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WO2018011405A1 - Anticorps il-13r alpha1 pour dans le traitement de l'inflammation atopique, de la septicémie et de la neutropénie. - Google Patents

Anticorps il-13r alpha1 pour dans le traitement de l'inflammation atopique, de la septicémie et de la neutropénie. Download PDF

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Publication number
WO2018011405A1
WO2018011405A1 PCT/EP2017/067872 EP2017067872W WO2018011405A1 WO 2018011405 A1 WO2018011405 A1 WO 2018011405A1 EP 2017067872 W EP2017067872 W EP 2017067872W WO 2018011405 A1 WO2018011405 A1 WO 2018011405A1
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neutrophils
mice
neutropenia
csf
treatment
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PCT/EP2017/067872
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English (en)
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Onur Boyman
Janine WOYTSCHAK
Daniela IMPELLIZZIERI
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Universität Zürich
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the use of antibody non-agonist ligands to IL-13Ra1 (UniProt P78552) in the treatment of atopic (allergic) inflammatory diseases, sepsis, and / or neutropenia.
  • neutrophils are the first innate immune cells to migrate to the site of action and rapidly exert several effector functions, including the secretion of cytokines and chemokines, thereby guiding and attracting additional innate and adaptive immune cells.
  • neutrophils Once neutrophils are in the circulation, they patrol the body in search of molecular cues of tissue inflammation, which induce neutrophils to leave the blood vessel and infiltrate the affected tissue. All these steps of neutrophil mobilization are driven by cytokines and chemokines, most notably granulocyte colony-stimulating factor (G-CSF) and the C-X-C chemokine receptor 2 (CXCR2)-binding chemokines CXCL1 and CXCL2, also termed keratinocyte chemoattractant (KC) and macrophage inflammatory protein-2 (MIP-2), respectively.
  • G-CSF granulocyte colony-stimulating factor
  • CXCR2 C-X-C chemokine receptor 2
  • CXCL1 and CXCL2 also termed keratinocyte chemoattractant (KC) and macrophage inflammatory protein-2 (MIP-2
  • G-CSF is synthesized by stromal and immune cells upon acute inflammation and infection and leads to expansion and mobilization of myeloid cells in the BM, most prominently neutrophils. While G-CSF directly stimulates neutrophil precursors to proliferate, G-CSF's influence on neutrophil mobilization relies on CXCR2 and CXCR4. Notably, neutrophil release from the BM is controlled by signals from CXCR4 and its ligand CXCL12, which cause neutrophils to remain in their BM niche, while CXCR2 and its ligands CXCL1 and CXCL2 mediate neutrophil egress from the BM. G-CSF signals tip this balance towards egress by favoring signals via CXCR2 over CXCR4.
  • Type-2 cell immune responses are characterized by the cytokines interleukin-4 (IL-4), IL-5, IL-9, IL-13, IL-25, IL-33, and thymic stromal lymphopoietin, which in turn are produced by, stimulate, and recruit type-2 immune cells, such as T-helper-2 cells as well as different innate immune cells, including type-2 innate lymphoid cells, eosinophils, basophils, mast cells, and IL-4- and/or IL-13-activated macrophages.
  • IL-4 interleukin-4
  • IL-9 interleukin-9
  • IL-13 IL-25
  • IL-33 thymic stromal lymphopoietin
  • neutrophils are conspicuously absent in type-2 cell inflammation.
  • chronic type-2 cell-driven inflammatory disorders such as atopic dermatitis, are often associated with recurrent bacterial infections that are usually contained by neutrophils.
  • marked neutropenia in target organs is a common finding in many allergic diseases.
  • patients suffering from atopic dermatitis contain normal counts of blood neutrophils but show a paucity of neutrophils in skin lesions, even though their skin is colonized with bacteria known to induce neutrophil recruitment.
  • a dominant type-2 cell immune signature characterizes the skin of atopic dermatitis patients, with IL-4 being prominently expressed.
  • the objective of the present invention is to provide means and methods for the treatment of conditions or diseases impacted by neutrophil dysregulation, particularly for the treatment of neutropenia and atopic inflammatory disease. This objective is attained by the subject matter of the independent claims of the present specification.
  • IL-4 is instrumental in initiating, polarizing and maintaining type-2 immunity motivated the inventors to inquire whether IL-4 signals directly affect neutrophil expansion, migration or function.
  • neutropenia is a life-threatening condition as it is accompanied by an increased risk of infections that can rapidly lead to death.
  • the most severe form of neutropenia is called agranulocytosis.
  • causes of neutropenia are manifold, most commonly including medications (chemotherapy, indomethacin and other drugs), cancer (especially leukemias), radiation, autoimmune diseases, genetic diseases, hemodialysis, and vitamin deficiency (vitamin B12 or folic acid).
  • neutrophils reduces their expansion and egress from BM, and dampens their recruitment to peripheral tissues.
  • type II IL-4R signalling can directly impair neutrophil recruitment during infection and inflammation, and that a non-agonist ligand specifically reactive to IL-13Ra1 is of use in the treatment of use in the treatment of a condition selected from neutropenia, allergic inflammation and sepsis.
  • Amino acid sequences are given from amino to carboxyl terminus.
  • Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3 rd ed. p. 21 ).
  • “Capable of forming a hybrid” in the context of the present invention relates to sequences that under the conditions existing in the extracellular bodily compartments or within the cytosol of a mammalian cell, are able to bind selectively to their target sequence.
  • Such hybridizing sequences may be contiguously reverse-complimentary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides.
  • the minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T/U nucleotides, and the backbone chemistry.
  • the hybridizing sequence is at least 80% identical, particularly 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reverse complimentary sequence of a DNA encoding SEQ ID 1 or SEQ ID 2.
  • the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.
  • antibody in its meaning known in the art of cell biology and immunology; it refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen binding fragment or single chains thereof and related or derived constructs.
  • a whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (V H ) and a heavy chain constant region (C H ).
  • the heavy chain constant region is comprised of three domains, C H 1 , C H 2 and C H 3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region (C L ).
  • the light chain constant region is comprised of one domain, C L .
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.
  • antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule (its "target") with high affinity, particularly with a coefficient of dissociation Kd ⁇ 10E-8 mol/l.
  • An antibody-like molecule binds to its target in a similar fashion as an antibody binds specifically to its target.
  • antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins.
  • antibody-like molecule further encompasses a polypeptide derived from protein A domains, a polypeptide derived from fibronectin domain FN3, a polypeptide derived from consensus fibronectin domains, a polypeptide derived from lipocalins, a polypeptide derived from Zinc fingers, a polypeptide derived from Src homology domain 2 (SH2), a polypeptide derived from Src homology domain 3 (SH3), a polypeptide derived from PDZ domains, a polypeptide derived from gamma-crystallin, a polypeptide derived from ubiquitin, a polypeptide derived from a cysteine knot polypeptide and a polypeptide derived from a knottin.
  • SH2 Src homology domain 2
  • SH3 polypeptide derived from Src homology domain 3
  • PDZ domains a polypeptide derived from gamma-crystallin
  • protein A domains derived polypeptide refers to a molecule that is a derivative of protein A and is capable of specifically binding the Fc region and the Fab region of immunoglobulins.
  • armadillo repeat protein refers to a polypeptide comprising at least one armadillo repeat, wherein an armadillo repeat is characterized by a pair of alpha helices that form a hairpin structure.
  • humanized camelid antibody in the context of the present specification refers to an antibody consisting of only the heavy chain or the variable domain of the heavy chain (VHH domain) and whose amino acid sequence has been modified to increase their similarity to antibodies naturally produced in humans and, thus show a reduced immunogenicity when administered to a human being.
  • VHH domain variable domain of the heavy chain
  • the term "specifically reactive" when used in the context of describing the binding of a ligand to its target refers to non-covalent binding of the ligand to the target characterized by a Kd ⁇ 10 "7 mol/l, typically even ⁇ 10 "8 mol/l or ⁇ 10 "9 mol/l, and a binding characteristic of the ligand to other biomolecules present in the biological context of the target characterized by a Kd > 10 "4 , even > 10 "3 .
  • specific binding of the ligand refers to the ability of the ligand to selectively bind to the target but no other structure.
  • a first aspect of the invention relates to the use of a non-agonist polypeptide ligand specifically reactive to IL-13Ra1 (UniProt ID P78552) in the treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis.
  • the non-agonist polypeptide ligand of IL-13Ra1 is capable of binding to IL-13Ra1 , thereby inhibiting the biological effect of IL-4 and IL-13 on cells on which the IL-13Ra1 is expressed.
  • the protein sequence of the IL-13 receptor alpha 1 canonical isoform is
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is specifically reactive to the extracellular part of the receptor molecule, which is described by the sequence
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of chronic neutropenia.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of chronic neutropenia caused by aplastic anemia, particularly aplastic anemia caused by viral infection, exposure to toxic chemicals or radiation.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of acute neutropenia, particularly neutropenia caused or associated with infection such as infection with Salmonella typhi, Mycobacterium (tuberculosis), or cytomegalovirus.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of neutropenia caused or associated with administration of a pharmaceutical drug, particularly administration of indomethacin, propylthiouracil, levamisole, clozapine, valproate, penicillamine, or trimethoprim / sulfamethoxazole combination therapy.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of agranulocytosis. In certain embodiments, the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of neutropenia caused or associated with deficiency of vitamin B12 or folic acid.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of allergic inflammation, particularly allergic asthma, atopic dermatitis, allergic rhinitis or allergic inflammation of the eye or conjunctivitis.
  • allergic inflammation particularly allergic asthma, atopic dermatitis, allergic rhinitis or allergic inflammation of the eye or conjunctivitis.
  • Further allergic disease conditions amenable to such treatment are eosinophilic disorders, food allergy, urticarial and allergic bronchopulmonary aspergillosis.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of bacteremia and sepsis, particularly sepsis caused by bacterial infection.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is used for the treatment of sepsis caused by or associated with a bacterial infection of the skin, a bacterial infection of the lung, a bacterial infection of the urinary tract, a bacterial infection of a joint, a bacterial infection of the gut, a bacterial infection caused by intravenal catheterization or injection, or an infection of a prosthesis.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 for use in treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis is selected from the group consisting of an antibody, an antibody fragment and an antibody-like molecule.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 for use in treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis is an antibody.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 for use in treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis is a human or humanized gamma immunoglobulin.
