11507824A (fr)
AU (1) AU723595B2 (fr)
CA (1) CA2223402C (fr)
IL (1) IL122338A0 (fr)
WO (1) WO1996040199A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1006183A1 (fr) * 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
WO2000023477A3 (fr) * 1998-10-20 2001-04-12 Univ Sheffield Variants d'immunoglobuline
WO2002089832A3 (fr) * 2001-05-09 2004-03-25 Alk Abello As Compositions pharmaceutiques destines a prevenir ou traiter les maladies associees aux cellules th1 et th2 par modulation du rapport th1/th2
WO2005049832A3 (fr) * 2003-11-14 2006-04-20 Univ Yale Interference d'acides nucleiques specifiques a fcgriia
US7192615B2 (en) 2001-02-28 2007-03-20 J&J Consumer Companies, Inc. Compositions containing legume products
US7309688B2 (en) 2000-10-27 2007-12-18 Johnson & Johnson Consumer Companies Topical anti-cancer compositions and methods of use thereof
US7985404B1 (en) 1999-07-27 2011-07-26 Johnson & Johnson Consumer Companies, Inc. Reducing hair growth, hair follicle and hair shaft size and hair pigmentation
US8039026B1 (en) 1997-07-28 2011-10-18 Johnson & Johnson Consumer Companies, Inc Methods for treating skin pigmentation
US8093293B2 (en) 1998-07-06 2012-01-10 Johnson & Johnson Consumer Companies, Inc. Methods for treating skin conditions
US8106094B2 (en) 1998-07-06 2012-01-31 Johnson & Johnson Consumer Companies, Inc. Compositions and methods for treating skin conditions
US8431550B2 (en) 2000-10-27 2013-04-30 Johnson & Johnson Consumer Companies, Inc. Topical anti-cancer compositions and methods of use thereof
EP2662446A3 (fr) * 2006-04-13 2014-04-02 Alcon Research, Ltd. Inhibition véhiculée par ARNi de conditions inflammatoires de la rate liées à la kinase de tyrosine
WO2016193696A1 (fr) * 2015-06-01 2016-12-08 Ucl Business Plc Cellule

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL111105A (en) * 1993-09-30 2009-05-04 Univ Pennsylvania Use of a molecule capable of inhibiting the expression of syk kinase to prepare a pharmaceutical composition for inhibiting phagocytosis
US5641863A (en) * 1993-09-30 1997-06-24 University Of Pennsylvania Chimeric IgG Fc receptors
EP0726965A1 (fr) * 1993-11-23 1996-08-21 Ciba Corning Diagnostics Corp. Utilisation d'oligomeres antisens dans un procede destine a limiter la contamination dans les reactions d'amplification de l'acide nucleique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BLOOD, Volume 83, Number 8, issued 15 April 1994, INDIK et al., "Insertion of Cytoplasmic Tyrosine Sequences into the Nonphagocytic Receptor FcgammaRIIB Establishes Phagocytic Function", pages 2072-2080. *
BLOOD, Volume 996, issued December 1992, PARK et al., "Mapping the Structure of the Fc Receptor FcgammaRIIIA Required for Phagocytosis", page 251A. *
See also references of EP0831875A4 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8039026B1 (en) 1997-07-28 2011-10-18 Johnson & Johnson Consumer Companies, Inc Methods for treating skin pigmentation
US8106094B2 (en) 1998-07-06 2012-01-31 Johnson & Johnson Consumer Companies, Inc. Compositions and methods for treating skin conditions
US8093293B2 (en) 1998-07-06 2012-01-10 Johnson & Johnson Consumer Companies, Inc. Methods for treating skin conditions
WO2000023477A3 (fr) * 1998-10-20 2001-04-12 Univ Sheffield Variants d'immunoglobuline
WO2000032767A1 (fr) * 1998-12-03 2000-06-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RECEPTEURS SOLUBLES DE RECOMBINAISON DU Fc
US8666680B2 (en) 1998-12-03 2014-03-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Recombinant soluble FC receptors
US7074896B1 (en) 1998-12-03 2006-07-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Recombinant soluble Fc receptors
EP1006183A1 (fr) * 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
US7985404B1 (en) 1999-07-27 2011-07-26 Johnson & Johnson Consumer Companies, Inc. Reducing hair growth, hair follicle and hair shaft size and hair pigmentation
US8431550B2 (en) 2000-10-27 2013-04-30 Johnson & Johnson Consumer Companies, Inc. Topical anti-cancer compositions and methods of use thereof
US7309688B2 (en) 2000-10-27 2007-12-18 Johnson & Johnson Consumer Companies Topical anti-cancer compositions and methods of use thereof
US7192615B2 (en) 2001-02-28 2007-03-20 J&J Consumer Companies, Inc. Compositions containing legume products
US7897144B2 (en) 2001-02-28 2011-03-01 Johnson & Johnson Comsumer Companies, Inc. Compositions containing legume products
WO2002089832A3 (fr) * 2001-05-09 2004-03-25 Alk Abello As Compositions pharmaceutiques destines a prevenir ou traiter les maladies associees aux cellules th1 et th2 par modulation du rapport th1/th2
US7368559B2 (en) 2003-11-14 2008-05-06 Diana Beardsley FcγRIIA-specific nucleic acid interference
WO2005049832A3 (fr) * 2003-11-14 2006-04-20 Univ Yale Interference d'acides nucleiques specifiques a fcgriia
US9371529B2 (en) 2006-04-13 2016-06-21 Arrowhead Research Corporation RNAi-mediated inhibition of spleen tyrosine kinase-related inflammatory conditions
US8865671B2 (en) 2006-04-13 2014-10-21 Alcon Research, Ltd. RNAi-mediated inhibition of spleen tyrosine kinase-related inflammatory conditions
EP2662446A3 (fr) * 2006-04-13 2014-04-02 Alcon Research, Ltd. Inhibition véhiculée par ARNi de conditions inflammatoires de la rate liées à la kinase de tyrosine
WO2016193696A1 (fr) * 2015-06-01 2016-12-08 Ucl Business Plc Cellule
CN107667170A (zh) * 2015-06-01 2018-02-06 Ucl商务股份有限公司 细胞
RU2729158C2 (ru) * 2015-06-01 2020-08-04 ЮСиЭл БИЗНЕС ЛТД Клетка
EP3730609A1 (fr) * 2015-06-01 2020-10-28 UCL Business Ltd Cellule
CN107667170B (zh) * 2015-06-01 2021-09-03 奥托路斯有限公司 细胞
US11345734B2 (en) 2015-06-01 2022-05-31 Autolus Limited Chimeric T cell

