WO2021117037A1 - Targeting il-13 receptor alpha 1 in atopic dermatitis and allergic diseases - Google Patents

Targeting il-13 receptor alpha 1 in atopic dermatitis and allergic diseases Download PDF

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WO2021117037A1
WO2021117037A1 PCT/IL2020/051269 IL2020051269W WO2021117037A1 WO 2021117037 A1 WO2021117037 A1 WO 2021117037A1 IL 2020051269 W IL2020051269 W IL 2020051269W WO 2021117037 A1 WO2021117037 A1 WO 2021117037A1
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antibody
seq
mice
isolated polypeptide
cells
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PCT/IL2020/051269
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French (fr)
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Ariel Munitz
Itai Benhar
Danielle KARO-ATAR
Almog BITTON
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Ramot At Tel-Aviv University Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—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
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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 in some embodiments thereof, relates to agents which bind to IL- 13R ⁇ 1 receptor for the treatment of allergic diseases and, more particularly, but not exclusively, for the treatment of atopic dermatitis.
  • AD Atopic dermatitis
  • Extrinsic AD manifests with high serum IgE levels, whereas intrinsic AD has no IgE upregulation 1 .
  • T cell-targeting drugs such as cyclosporine 2, 3 .
  • Thl, Thl7, and Th22 polarizations differ between AD endotypes, a strong Th2 axis is associated with both extrinsic and intrinsic AD 4 .
  • IL-4 and IL-13 are produced at elevated levels in the lesional skin and are central regulators of many of the hallmark features of AD, including epidermal hyperplasia, skin barrier dysfunction, and production of eosinophil and T cell chemokines 5, 6 .
  • the importance of IL-4, IL-13 and their associated receptors in AD is best exemplified by the ongoing pursuit to pharmacologically target these cytokines and/or their signaling components in AD 5,7 .
  • an isolated polypeptide comprising a binding domain which binds specifically to human Interleukin 13 receptor, alpha 1 (IL-13R ⁇ 1), wherein the polypeptide downregulates an activity of Interleukin 13 (IL-13), the polypeptide comprising each of the following amino acid sequences:
  • composition comprising the isolated polypeptide described herein as the active agent and a pharmaceutically acceptable carrier.
  • a method of treating a disease mediated by IL-13 in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the isolated polypeptide described herein, thereby treating the disease.
  • the activity comprises induction of CCL26 secretion.
  • the polypeptide is an antibody.
  • the polypeptide is an antibody having an antigen recognition domain which comprises the CDR sequences of an antibody having a heavy chain as set forth in SEQ ID NO: 55, wherein the CDR sequences are in the same orientation as the antibody having the heavy chain as set forth in SEQ ID NO: 55.
  • the isolate polypeptide further comprises a light chain.
  • the CDR1 of a heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 28
  • CDR2 of the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 29
  • CDR3 of the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:
  • the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 55.
  • the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 56.
  • the polypeptide has an affinity between O.lnM -lOnM for the human IL-13R ⁇ 1 as measured by ELISA.
  • the antibody is selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE antibody.
  • the antibody is a bi-specific antibody.
  • the first target of the bi-specific antibody is human IL-13R ⁇ 1
  • the second target of the bi-specific antibody is human IL-4.
  • the isolated polypeptide described herein is for use in treating a disease mediated by IL-13.
  • the disease mediated by IL-13 is selected from the group consisting of atopic dermatitis, asthma, eosinophilia, urticaria, and allergic rhinitis fibrotic diseases, COPD and cancer.
  • the disease is atopic dermatitis.
  • the method further comprises administering to the subject an agent which downregulates the amount and / or activity of IL-4.
  • the agent is an antibody directed against the IL-4.
  • FIGs. 1 A-K The type 2 IL-4 receptor is required for oxazolone-induced atopic dermatitis.
  • Wild type (WT) and IlI3r ⁇ 1 -/- mice were challenged with acetone or oxazolone for 3 weeks. Thereafter, ear thickness was measured (A). Quantitation of epidermal thickness (B) and representative photomicrographs of hematoxylin and eosin stained slides are shown (C). Skin protein levels of TNF-a (D), IL-4 (E), CXCL1 (G), CCL11 (H), CCL24 (I), CCL17 (J), as well as serum IgE (F) are shown.
  • FIGs.2A-Q Differential roles for IL-4 and IL-13 via the type 2 IL-4R in oxazolone-driven atopic dermatitis.
  • Wild type (WT) and IlI3r ⁇ 1 -/- mice were challenged with oxazolone and treated with anti-IL-4 or isotype control antibody (Isotype).
  • ear thickness was measured and representative photomicrographs (B) and quantitation of epidermal thickness (D) of hematoxylin and eosin stained slides are shown.
  • Skin protein levels of TNF-a (E), IL-4 (F), CXCL1 (H), CCL11 (I), CCL24 (J), CCL17 (K) as well as serum IgE (G) are shown.
  • FIGs. 3A-J Neutralization of IL-13 and TNF-a in oxazolone-treated wild type mice demonstrates a key role for IL-13 in oxazolone-induced dermatitis, CXCL1 expression and infiltration of neutrophils.
  • Wild type (WT) mice were challenged with oxazolone and treated with isotype control or with neutralizing antibodies targeting IL-13 (A-G) or TNF-a (H-J). Subsequently, ear thickness (B, H) and epidermal thickening (C) were measured.
  • FIGs. 4A-N Dissecting the contribution of the type 2 IL-4R expressed by hematopoietic and non-hematopoietic cells to oxazolone-induced allergic skin disease.
  • Wild type (WT) and IlI3r ⁇ 1 -/- mice were irradiated. Thereafter, recipient mice received bone marrow cells from WT or IlI3r ⁇ 1 -/- mice.