  • the antibody fragment is a Fab domain or an Fv domain of an antibody, or a single-chain antibody fragment, which is a fusion protein consisting of the variable regions of light and heavy chains of an antibody connected by a peptide linker.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is a single domain antibody, consisting of an isolated variable domain from a heavy or light chain.
  • the non-agonist polypeptide ligand specifically reactive to IL- 13Ra1 is a heavy-chain antibody consisting of only heavy chains such as antibodies found in camelids.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 antibody-like molecule is a repeat protein, such as a designed ankyrin repeat protein.
  • the non-agonist polypeptide ligand specifically reactive to IL-13Ra1 for use in treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis does not specifically bind to IL-4Ra.
  • the non-agonist anti-IL-13Ra1 polypeptide ligand is selected from the group consisting of an antibody, an IL-13Ra1 -specific antibody fragment, an antibody-like molecule and a protein A domains derived polypeptide.
  • Non-antibody scaffolds targeting IL-13Rct1 (antibody-like molecules)
  • Non-limiting examples of such targeting proteins are camelid antibodies, protein scaffolds derived from protein A domains (termed “Affibodies”, Affibody AB), tendamistat (an alpha-amylase inhibitor, a 74 amino acid beta-sheet protein from Streptomyces tendae), fibronectin, lipocalin ("Anticalins", Pieris), T-cell receptors, ankyrins (designed ankyrin repeat proteins termed "DARPins", Univ.
  • IL-13Ra1 can be expressed in insect cells, using a baculovirus expression system (Frei et al., Nat Biotechnol. 2012 30:997-1001 ). The IL-13Ra1 can then be biotinylated and thus be immobilized on streptavidin-coated magnetic beads or on microtiter plates coated with streptavidin or neutravidin (Steiner et al. (2008) J. Mol. Biol.
  • Phage display is a suitable format for antibody fragments (Fab fragments, scFv fragments or single domain antibodies s) (Hoogenboom Nature Biotechnology.
  • yeast display (Pepper et al., Combinatorial Chemistry & High Throughput Screening. 1 1 (2):127- 134, 2008 Feb.).
  • a library of the binding protein of interest is displayed on the surface of yeast, and the target epitope is either directly labeled with a fluorescent dye or its polyhistidine (his 6 )-tag is detected with an anti-his-tag antibody, which is in turn detected with a secondary antibody.
  • a fluorescent dye or its polyhistidine (his 6 )-tag is detected with an anti-his-tag antibody, which is in turn detected with a secondary antibody.
  • Such methods are well known to the practitioners in the field (Boder et al., Methods in Enzymology. 328:430-44, 2000.).
  • the non-agonist anti-IL-13Ra1 polypeptide ligand is an immunoglobulin consisting of two heavy chains and two light chains. In some embodiments, the non-agonist anti-IL-13Ra1 polypeptide ligand is a single domain antibody, consisting of an isolated variable domain from a heavy or light chain. In some embodiments, the non- agonist anti-IL-13Ra1 polypeptide ligand is a heavy-chain antibody consisting of only heavy chains such as antibodies found in camelids.
  • a pharmaceutical composition comprising a non-agonist polypeptide ligand specifically reactive to IL-13Ra1 is provided for use in treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis.
  • the non-agonist anti-IL-13Ra1 polypeptide ligand according to the above described aspects and embodiments is used in a method of manufacture of a medicament for the treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis.
  • Medicaments according to the invention are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing.
  • Also within the scope of the present invention is a method or treating or preventing a condition selected from neutropenia, allergic inflammation and sepsis in a patient in need thereof, comprising administering to the patient a non-agonist polypeptide ligand specifically reactive to IL-13Ra1 as specified in any of the aspects or embodiments of the invention above.
  • Such treatment according to the invention may be for prophylactic or therapeutic purposes.
  • the inhibitor or compound is preferably in the form of a pharmaceutical preparation comprising the inhibitor or compound in chemically pure form and, optionally, a pharmaceutically acceptable carrier or adjuvants.
  • the dosage of the inhibitor or compound depends upon the species, its age, weight, individual condition, the individual pharmacokinetic data, the mode of administration, and whether the administration is for prophylactic or therapeutic purposes.
  • the daily dose administered may range from approximately 0.1 mg/kg to approximately 1000 mg/kg, preferably from approximately 0.5 mg/kg to approximately 100 mg/kg, of an inhibitor or compound according to the above aspects or embodiments of the invention.
  • the effect underlying the instant invention namely the suppression of IL-13Ra1 signaling on neutrophils and related cells in the treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis, is achieved by inhibiting of IL-13Ra1 gene expression mediated by a single-stranded or double-stranded interfering ribonucleic acid oligomer or a precursor thereof, comprising a sequence tract complementary to, in other word capable of forming a hybrid with, IL-13Ra1 mRNA.
  • siRNA silencing or "knocking down" genes, by degradation of mRNA or other effects, is well known.
  • technologies developed for this purpose are siRNA, miRNA, shRNA, shmiRNA, or dsRNA.