Also Published As

Publication number Publication date
JPH11507824A (ja) 1999-07-13
AU723595B2 (en) 2000-08-31
CA2223402C (fr) 2012-07-31
EP0831875A1 (fr) 1998-04-01
AU6386996A (en) 1996-12-30
CA2223402A1 (fr) 1996-12-19
EP0831875A4 (fr) 2002-07-31
IL122338A0 (en) 1998-04-05

Similar Documents

Publication Publication Date Title
US7148014B2 (en) Methods of inhibiting phagocytosis
Tiwari-Woodruff et al. OSP/claudin-11 forms a complex with a novel member of the tetraspanin super family and β1 integrin and regulates proliferation and migration of oligodendrocytes
US8114603B2 (en) TREM-1 splice variant for use in modifying immune responses
US5821071A (en) Methods of stimulating phagocytosis
JPH04502859A (ja) 内皮細胞―白血球付着分子（ｅｌａｍ）および白血球付着に関与する分子（ｍｉｌａ）
CA2223402C (fr) Procedes d'inhibition de la phagocytose
AU701919B2 (en) Methods of inhibiting phagocytosis
WO1996040199A9 (fr) Procedes d'inhibition de la phagocytose
Davidson et al. Distribution and immunoregulatory properties of antisecretory factor
US20020081683A1 (en) Novel RGS-containing molecules and uses thereof
Rother et al. Expression of recombinant transmembrane CD59 in paroxysmal nocturnal hemoglobinuria B cells confers resistance to human complement
AU724246B2 (en) Cytoplasmic modulators of integrin regulation/signaling
US20040266990A1 (en) Cd109 nucleic acid molecules polypeptides and methods of use
MXPA01009491A (es) Moleculas de acido desoxirribonucleico (adn) que codifican para proteinas clax humanas y sus proteinas de fusion solubles.
Douhan III Noncoordinate regulation of the human and murine major histocompatibility complex class II genes
AU2002246642A1 (en) TREM-1 Splice variant for use in modifying immune responses
Bannish Transcriptional regulation of MHC class II expression in class II negative B cell lines

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA IL JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

COP Corrected version of pamphlet

Free format text: PAGES 1-57,DESCRIPTION,AND PAGES 58-69,CLAIMS,REPLACED BY NEW PAGES BEARING THE SAME NUMBER;PAGES 1/11-11/11,DRAWINGS,REPLACED BY NEW PAGES 1/15-15/15;DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2223402

Country of ref document: CA

Ref country code: CA

Ref document number: 2223402

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1997 502301

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1996923327

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996923327

Country of ref document: EP