  • WT or IlI3r ⁇ 1 -/- mice were challenged with oxazolone and ear thickness was measured (A).
  • Representative photomicrographs (C) and quantitation of epidermal thickness (B) of hematoxylin and eosin stained slides are shown.
  • FIGs. 5A-H Assessment of an anti-mouse IL-13R ⁇ 1 neutralizing antibody. Binding properties of MSS mAb to mouse (m) or human (h) IL-13R ⁇ 1 ECD, or streptavidin (A). ELISA- estimated affinity (Kd) to mIL-13R ⁇ 1 was calculated (A). MC38 cells were treated with lOng/ml of IL-13 (B), IL-4 (C), or TNF-a (D) in the presence of anti-IL-13R ⁇ 1 (MSS) or isotype control mAb at decreasing 2-fold concentrations.
  • FIGs. 6A-J Pharmacological neutralization of the type 2 IL-4R alleviates oxazolone- induced atopic dermatitis.
  • Wild type mice were challenged with oxazolone for 3 weeks and treated with isotype control or anti-IL-13R ⁇ 1 (MSS). Thereafter, ear thickness was measured (A).
  • Representative photomicrographs (C) and quantitation of epidermal thickness (B) of hematoxylin and eosin stain slides are shown. Skin protein levels of TNF-a (D), IL-4 (E), CXCL1 (G), CCL11
  • FIGs.7A-E Generation and assessment of an anti-human IL-13R ⁇ 1 neutralizing antibody. Binding properties of 2HA6 mAb to mouse (m) or human (h) IL-13R ⁇ 1 ECD, or streptavidin (A). ELISA-estimated affinity (Kd) to hIL-13R ⁇ 1 was calculated (A). Data are shown as mean ⁇ SD. A549 cells were treated with lOng/ml of IL-13 (B), IL-4 (C), or TNF-a (D) in the presence of anti- IL-13R ⁇ 1 (2HA6) or isotype control mAb. Thereafter, the levels of CCL26 (B-C) and IL-6 (D) in the culture supernatants were measured.
  • FIG. 8 Expression of Il-13R ⁇ 1 in the skin. Wild type mice were treated with acetone (Control) or challenged with oxazolone. Thereafter, the ears were enzymatically digested and the expression of IL-13R ⁇ 1 was determined by flow cytometry. Data are shown as mean ⁇ SEM; * ⁇ p 0.05, **p ⁇ 0.01.
  • FIGs. 9A-B IL-13R ⁇ 1 regulates oxazolone-induced dermatitis.
  • Wild type (WT) C57BL/6 and III 3 ra1 -/- mice were challenged with oxazolone and euthanized on Day 25. Representative ear photographs of mice from each group are shown.
  • FIGs. lOA-O The type 2 IL-4 receptor is required for DFNB-induced atopic dermatitis.
  • Wild type (WT) and IlI3r ⁇ 1 -/- mice were challenged with acetone or DNFB for 3 weeks. Thereafter, ear thickness was measured (A).
  • Quantitation of epidermal thickness (B) and representative photomicrographs of hematoxylin and eosin stained slides of acetone- and oxazolone-challenged skin are shown (C).
  • the levels of skin protein levels of TNF-a (D), IL-4 (E), CXCL1 (G), CCL11 (H), CCL24 (I), CCL17 (J), as well as serum IgE (F) are shown.
  • J skin leukocytes
  • K macrophages
  • L T cells
  • M neutrophils
  • N monocytes
  • O eosinophils
  • FIGs. 11A-B Assessing the cellular source for IL-4 and IL-13 in oxazolone-induced dermatitis.
  • IL-4 reporter (4Get) and IL-13 reporter ( IU3 Smart ) mice were treated with oxazolone to induce experimental atopic dermatitis. Twenty for hours after the last oxazolone challenge, the ears were obtained, enzymatically digested and single cell suspensions stained to identify CD4 + , CD8 + , NKT and ⁇ T cells. The percentage of cells (out of the total gated population) that are positive for GFP or stained positive for human CD4 (hCD4), which represents IL-13 expressing cells in shown (A-B). Each dot represents a different mouse.
  • FIGs. 12A-D Engraftment efficiency of following adoptive transfer of wild type and ⁇ 113 ⁇ 1 -/- bone marrow cells.
  • Wild type (WT) C56BL/6 mice, WT B6 CD45.1 mice, and H13ral ' /- mice were irradiated with 9 Gy total body irradiation (TBI). Twenty-four hours post-TBI, recipients received 5xl0 6 bone-marrow cells obtained from donor CD45.1 or IlI3r ⁇ 1 -/- mice. Two weeks later, bloods were drawn from representative mice (one from each group, A-D) and blood cells were stained with CD45. l-APC/eF780 and CD45.2-PE antibodies by flow cytometry.
  • FIGs. 13A-C IL-13R ⁇ 1 antisera inhibit IL-13-induced IL-13Ra2 secretion by MC38 cells
  • Wild type mice were immunized with the recombinant extracellular domain of mouse mouse (m) IL-13R ⁇ 1 and boosted fortnightly three times. Prior to immunization and following the 3 rd boost, blood was drawn and sera were examined for binding mIL-13R ⁇ 1 (A), or streptavidin (control antigen) (B).
  • MC38 cells were treated with a 1:100 dilution in saline of serum from pre-immunized mice (Pre Immunization) or antisera (Post Immunization) in presence of lOOng/ml recombinant mouse IL-13 (C).