  • siRNA miRNA, shRNA, shmiRNA, or dsRNA.
  • IL-13Ra1 gene expression is effected through a single-stranded or double- stranded antisense ribonucleic or deoxyribonucleic acid, comprising sequences complementary to a sequence comprised in an operon that expresses the IL-13Ra1 encoding gene.
  • an operon sequence may include, without being restricted to, an intron, an exon, an operator, a ribosome binding site or an enhancer sequence.
  • antisense molecules may for example be 12-50 nucleotides in length.
  • suppression of IL-13Ra1 signaling on neutrophils and related cells in the treatment or prevention of a condition selected from neutropenia, allergic inflammation and sepsis is achieved by an expression vector, comprising a sequence encoding an interfering ribonucleic acid oligomer or precursor thereof, as is described in the preceding paragraphs.
  • the sequence is under the control of an RNA-polymerase promoter sequence operable in a mammalian cell.
  • Such an expression vector allows for the production of an interfering RNA within the cell. Methods for making and using such expression vectors are known in the art.
  • the invention in another aspect relates to a dosage form for the prevention or treatment of neutropenia, allergic inflammation or sepsis.
  • the dosage form comprises a non- agonist polypeptide ligand specifically reactive to IL-13Ra1 according to one of the above aspects of the invention.
  • the active agent is applied by parenteral administration, such as by subcutaneous, intravenous, intrahepatic or intramuscular injection forms.
  • a pharmaceutically acceptable carrier and/or excipient is present.
  • the pharmaceutical compositions comprise from approximately 1 % to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient.
  • a non-agonist polypeptide ligand specifically reactive to IL-13Ra1 can be administered alone or in combination with one or more other therapeutic agents.
  • Possible combination therapies can take the form of fixed combinations of the inhibitor, or compound with one or more other therapeutic agents known in the prevention or treatment of neutropenia, allergic inflammation or sepsis.
  • the administration can be staggered or the combined agents can be given independently of one another or in the form of a fixed combination.
  • a pharmaceutical composition comprises an non-agonist polypeptide ligand specifically reactive to IL-13Ra1 according to the above aspects or embodiments of the invention, in combination with a therapeutically active amount of the granulocyte colony-stimulating factor (G-CSF, UniProt ID P09919).
  • G-CSF granulocyte colony-stimulating factor
  • Fig. 1 shows that neutrophil influx during skin infection is modulated by IL-4 signals: (A- E) 3x10 7 colony-forming units (cfu) of Group A Streptococcus (GAS) M1 were injected subcutaneously into the shaved flank of C57BL/6 mice pretreated with
  • Data are pooled from 2-3 independent experiments with a total of 4 (D) and 8-12 mice per condition (A-C, and E; 2-3 mice for uninfected) and represented as mean ⁇ SEM. *P ⁇ 0.05; **P ⁇ 0.01 ; ***P ⁇ 0.001.
  • Fig. 2 shows that flood neutrophilia upon systemic infection is suppressed by IL-4:
  • C57BL/6 mice received either PBS or 10 5 cfu LM intravenously (i.v.) with or without a neutralizing anti-G-CSF mAb (anti-G-CSF) on days -1 and 0 of infection. Shown are flow cytometric analysis 24 hours postinfection of CD3 " CD1 1 b + Ly6G + neutrophil frequencies in blood (A) and neutrophil counts in blood and spleen (B).
  • C C57BL/6 mice were treated with PBS, G-CSF, IL-4, or G-CSF plus IL-4 for 3 consecutive days. Shown is quantification of CD1 1 b + Ly6G + neutrophils in BM, blood and spleen 16 hours after last injection.
  • D-F C57BL/6 mice were pretreated for 3 days with PBS, G- CSFcx, IL-4cx, or G-CSFcx plus IL-4cx, followed by systemic (i.v.) infection with 10 5 cfu LM the next day:
  • D Quantification of LM cfu in spleen and liver 24 and 72 hours postinfection.
  • E Mice were assessed for weight change.
  • F Mice were monitored for survival.
  • IL-4 acts directly on neutrophils via type I I IL-4R:
  • a and B Wild-type mice received 3 injections of PBS, G-CSFcx, or G-CSFcx plus IL-4cx.
  • Histograms show IL-4 receptor (IL-4R) subunit expression compared to isotype control (gray shaded area) of mice receiving PBS (black line) or G-CSFcx (blue line) and quantification by mean fluorescence intensity (MFI) of IL-4Ra, common ⁇ -chain (y c ) and IL-13Ra1 expression on CD1 1 b + Ly6G + neutrophils isolated from BM (A) or spleen (B).
  • MFI mean fluorescence intensity
  • C Stimulation of splenocytes from WT, IL-4Ra-deficient (Il4ra ⁇ ' ⁇ ), y c -deficient (//2rg "/_ ), and IL-13Ra1 -deficient (Il13ra1 ⁇ ' ⁇ ) mice with PBS or IL-4 (500 ng/ml) for 15 minutes, followed by quantification of phosphorylated STAT6 (pSTAT6) in CD1 1 b + Ly6G + neutrophils. Shown is change in percentage of MFI of pSTAT6 compared to PBS.