  • Pre Immunization pre-immunized mice
  • antisera post Immunization
  • C lOOng/ml recombinant mouse IL-13
  • FIGs. 14A-B Schematic representation of the Yeast Display system used in this study to isolate IL-13R ⁇ 1-binding antibodies.
  • scFv single chain variable fragment
  • the fusion Alpha-agglutinin (Aga)2-scFv protein forms disulfide bonds with the membrane protein Agal, resulting in scFv display on the cell surface, thus allowing it to bind soluble antigens (e.g. biotinylated IL-13R ⁇ 1).
  • Antigen binding ability is evaluated by detection of the scFv-bound biotinylated antigen by Allophycocyanin (APC)- conjugated streptavidin (SA). Furthermore, a c-myc tag located at the C-terminus of the Aga2- scFv fusion protein, allows to assess the efficiency of scFV display by detecting a complex comprised of mouse anti-myc and Alexa flour 488 (AF488)-conjugated anti-mouse IgG antibodies.
  • the yeast library was analyzed by flow cytometry (Galios, Beckman Coulter) (B) and APC7AF488 + clones were enriched by repetitive sorting cycles (B). Thereafter, the output scFvs were sequenced.
  • B Representative enrichment procedure of clones binding mouse (m) IL- 13Ra 1.
  • FIGs. 15A-C IL-13R ⁇ 1-specific antisera inhibit IL-13-induced CCL26 secretion by A549 cells.
  • Wild type C57BL/6 mice were immunized with the recombinant extracellular domain of human (h) IL-13R ⁇ 1 and boosted fortnightly three times. Prior to immunization and nine days following the 3 rd boost, blood was drawn and sera were examined for binding hIL-13R ⁇ 1 (A), or streptavidin (control antigen) (B).
  • A549 cells were treated with a 1:100 dilution in saline of serum from pre-immunized mice (Pre Immunization) or antisera (Post Immunization) in presence of 100ng/ml recombinant mouse IL-13 (C).
  • Pre Immunization pre-immunized mice
  • Post Immunization post Immunization
  • C recombinant mouse IL-13
  • the ability of antisera to block IL-13 -induced CCL26 secretion levels in the culture supernatants were determined. Data are shown as mean ⁇ SD; **p ⁇ 0.01. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
  • the present invention in some embodiments thereof, relates to agents which bind to IL- 13R ⁇ 1 receptor for the treatment of allergic diseases and, more particularly, but not exclusively, for the treatment of atopic dermatitis.
  • IL-13 and IL-4 are potent mediators of type 2-associated inflammation such as those found in atopic dermatitis (AD).
  • IL-4 shares overlapping biological functions with IL-13, a finding that is mainly explained by their ability to signal via the type 2 IL-4 receptor (R), which is comprised of IL-4Ra in association with IL-13R ⁇ 1.
  • the present inventors sought to define the role of the type 2 IL-4R in AD.
  • Two distinct models of experimental AD in IlI3r ⁇ 1 -/- mice, which lack the type 2 IL-4R were set up.
  • expression of TNF-a was dependent on the type 2 IL-4R, whereas, induction of IL-4, IgE, CCL24, and skin eosinophilia were dependent on the type 1 IL-4R.
  • the present inventors also generated an antibody which recognizes human IL-13R ⁇ 1 ( Figures 7A-E) and propose that this antibody can be used for the treatment of allergic diseases including AD. Furthermore, the antibody can serve as a basis for the generation of additional antibodies for the treatment of such diseases.
  • an isolated polypeptide comprising a binding domain which binds specifically to human Interleukin 13 receptor, alpha 1 (IL-13R ⁇ 1), wherein the polypeptide downregulates an activity of IL-13, the polypeptide comprising each of the following amino acid sequences:
  • polypeptide encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drag Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • Peptide bonds (-CO-NH-) within the polypeptide may be substituted, for example, by N- methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-0-0-C(R)-N-), ketomethylen bonds (- CO-CH2-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted for synthetic nonnatural acid such as Phenylglycine, Tic, naphtylalanine (Nal), phenylisoserine, threoninol, ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • synthetic nonnatural acid such as Phenylglycine, Tic, naphtylalanine (Nal), phenylisoserine, threoninol, ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • polypeptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc.).
  • modified amino acids e.g. fatty acids, complex carbohydrates etc.
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids. Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used in the polypeptides of the present invention.
  • IL-13R ⁇ 1 refers to a subunit of the IL-13 receptor which dimerizes with the IL-4Ra subunit. Together with IL-4Ra subunit, the IL-13R ⁇ 1 subunit mediates the biological effects of IL-13.
  • An exemplary mRNA which encodes human is set forth in RefSeq No. NM_001560.3.
  • An exemplary amino acid sequence of human IL-13R ⁇ 1 is set forth in SEQ ID NO: 65 (UniProtKB - P78552).
  • polypeptides described herein bind specifically to human Interleukin 13 receptor, alpha 1 (IL-13R ⁇ 1). According to a specific embodiment, the polypeptides bind with at least 2 fold, 5 fold, 10 fold or even higher affinity to human IL-13R ⁇ 1 as compared to mouse IL-13R ⁇ 1, as measured by ELISA. According to a specific embodiment, the polypeptides bind with at least 2 fold, 5 fold, 10 fold or even higher affinity to human type 2 IL-4 receptor as compared to human type 1 IL-4 receptor, as measured by ELISA.
  • the polypeptides described herein may have an apparent affinity between O.lnM -lOnM for the human IL-13R ⁇ 1 as measured by ELISA.
  • the affinity is equivalent to the Kd.
  • the polypeptides may have an affinity (or Kd) between O.SnM-SnM for the human IL-13R ⁇ 1 as measured by ELISA.
  • Additional contemplated affinities (Kds) are between 500 nM- 0.5 nM, 100 nM-1 nM, 50 nM-1 nM, 20 nM-1 nM, 10 nM-1 nM, as measured by ELISA.
  • K D refers to the equilibrium dissociation constant between the polypeptide and its respective target.
  • the polypeptides of the present invention are capable of inhibiting one or more functional activities of hIL-13R ⁇ 1.
  • the polypeptides inhibit (or downregulate) CCL26 secretion from epithelial cells, as measured by ELISA.
  • the polypeptides may inhibit IL-13-induced eotaxin release in fibroblast cells.
  • the polypeptides may inhibit IL-13-induced STAT6 phosphorylation in fibroblast cells.
  • the polypeptides may inhibit IL-4-induced eotaxin release in fibroblast cells.
  • the polypeptides may inhibit IL-4-induced STAT6 phosphorylation in fibroblast cells.
  • the polypeptides inhibit all of the above functional activities of hIL-13R ⁇ 1.
  • the polypeptides of the instant invention may or may not inhibit the binding of IL-13 to isolated IL-13R ⁇ 1 (i.e., IL-13R ⁇ 1 that is not part of a dimeric receptor with IL4Ra). In one embodiment, the polypeptides of the instant invention prevent dimerization of IL-13R ⁇ 1 with IL- 4Ra. In another embodiment, the polypeptides described herein compete for the binding of IL-13 with IL-13R ⁇ 1.
  • the polypeptide may be selected from the group consisting of TCR, CAR-T and an antibody.
  • the polypeptide is an antibody.
  • the reference to antibodies is in more details as compared to other polypeptides having affinity binding entities, the description of this embodiment should not be construed as limiting and the present invention is equally related to binding entities as described herein especially in the sense of cell therapy as further described hereinbelow.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VL that are capable of binding to an epitope of an antigen in an MHC restricted manner.
  • antibody aims to encompass any affinity binding entity which binds a cell surface presented molecule with an MHC restricted specificity.
  • CDRs of the antibodies of some embodiments of the present invention may be implanted in artificial molecules such as T cell receptors or CARs as further described hereinbelow.
  • Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.
  • CDR complementarity-determining region
  • light chain referred to herein as “light chain”
  • heavy chain a complementarity-determining region of an immunoglobulin heavy chain
  • variable region of a light chain a variable region of a heavy chain
  • a light chain a variable region of
  • CDR complementarity-determining region
  • VH VH
  • VL VL
  • Examples of heavy chain CDR sequences are provided by SEQ ID NOs: 28, 29, 30, 34, 35 and 36.
  • Examples of light chain CDR sequences are provided by SEQ ID NOs: 40, 41, 42, 46, 47 or 48.
  • the identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by IMGT (see Giudicelli V, Chaume D, Bodmer J, Miiller W, Busin C, Marsh S, et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206-11), Kabat et al. (See, e.g., Rabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al.
  • variable regions and CDRs may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches. According to a specific embodiment, the CDRs are determined according to Rabat et al. (supra).
  • the antibodies provided herein have an antigen recognition domain which comprises the CDR sequences of an antibody having a heavy chain as set forth in SEQ ID NO: 55, wherein the CDR sequences are in the same orientation as the antibody having the heavy chain as set forth in SEQ ID NO: 55.
  • an antibody comprising SEQ ID NO: 28 (as CDR1 of the heavy chain), SEQ ID NO: 29 (as CDR2 of the heavy chain) and SEQ ID NO: 30 (as CDR3) of the heavy chain is contemplated, wherein the CDRs are determined according to the IGMT method.
  • SEQ ID NO: 34 (as CDR1 of the heavy chain), SEQ ID NO: 35 (as CDR2 of the heavy chain) and SEQ ID NO: 36 (as CDR3) of the heavy chain is contemplated, wherein the CDRs are determined according to the Rabat method.
  • the antibodies provided herein have an antigen recognition domain which comprises the CDR sequences of an antibody having a light chain as set forth in SEQ ID NO: 56, wherein the CDR sequences are in the same orientation as the antibody having the heavy chain as set forth in SEQ ID NO: 56.
  • an antibody comprising SEQ ID NO: 40 (as CDR1 of the light chain), SEQ ID NO: 41 (as CDR2 of the light chain) and SEQ ID NO: 42 (as CDR3) of the light chain is contemplated, wherein the CDRs are determined according to the IGMT method.
  • SEQ ID NO: 46 (as CDR1 of the light chain), SEQ ID NO: 47 (as CDR2 of the light chain) and SEQ ID NO: 48 (as CDR3) of the light chain is contemplated, wherein the CDRs are determined according to the Rabat method.
  • the light chain comprises the CDR sequences SEQ ID NO: 40, SEQ ID NO: 47 and SEQ ID NO: 42.
  • variable region of the heavy chain of the antibodies of this aspect of the present invention may have an amino acid sequence at least 90 % identical, 91 % identical, 92 % identical, 93 % identical, 94 % identical, 95 % identical, 96 % identical, 97 % identical, 98 % identical, 99 % identical to SEQ ID NO: 55, wherein the CDR sequences remain as defined herein above.
  • Fv defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;
  • scFv single chain Fv
  • dsFv disulfide-stabilized Fv
  • Fab a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CHI domains thereof;
  • Fab a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);
  • F(ab’)2 a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and
  • Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
  • Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the heavy and light chains of an antibody of the invention may be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, or two, complete light chains) or may include an antigen-binding portion (a Fab, F(ab').sub.2, Fv or a single chain Fv fragment ("scFv")).
  • the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2,
  • the immunoglobulin isotype is selected from IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl (e.g., human IgGl) or IgG4 (e.g., human IgG4).