  • BM chimeric mice were injected for 3 consecutive days with PBS, G-CSFcx, or G-CSFcx plus IL-4cx and change in ratios of CD45.2 + to CD45.1 + cells within CD3 " CD1 1 b + Ly6G + blood neutrophils were determined by flow cytometry 16 hours after last injection.
  • H WT and H4ra ⁇ ' ⁇ mice were infected i.v. with 10 5 cfu LM and monitored for survival.
  • IL-4 induces a BM-resident phenotype in neutrophils.
  • A-D WT mice were treated with PBS, G-CSFcx or IL-4cx for 3 consecutive days. BM and spleen were analyzed 16 hours after last injection.
  • A Quantification of geometric MFI of CXCR2 and CXCR4 in neutrophils from BM.
  • B Histogram showing CXCR4 expression on neutrophils in BM.
  • C Quantification of geometric MFI of CXCR2 and CXCR4 in neutrophils from spleen.
  • IL-4 inhibits CXCR2-mediated migration in vitro and in vivo.
  • A Purified BM-derived CD1 1 b + Ly6G + neutrophils were pretreated with either PBS or IL-4 (30 ng/ml), followed by migration towards CXCL2 (100 ng/ml) over 240 minutes.
  • B Purified BM neutrophils were pretreated with either PBS or IL-4 (30 ng/ml), followed by migration towards titrated concentrations of CXCL2 for 2 hours.
  • C and D Purified BM neutrophils were pretreated titrated amounts of IL- 4, followed by migration towards a fixed concentration of CXCL1 (100 ng/ml) (C) or CXCL2 (100 ng/ml) (D) for 2 hours.
  • E Purified BM neutrophils were pretreated in vitro with PBS, IL-2, IL-4, IL-7, IL-13, or IL-15 followed by migration towards CXCL1 (100 ng/ml) for 2 hours.
  • A CXCL1- induced migration of purified neutrophils was analyzed upon treatment in vitro with PBS, phosphoinositide 3-kinase (PI3K) inhibitor LY294002, or p38 mitogen- activated protein kinase (MAPK) inhibitor SB203580.
  • B WT splenocytes were stimulated with either PBS (gray shaded area) or IL-4 (30 ng/ml) for 5 (orange line) or 15 minutes (red line), followed by assessment by flow cytometry of p38 MAPK phosphorylation in CD3 " CD1 1 b + Ly6G + neutrophils.
  • C WT splenocytes were stimulated with either PBS, IL-4 plus DMSO, or IL-4 plus p38 inhibitor SB203580, followed by assessment by flow cytometry of p38 MAPK phosphorylation in CD3 " CD1 1 b + Ly6G + neutrophils. Shown are histograms of phosphorylated p38 MAPK expression in CD3 " CD1 1 b + Ly6G + neutrophils (left) and quantification of MFI values of phosphorylated p38 MAPK (right).
  • D CXCL1-induced migration of purified neutrophils incubated with PBS, IL-4 plus DMSO, IL-4 plus SB203580, or IL-4 plus LY294002.
  • FIG. 1 shows that migration of human neutrophils towards CXCL8 is impaired by stimulation with IL-4 or IL-13.
  • a and B Human neutrophils were isolated from peripheral blood of a healthy donor and pre-treated in vitro for 1 hour with different concentrations (ng/mL) of (A) recombinant human interleukin-4 (IL-4) or (B) recombinant human IL-13, followed by migration towards a fixed concentration of recombinant human CXCL8 (IL-8; 100ng/mL) for 90 minutes. Shown are the counts of migrated neutrophils.
  • C-F Representative histograms (left panels) of expression levels of the indicated IL-4 receptor (IL-4R) subunits (C) CD124 (IL-4Ra), (D) CD132 (common ⁇ chain, yc), (E) CD213a1 (IL-13Ra1 ), and (F) CD213a2 (IL-13Ra2) on human neutrophils isolated from peripheral blood of a healthy donor at basal level (black filled histograms), indicated as to for time-point 0, and upon in vitro stimulation with recombinant human G-CSF for 24 hours (black lines).
  • IL-4R IL-4 receptor subunits
  • Bars show the mean fluorescence intensity (MFI) ⁇ standard deviation of either basal level (indicated as to for time-point 0; open bars) or upon stimulation with G-CSF (grey filled bars). Statistical significance was calculated using Student's f-test.
  • mice The skin of patients with atopic dermatitis usually expresses higher IL-4 concentrations, shows some degree of neutropenia, and is the target of recurrent bacterial infections.
  • GAS Group A Streptococcus
  • Fig. 1A Subcutaneous inoculation of GAS led to a large skin lesion within 48 hours, with further progression after 72 hours (Fig. 1A). This was accompanied by a prominent inflammatory swelling of the infected leg (Fig. 1 B), which was paralleled by a significant influx of CD1 1 b + Ly6G + cells (Fig. 1 C), with Ly6G being specific for neutrophils.
  • IL-4cx anti-IL-4 monoclonal antibody complexes
  • mice receiving IL-4cx demonstrated an attenuated neutrophil response to GAS skin infection by showing lower neutrophil influx into the skin (Fig. 1C and 1 D). Conversely, bacterial loads of GAS were 4-5-times higher in the skin of mice receiving IL-4cx (Fig. 1 E).