  • IgGl e.g., human IgGl
  • IgG4 e.g., human IgG4
  • the choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.
  • the antibody is an IgGl antibody having a heavy chain constant domain as set forth in SEQ ID NO: 62.
  • the antibody is an IgGl antibody having a light chain constant domain as set forth in SEQ ID NO: 64.
  • a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen.
  • the BsMAb is engineered to simultaneously bind to IL-13R ⁇ 1 and IL-4.
  • chimeric antigen receptor refers to a recombinant or synthetic molecule which combines antibody-based specificity for a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits cellular immune activity to the specific antigen.
  • T Cell Receptor or “TCR” refers to soluble and non-soluble forms of recombinant T cell receptor.
  • polypeptides e.g. antibodies
  • a functional moiety also referred to as an “immunoconjugate”
  • the immunoconjugate molecule can be an isolated molecule such as a soluble and/or a synthetic molecule.
  • detectable or reporter moieties may be conjugated to the antibody of the invention. These include, but not are limited to, a radioactive isotope (such as [125] iodine), a phosphorescent chemical, a chemiluminescent chemical, a fluorescent chemical (fluorophore), an enzyme, a fluorescent polypeptide, an affinity tag, and molecules (contrast agents) detectable by Positron Emission Tomagraphy (PET) or Magnetic Resonance Imaging (MRI).
  • fluorophores examples include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FTTC), Cy-chrome, ihodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red, PE-Cy5, and the like.
  • PE phycoerythrin
  • FTTC fluorescein isothiocyanate
  • Cy-chrome Cy-chrome
  • ihodamine green fluorescent protein
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • Texas red PE-Cy5, and the like.
  • fluorophore selection methods of linking fluorophores to various types of molecules see Richard P. Haugland, “Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992- 1994”, 5th ed., Molecular Probes, Inc. (1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.;
  • Fluorescence detection methods which can be used to detect the antibody when conjugated to a fluorescent detectable moiety include, for example, fluorescence activated flow cytometry (FACS), immunofluorescence confocal microscopy, fluorescence in-situ hybridization (FISH) and fluorescence resonance energy transfer (FRET).
  • FACS fluorescence activated flow cytometry
  • FISH fluorescence in-situ hybridization
  • FRET fluorescence resonance energy transfer
  • enzymes may be attached to the antibody of the invention [e.g., horseradish peroxidase (HPR), beta-galactosidase, and alkaline phosphatase (AP)] and detection of enzyme-conjugated antibodies can be performed using ELISA (e.g., in solution), enzyme-linked immunohistochemical assay (e.g., in a fixed tissue), enzyme-linked chemiluminescence assay (e.g., in an electrophoretically separated protein mixture) or other methods known in the art [see e.g., Khatkhatay MI. and Desai M., 1999. J Immunoassay 20: 151-83; wisdom GB., 1994. Methods Mol Biol.
  • HPR horseradish peroxidase
  • AP alkaline phosphatase
  • the affinity tag (or a member of a binding pair) can be an antigen identifiable by a corresponding antibody [e.g., digoxigenin (DIG) which is identified by an anti-DIG antibody) or a molecule having a high affinity towards the tag [e.g., streptavidin and biotin].
  • DIG digoxigenin
  • the antibody or the molecule which binds the affinity tag can be fluorescently labeled or conjugated to enzyme as described above.
  • a streptavidin or biotin molecule may be attached to the antibody of the invention via the recognition sequence of a biotin protein ligase (e.g., BirA) as described in the Examples section which follows and in Denkberg, G. et al, 2000. Eur. J. Immunol. 30:3522-3532.
  • a streptavidin molecule may be attached to an antibody fragment, such as a single chain Fv, essentially as described in Cloutier SM. et al, 2000. Molecular Immunology 37:1067-1077; Dubel S. et al, 1995.
  • biotin conjugated antibodies are bound to a streptavidin molecule to form a multivalent composition (e.g., a dimer or tetramer form of the antibody).
  • the antibody may be conjugated to a therapeutic moiety.
  • the therapeutic moiety can be, for example, a cytotoxic moiety, a toxic moiety, a cytokine moiety and a second antibody moiety comprising a different specificity to the antibodies of the invention.
  • the functional moiety (the detectable or therapeutic moiety of the invention) may be attached or conjugated to the antibody of the invention in various ways, depending on the context, application and purpose.
  • the immunoconjugate may be produced by recombinant means.
  • the nucleic acid sequence encoding a toxin e.g., PE38KDEL
  • a fluorescent protein e.g., green fluorescent protein (GFP), red fluorescent protein (REP) or yellow fluorescent protein (YfP)
  • GFP green fluorescent protein
  • REP red fluorescent protein
  • YfP yellow fluorescent protein
  • the functional moiety may be chemically synthesized by, for example, the stepwise addition of one or more amino acid residues in defined order such as solid phase peptide synthetic techniques.
  • a functional moiety may also be attached to the antibody of the invention using standard chemical synthesis techniques widely practiced in the art [see e.g., hypertexttransferprotocol://worldwideweb (dot) chemistry (dot) org/portal/Chemistry)], such as using any suitable chemical linkage, direct or indirect, as via a peptide bond (when the functional moiety is a polypeptide), or via covalent bonding to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer.
  • Chimeric peptides may be linked via bonding at the carboxy (C) or amino (N) termini of the peptides, or via bonding to internal chemical groups such as straight, branched or cyclic side chains, internal carbon or nitrogen atoms, and the like.
  • Description of fluorescent labeling of antibodies is provided in details in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110.
  • the present inventors have shown that the antibodies of the present invention may be used to treat diseases mediated by IL-13.
  • diseases include, but are not limited to asthma, allergy, allergic rhinitis, chronic sinusitis, hay fever, atopic dermatitis, chronic obstructive pulmonary disease ("COPD"), pulmonary fibrosis, esophageal eosinophilia, scleroderma, psoriasis, psoriatic arthritis, fibrosis, inflammatory bowel disease (particularly, ulcerative colitis), anaphylaxis, and cancer (particularly, Hodgkin's lymphoma, glioma, and renal carcinoma), and general Th2-mediated disorders/conditions .
  • COPD chronic obstructive pulmonary disease
  • Additional contemplated cancers that can be treated with the polypeptides described herein include but are not limited to adrenocortical carcinoma, hereditary; bladder cancer; breast cancer, breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptibility to; breast cancer, type 4; breast cancer, type 4; breast cancer- 1; breast cancer-3; breast-ovarian cancer, Buridtt’s lymphoma; cervical carcinoma; colorectal adenoma; colorectal cancer, colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type 3; colorectal cancer, hereditary nonpolyposis, type 6; colorectal cancer, hereditary nonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrial carcinoma; esophageal cancer; gastric cancer
  • the disease is atopic dermatitis.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term “subject” includes mammals, preferably human beings at any age which suffer from the pathology.
  • the antibodies of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the antibody accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • Conventional approaches for drag delivery to the central nervous system include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB ; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (TCRLrantibody) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., dermatitis) or prolong the survival of the subject being treated.
  • TCRLrantibody active ingredients
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-1) ⁇
  • Dosage amount and interval may be adjusted individually to provide antibody levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit (diagnostic or therapeutic), which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S . Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the present invention further provides for the administration of the disclosed anti-hlL- 13R ⁇ 1 antibody molecules for purposes of gene therapy.
  • the cells of a subject would be transformed with nucleic acid encoding the antibody molecules of the invention.
  • Subjects comprising the nucleic acids will then produce the antibody molecules endogenously.
  • Alvarez, et al, Clinical Cancer Research 3:181-3087, 2000 introduced single-chain anti-ErbB2 antibodies to subjects using a gene therapy approach.
  • Nucleic acids encoding any polypeptide or antibody molecule of the invention may be introduced to a subject.
  • the antibody molecule is a human, single-chain antibody.
  • the nucleic acids may be introduced to the cells of a subject by any means known in the art.
  • the nucleic acids are introduced as part of a viral vector. Examples of particular viruses from which the vectors may be derived include lentiviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphavirus, influenza virus, and other recombinant viruses with desirable cellular tropism.
  • viral vectors commercially, including, but by no means limited to, AVIGEN, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, and lentiviral vectors), CLONTECH (retroviral and baculoviral vectors), Genovo, Inc.
  • adenoviral and AAV vectors GENVEC (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously, and thus are not infectious, in the target cell.
  • the replication defective virus may be a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsulating the genome to produce viral particles.
  • Defective viruses which entirely or almost entirely lack viral genes may be used as well. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted.
  • vectors comprising attenuated or defective DNA virus sequences include, but are not limited to, a defective herpes virus vector (Kanno et al, Cancer Gen. Ther. 6: 147-154, 1999; Kaplitt et al, J. Neurosci. Meth. 71:125-132, 1997 and Kaplitt et al, J. Neuro One. 19:137-147, 1994).
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Attenuated adenovirus vectors, such as the vector described by Strafford-Perricaudet et al, J. Clin. Invest. 90:626-630, 1992 are desirable in some instances. Various replication defective adenovirus and minimum adenovirus vectors have been described (see, e.g., W094/26914, W094/28938, W094/28152, W094/12649, WO95/02697 and W096/22378).
  • the replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al, Gene 101:195, 1991; EP 185573; Graham, EMBO J. 3:2917, 1984; Graham et al, J. Gen. Virol. 36:59, 1977).
  • the adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
  • the use of vectors derived from the AAV s for transferring genes in vitro and in vivo has been described
  • the gene can be introduced in a retroviral vector, e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289, and 5,124,263; Mann et al, Cell 33:153, 1983; Markowitz et al, J. Virol., 62:1120, 1988; EP 453242 and EP178220.
  • the retroviruses are integrating viruses which infect dividing cells.
  • Lentiviral vectors can be used as agents for the direct delivery and sustained expression of nucleic acids encoding an antibody molecule of the invention in several tissue types, including brain, retina, muscle, liver and blood.
  • the vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the antibody molecule.
  • Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for gene therapy.
  • An example is a tetracycline-inducible VS V-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 10.sup.6 IU/ml for at least 3 to 4 days; see Kafri et al, J. Virol. 73:576-584, 1999.
  • the vector produced by the inducible cell line can be concentrated as needed for efficiently transducing nondividing cells in vitro and in vivo.
  • Sindbis virus is a member of the alphavirus genus and has been studied extensively since its discovery in various parts of the world beginning in 1953.
  • Gene transduction based on alphavirus, particularly Sindbis virus has been well-studied in vitro (see Straus et al, Microbiol. Rev., 58:491-562, 1994; Bredenbeek et al, J. Virol., 67:6439-6446, 1993; Ijima et al, Int. J. Cancer 80:110-118, 1999 and Sawai et al, Biochim. Biophyr. Res. Comm. 248:315-323, 1998.
  • alphavirus vectors make them a desirable alternative to other virus-derived vector systems being developed, including rapid engineering of expression constructs, production of high-titered stocks of infectious particles, infection of nondividing cells, and high levels of expression (Strauss et al, 1994 supra).
  • Use of Sindbis virus for gene therapy has been described. (Wahlfors et al, Gene. Ther. 7:472-480, 2000 and Lundstrom, J. Recep. Sig. Transduct. Res. 19(1- 4):673-686, 1999.