  • IL-4 suppresses blood neutrophilia following systemic infection
  • LM infection- induced blood neutrophilia was dependent on G-CSF secretion.
  • LM infection caused a significant surge in G-CSF concentration, as measured using serum of LM-infected mice to stimulate a G-CSF-sensitive cell line, the proliferation of which was abrogated by using a neutralizing anti-G-CSF mAb in vivo or in vitro.
  • IL-4 antagonizes G-CSF effects on neutrophils
  • G-CSF has a short in vivo half-life, unless it is coupled to polyethylene glycol or a particular anti-G-CSF mAb, thus forming G-CSFcx with extended biological half-life (Rubinstein et al., 2013, J Hematol Oncol 6, 75), similar to IL-4cx.
  • G-CSFcx caused a prominent increase in CD1 1 b + Ly6G + neutrophils in BM, blood, and spleen (Fig. 3A and 3B).
  • G-CSFcx-mediated neutrophilia was abrogated by concomitant injection of IL-4cx Fig. 3A and 3B).
  • administration of IL-4cx alone led to a slight decrease of neutrophil percentages and counts in the assessed compartments (Fig. 3A and 3B).
  • mice received a lethal dose of 10 5 cfu LM intravenously, leading to high bacterial titers in the spleen already 24 hours later and in the liver at 72 hours after infection (Fig. 3D). This was accompanied by rapid weight loss and death of animals around day 4 after infection (Fig. 3E and 3F). Notably, injection of G-CSFcx rapidly and significantly lowered bacterial loads in spleen and liver of infected mice (Fig. 3D), reversed the weight loss and prevented death of animals (Fig. 3E and 3F).
  • IL-4 acts directly on neutrophils via type II IL-4R
  • IL-4 exerts its pleotropic effects by binding to two types of IL-4Rs, both of which subsequently lead to phosphorylation of signal transducer and activator of transcription 6 (STAT6).
  • Heterodimerization of IL-4Ra (also termed CD124) with the common ⁇ -chain (y c , also known as CD132) forms the type I IL-4R, which is expressed typically by hematopoietic cells, including B and T cells.
  • type II IL-4Rs are thought to be predominant on non-hematopoietic cells as well as macrophages and consist of heterodimers of IL-4Ra and IL-13Ra1 .
  • IL-13 can signal via type II IL-4Rs by binding to IL-13Ra1 and subsequently IL-4Ra.
  • CD1 1 b + Ly6G + neutrophils isolated from the BM of control mice showed low expression of IL- 4Ra, background expression of y c and IL-13Ra1 (Fig. 4A).
  • IL-4Ra significantly increased by 8.5-fold along with a marked 3.8-fold increase in expression of IL-13Ra1 , both as measured by mean fluorescence intensity (MFI), whereas y c remained unchanged (Fig. 4A).
  • MFI mean fluorescence intensity
  • y c remained unchanged (Fig. 4A).
  • the addition of IL-4cx to G-CSFcx treatment of mice did not alter the effects of G-CSFcx on upregulation of these receptor subunits (Fig. 4A).
  • IL-4 induces a BM-resident, non-migratory phenotype of neutrophils
  • IL-4cx increased CXCR4 in BM neutrophils with CXCR2 remaining low or slightly decreasing (Fig. 5A and 5B), thus promoting BM retention of neutrophils.
  • IL-4cx significantly reduced CXCR2 expression to 8% of PBS (Fig. 5C and 5D), which disfavors migration of neutrophils towards the CXCR2-binding chemokines CXCL1 and CXCL2.
  • IL-4 inhibits CXCR2-mediated migration in vitro and in vivo
  • Neutrophil chemotaxis is intracellular ⁇ regulated by two antagonistic signaling pathways, involving phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK). CXCR2 binding activates PI3K to cause migration of neutrophils. Conversely, once neutrophils arrive to a site of inflammation and infection, so-called end-target chemoattractants, such as N-formyl-Met-Leu-Phe (fMLP) from bacteria or complement factor C5a, stimulate the p38 MAPK pathway, which overrides PI3K-mediated signals.
  • end-target chemoattractants such as N-formyl-Met-Leu-Phe (fMLP) from bacteria or complement factor C5a
  • IL-4 signaling has previously been shown to activate p38 MAPK in human polymorpho- nuclear cells, including neutrophils ( Ratthe et al. (2007), J Leukoc Biol 81, 1287-1296). We thus hypothesized that the inhibitory effect of IL-4 on neutrophil migration might rely on p38 MAPK-mediated interference with PI3K signaling. Stimulation of purified CD1 1 b + Ly6G + neutrophils with IL-4 in vitro caused phosphorylation of p38 MAPK within 5 minutes, which further increased after 15 minutes (Fig. 7B). Concomitant use of the selective ⁇ 38 ⁇ inhibitor SB203580 completely abrogated the appearance of phospho-p38 MAPK in neutrophils (Fig. 7C).
  • IL-4 induces phosphorylation of the p38a MAPK family member in neutrophils, as only p38a and ⁇ 38 ⁇ have been found in neutrophils, whereas ⁇ 38 ⁇ was absent in these cells (Hale et al. (1999), J Immunol 162, 4246-4252).