  • a vector can be introduced to cells by lipofection or with other transfection facilitating agents (peptides, polymers, etc.).
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo and in vitro transfection of a gene encoding a marker (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987 and Wang et al, Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).
  • Useful lipid compounds and compositions for transfer of nucleic acids are described in WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127.
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al, J. Biol. Chem. 267:963-967, 1992; Williams et al, Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991).
  • Receptor- mediated DNA delivery approaches can also be used (Wu et al, J. Biol. Chem.
  • U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Vilquin et al, Gene Ther. 8:1097, 2001; Payen et al, Exp. Hematol. 29:295-300, 2001; Mir, Bioelectrochemistry 53:1-10, 2001; WO 99/01157, WO 99/01158 and WO 99/01175).
  • compositions suitable for such gene therapy approaches and comprising nucleic acids encoding an anti-hIL-13Ralphal antibody molecule of the present invention are included within the scope of the present invention.
  • the present invention provides a method for identifying, isolating, quantifying or antagonizing IL-13Ralphal in a sample of interest using an antibody molecule of the present invention.
  • the antibody molecules may be utilized as a research tool in immunochemical assays, such as western blots, ELISAs, radioimmunoassay, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art (see, e.g., Immunological Techniques Laboratory Manual, ed. Goers, J. 1993, Academic Press) or various purification protocols.
  • the antibody molecules may have a label to facilitate ready identification or measurement of the activities associated therewith.
  • detectable labels e.g., enzymes, dyes, or other suitable molecules which are either readily detectable or cause some activity/result that is readily detectable
  • polypeptides of the present invention may be co-administered with additional agents to increase its therapeutic effect.
  • agents that down- regulate (or inhibit) IL-4 include antibodies, small molecule inhibitors, polynucleotide agents directed against genes or RNA that encode IL-4.
  • the present invention contemplates co-administration (or coformulation) with Tofacitinib or Ruxolitinib, which blocks IL-4 signaling.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • the term "method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • any Sequence Identification Number can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.
  • mice Male and female IlI3r ⁇ 1 -/- mice (backcrossed >F9 to C57BL/6) were generated as previously described 19 .
  • C57BL/6 wild-type mice were obtained from Harlan Laboratories (Rehovot, Israel). All experiments were reviewed and approved by the Animal Care Committee of Tel Aviv University and were performed in accordance with its regulations and guidelines regarding the care and use of animals for experimental procedures. All of the experiments were conducted in the specific pathogen free facilities of the Tel Aviv University. In all experiments, age-, weight-, and sex-matched mice were used.
  • Oxazolone-induced dermatitis Mice (6- to 8-week-old, male and female) were sensitized on day 0 by the application of 15 ⁇ of 1% oxazolone (OX) (Sigma- Aldrich, Rehovot, Israel) in acetone on both flanks of both ears (60 ⁇ per mouse). Starting on day 7, mice were challenged in the same manner with 0.5% oxazolone, three times per week, for up to 10 challenges. Control mice were similarly treated with acetone. Twenty-four hours after the last challenge, the mice were bled for obtaining sera and euthanized. Ear thickness was measured by a digital caliper, and total ear tissues were taken for further analyses.
  • OX oxazolone
  • mice were treated intraperitoneally twice a week with rat IgGl anti-IL-4 (120 pg/mouse, clone 11B11, BioXCell, Lebanon, NH) or rat IgGl isotype control (clone TNP6A7, BioXCell, Lebanon, NH).
  • mice were treated intraperitoneally three times a week with anti-IL-13R ⁇ 1 (clone MSS, produced in house, 200 pg/mouse) or mouse IgGl isotype control (clone MOPC-21, BioXCell, Lebanon, NH).
  • NASH 10 epidermal thickness measurements of each slide were made and average thickness was calculated per image. The end value per mouse represents an average of 3 independent measurements.
  • Ear lysate preparation Ears were placed in ice-cold IP Lysis Buffer (750 pi, Pierce #87787, Thermo Fisher Scientific, Waltham, MA) supplemented with a protease inhibitors cocktail (Sigma, Rehovot, Israel). Thereafter, the ears were homogenized, centrifuged (14,000g, 10 minutes, 4°C), and supernatants were collected, aliquoted, and stored at -20°C.
  • Enzyme-linked immunosorbent assay Cytokines and IgE levels were measured by ELISA according to manufacturer's instructions.
  • mouse proteins were used: IL-4 (BioLegend, CA, USA); IL-6, CCL17, CCL24, IL-13Ra2 (R&D systems, Minneapolis, MN, USA); TNF-o, CCL2, CCL11, CXCL1 (Peprotech, Rehovot, IL); IgE (BD Biosciences, San Jose, CA, USA).
  • the following kits for human proteins were used: CCL26 (R&D systems, Minneapolis, MN, USA); IL-6 (Peprotech, Rehovot, IL).
  • Flow cytometry Single-cell suspensions of enzymatically-digested ears were stained using the following antibodies: CD45-APC, CDllb-PerCP/Cy5.5, CD1 lc-FITC, CD8a-PE (eBioscience, San Diego, CA, USA); SiglecF-PE (BD Biosciences, San Jose, CA, USA); Ly6C- PE/Cy7, F4/80-AF700, CD4-AF488, CD3e-PE/Cy7 (BioLegend, San Diego, CA, USA); Ly6G- APC/Cy7, B220-PerCP/Cy5.5 (Biogems, Westlake Village, CA, USA).
  • DAPI (Sigma-Aldrich, Rehovot, IL) was used as a marker for cell death.