  • IL-4 inhibited neutrophil recruitment and migration during bacterial infection and inflammation via directly interacting with type II IL- 4Rs on neutrophils.
  • increased G-CSF production upon local and systemic bacterial infection or 'sterile' inflammation as mimicked by injection of long-lasting G-CSF usually expands BM neutrophils.
  • G-CSF is known to induce neutrophil egress from the BM by weakening CXCF -mediated retention signals and augmenting the neutrophils' sensitivity towards CXCR2-binding chemokines.
  • G-CSF signals also increased the expression of type II IL-4Rs, made of IL-4Ra and IL-13Ra1 , rendering neutrophils more sensitive to IL-4.
  • IL-4 signalling in neutrophils resulted in decreased G-CSF-mediated expansion and increased retention of neutrophils in the BM.
  • IL-4 signals also inhibited neutrophil migration by opposing CXCR2-PI3K-mediated signals via the activation of the PI3K antagonist p38 MAPK.
  • type II IL-4R-mediated signaling in neutrophils efficiently interferes with several central and peripheral mechanisms of neutrophil recruitment.
  • IL-4 signals dampen neutrophil or myeloid cell recruitment, which is in line with our herein described mechanism.
  • neutrophil recruitment to the liver is increased in animals lacking both IL-4 and IL-13.
  • inhibition of IL-4 by using a neutralizing anti-IL-4 mAb leads to increased neutrophil counts in the lungs Choy et al. (2015), Sci Transl Med 7, 301 ra129).
  • IL-4 intravenous injection of IL-4 is able to hamper IL-i p-mediated recruitment of neutrophils to an airpouch, although the mechanism by which IL-4 exerted its effect has been unclear and IL-4 is unable to inhibit CXCL8-mediated neutrophil chemotaxis in this publication (Perretti et al. (1995), Br J Pharmacol 116, 2251-2257).
  • systemic administration of IL-4 improves joint inflammation in three different arthritis models, including the rat adjuvant arthritis model, collagen-induced arthritis model in mice, and K/BxN-mediated joint inflammation in mice (Bober et al., 2000 (ibid.); Hemmerle et al.
  • type II IL-4R signaling antagonizes some of the actions of G-CSF on BM neutrophils, particularly those effects of G-CSF pertaining to expansion and egress of BM neutrophils.
  • G-CSF exerted both STAT3- dependent and STAT3-independent effects in neutrophils and also stimulated suppressor of cytokine signaling 3 (SOCS3), which in turn acts as an important negative feedback regulator of G-CSF receptor signaling by inhibiting STAT3 (Carow and Rottenberg, 2014, Front Immunol 5, 58; Nguyen-Jackson et al., 2010, Blood 115, 3354-3363).
  • SOCS3 cytokine signaling 3
  • IL-4R-p38 MAPK-CXCR2 axis The therapeutic implications of the herein described type II IL-4R-p38 MAPK-CXCR2 axis are manifold. As mentioned previously, individuals suffering from allergic disorders show a relative paucity of neutrophils in the affected organs. For example, the skin of patients with atopic dermatitis contains more IL-4 and lower counts of neutrophils and is more susceptible to bacterial infections that are usually contained by neutrophils. Thus, therapeutic approaches targeting IL-4Ra, IL-13Ra1 or p38 MAPK might lower the risk of recurrent bacterial infections.
  • IL-4 is well known to directly affect many immune cells, including macrophages, dendritic cells, B cells and T cells, thereby driving type 2 cell-mediated immunity.
  • IL-4Rs By showing a direct action of IL-4 on neutrophils via type II IL-4Rs we extend the range of target cells of IL-4 and also demonstrate that type II IL-4Rs can become very prominent on immune cells and potently affect their responses during immunity and immunopathology.
  • C57BL/6, CD45.1 (Ly5.1 )-congenic, Cxcr2 ⁇ ' ⁇ , l ⁇ 2rg ⁇ ' ⁇ , Rag1 ⁇ ' ⁇ (all on a C57BL/6 background), and Balb/c and H4ra ⁇ ' ⁇ (on a Balb/c background) were purchased from The Jackson Laboratory (Bar Harbor).
  • H13ra1 ⁇ ' ⁇ (on a Balb/c background) were provided by Regeneron Pharmaceuticals (Ramalingam et al., 2008, Nat Immunol 9, 25-33). Experiments were approved by the Cantonal Veterinary Office and performed in accordance with Swiss law.
  • mice were infected either systemically with 10 5 cfu Listeria monocytogenes (LM) or subcutaneously with 3x10 7 cfu Group A Streptococcus (GAS) M 1 as previously described (Nizet et al., 2001 (ibid.); Zinkernagel et al., 2008 (ibid.)).
  • LM Listeria monocytogenes
  • GAS Group A Streptococcus
  • mice received over three consecutive days daily injections of PBS, free cytokines (5 pg human G-CSF [Neupogen]; 7.5 pg mouse IL-4 [mlL-4, eBioscience]), or cytokine-anti-cytokine monoclonal antibody (mAb) complexes (1 pg human G-CSF complexed with 6 pg anti-human G-CSF mAb clone BVD1 1-37G10, SouthernBiotech; 1 .5 pg mlL-4 complexed with 7.5 pg anti-mlL-4 mAb clone 1 1 B1 1 , BioXcell) prior to infection, as previously published (Boyman et al., 2006 (ibid.); Finkelman et al., 1993 (ibid.); Rubinstein et al., 2013 (ibid.)).
  • free cytokines 5 pg human G-CSF [Neupogen]; 7.5 pg mouse IL-4 [m
  • mice received daily intraperitoneal injections of 100 ⁇ 9 neutralizing mAb against mG-CSF (MAB414; R&D) or mlL-4 (1 1 B1 1 ; BioXcell).
  • MAB414 neutralizing mAb against mG-CSF
  • mlL-4 (1 1 B1 1 ; BioXcell.
  • p38 MAPK activity was blocked in vivo by administering mice three injections of 300 ⁇ g of SB203580 (Calbiochem) (Heit et al., 2002 Nat Immunol 9, 743-752).
  • Murine skin samples were embedded in O.C.T. Compound (Sakura) and stained using anti- Ly6G mAb (1A8; BioXcell). Sections were analyzed using ImageScope for image acquisition (Aperio Technologies, Inc.).
  • splenocytes were stimulated with cytokines for 15 minutes and subsequently fixed by addition of paraformaldehyde and ice-cold methanol. Skin was cut into small pieces and incubated for 1 hour at 37°C with an enzymatic cocktail consisting of 5 pg/ml LiberaseTM (Roche), 1 pg/ml DNAase I (Sigma) and 5 pg/ml Dispase II (Roche) in RPMI media. Subsequently, cells were liberated by extensive pipetting and filtered. Cells were acquired on a BD FACSCantoTM II or BD LSR Fortessa flow cytometer and analyzed using FlowJo software (Tristar Inc.).
  • Immune lineage-negative (Lin " ) bone marrow (BM) cells of WT CD45.1 -congenic and H4ra ⁇ ' ⁇ CD45.2-congenic mice were purified by negative selection using magnetic beads (StemCell Technologies) and biotinylated mAbs against CD19, CD3, MHC class II, NK1.1 , and Ter1 19.
  • Lin " BM cells from WT and H4ra ⁇ ' ⁇ mice were mixed at a 1 :1 ratio and injected intravenously into irradiated (950 rad) H4ra ⁇ ' ⁇ CD45.2-congenic host mice.
  • BM chimeric mice received 1 mg/ml sulfamethoxazol and 0.2 mg/ml trimethoprim in their drinking water for two weeks and were left for three weeks in order to allow for reconstitution of neutrophils before use.
  • CD3 " CD1 1 b + Ly6G + neutrophils were obtained by positive selection using Ly6G microbeads (Miltenyi Biotec), yielding a purity of 92-95%. 10 5 purified neutrophils were pretreated for 20 minutes with PBS or cytokines (30 ng/ml), including IL-2, IL-4, IL-7, IL-13, or IL-15 (from eBioscience and Peprotech), before seeding into the upper chamber of a 5 ⁇ m-transwell (Corning Costar). Subsequently, migration of neutrophils was determined for 2 hours towards CXCL1 or CXCL2 (both 100 ng/ml; PeproTech) given to the lower chamber. Where indicated, SB203580 or Ly294002 (both 30 ⁇ ; Calbiochem) was added 15 minutes before pretreatment. Airpouch model
  • mice were left overnight and subsequently euthanized, followed by flushing of the airpouch with PBS plus 2 mM EDTA to collect cells within. The content of the airpouch was counted and analyzed by flow cytometry for leukocytes.

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Abstract

L'invention porte sur l'utilisation d'un ligand polypeptidique, non-agoniste, réagissant spécifiquement à IL -13 Rα1 dans le traitement, ou la prévention, de la neutropénie, l'inflammation allergique, la bactériémie et la septicémie.
PCT/EP2017/067872 2016-07-15 2017-07-14 Anticorps il-13r alpha1 pour dans le traitement de l'inflammation atopique, de la septicémie et de la neutropénie. WO2018011405A1 (fr)

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WO2021117037A1 (fr) * 2019-12-08 2021-06-17 Ramot At Tel-Aviv University Ltd. Ciblage du récepteur alpha 1 de l'il-13 dans la dermatite atopique et les maladies allergiques
WO2023285349A1 (fr) 2021-07-12 2023-01-19 2N Pharma Aps Inhibiteurs de la carnitine-palmitoyl-transférase-1 (cpt-1) destinés à être utilisés dans un procédé de prévention ou de traitement de la septicémie chez un sujet mammifère

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021117037A1 (fr) * 2019-12-08 2021-06-17 Ramot At Tel-Aviv University Ltd. Ciblage du récepteur alpha 1 de l'il-13 dans la dermatite atopique et les maladies allergiques
WO2023285349A1 (fr) 2021-07-12 2023-01-19 2N Pharma Aps Inhibiteurs de la carnitine-palmitoyl-transférase-1 (cpt-1) destinés à être utilisés dans un procédé de prévention ou de traitement de la septicémie chez un sujet mammifère

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