  • Cell counts were measured using Flow-Count Fluorospheres (Beckman Coulter, Brea, CA) according to the manufacturer’s instructions. Events were acquired using Gallios Flow Cytometer, and data were analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA).
  • mice Female C57BL/6 or IlI3r ⁇ 1 -/- mice were immunized subcutaneously with human or mouse IL-13R ⁇ 1 ECD (ACROBiosystems, Newark, DE, USA) in complete Freund's adjuvant (Sigma-Aldrich, Rehovot, IL) (30 ⁇ g/mouse). Three additional boosts were made fortnightly with human or mouse IL- 13R ⁇ 1
  • mice were euthanized and antigen-specific B cells and plasma cells from spleens and bone marrows were sorted using FACSAria Fusion (BD Biosciences, San Jose, CA, USA) using a biotinylated antigen and the following antibodies: B220-AF488 (eBioscience, San Diego, CA, USA); CD19-PE (BioLegend, San Diego, CA, USA); CD138-PE (BD Biosciences, San Jose, CA, USA). cDNA libraries were made from the sorted cells and mouse V-genes were amplified using the appropriate primers.
  • VH and VL chains were assembled into single chain Fv (scFv) antibodies by cloning into a pETcon2-based yeast display vector. Clones were selected for binding biotinylated human or mouse IL-13R ⁇ 1 ECD using Yeast Display (method described in 22 ). Finally, scFv clones were converted to full-size antibody heavy and light chains and expressed as human or mouse IgGls in Expi293F mammalian expression systems (Thermo Fisher, Waltham, MA, USA). IgGs were purified using affinity chromatography on protein A or protein G columns (GE Healthcare, Chicago, IL, USA).
  • MC38 or A549 cells were seeded (4x1 ⁇ 4 cells/well in 96-well plates and l.SxlO 5 cells/well in 24-well plates, respectively) and left to adhere.
  • medium Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and lx Penicillin/Streptomycin
  • DMEM Dulbecco's Modified Eagle Medium
  • the cell culture media were collected, centrifuged (3000 rpm, 2 min, 4 °C) and supernatants were collected. Thereafter, the levels of CCL26 (for A549 cells), IL-13Ra2 (for MC38 cells) and IL-6 (for both cell lines) were measured by ELISA.
  • IL-13-induced lung inflammation MSS (anti-mouse IL-13R ⁇ 1) or MOPC-21 (mouse IgGl isotype control) were administered to C57BL/6 mice (intraperitoneal, 200 pg/mouse, days 0 and 2).
  • IL-13 (1 ⁇ g/mouse, Peprotech, Rehovot, IL) or saline were administered intratracheally 2 hours after the injection of MSS.
  • the mice were euthanized and bronchoalveolar lavage fluid (BALF) was obtained.
  • BALF CCL24 levels measured by ELISA
  • eosinophil counts measured by flow cytometry
  • IL-13R ⁇ 1 is differentially expressed by hematopoietic and non-hematopoietic cells in the skin
  • IL-13R ⁇ 1 IL-13R ⁇ 1 was highly expressed by monocytes and neutrophils and to lesser extent by eosinophils and lymphocytes.
  • IL-13R ⁇ 1 The expression of IL-13R ⁇ 1 was observed on the surface of non-hematopoietic cells as well, with nearly no expression by fibroblasts (defined as CD45 " /PDGFRa* cells), which may express the type 2 IL-4R(26).
  • OX- and DNFB-induced dermatitis are mediated via the type 2 IL-4 receptor:
  • OX-challenged WT mice displayed increased ear thickening (Fig. 1A and Figure 9).
  • ear thickness in OX-challenged H13ral '-/- mice was comparable to acetone-treated mice ( Figure 1A).
  • Histopathological assessment of the skin obtained from OX-challenged WT mice revealed hyperkeratosis, increased numbers of nuclei and epidermal thickening ( Figures 1B-C).
  • OX-challenged lll3ral '-/ mice displayed decreased numbers of nuclei and decreased epidermal thickening ( Figures 1B-C).
  • WT and IlI3r ⁇ 1 -/- mice were treated with 2,4-dinitrofluorobenzene (DNFB)(27).
  • DNFB- challenged IlI3r ⁇ 1 -/- mice displayed decreased dermatitis as observed by decreased ear and epidermal thickening in comparison with DNFB -treated WT mice ( Figures lOA-O).
  • TNF-a proinflammatory cytokines
  • OX- and DNFB-challenge were analyzed following OX- and DNFB-challenge, ( Figure ID and Figures lOA-O).
  • IL-4 is a key cytokine mediating the induction of IgE, which serves as an important biomarker and effector of Th2 immunity and atopy(29).
  • Assessment of skin IL-4 levels revealed that although IL-4 was decreased in OX-challenged IlI3r ⁇ 1 -/- mice ( Figure IE), an IL-13R ⁇ 1- independent pathway exists for induction of IL-4.
  • Serum IgE levels in WT mice were elevated following OX treatment ( Figure IF).
  • OX-challenged IlI3r ⁇ 1 -/- mice exhibited slightly higher serum IgE levels in comparison with OX-challenged WT mice ( Figure IF).
  • the inflammatory milieu in AD is characterized by the induction of various chemokines promoting cellular migration(iO). Therefore, the present inventors set about to determine whether the type 2 IL-4 receptor regulates chemokine production and subsequent cellular influx into the skin.
  • OX-challenged WT mice displayed increased expression of CXCL1, CCL11, and CCL24 ( Figures 1G-I). Induction of CXCL1 and CCL11 in response to OX challenge was dependent on the type 2 IL-4 receptor ( Figures 1G-H), whereas OX-induced CCL24 expression was independent of the type 2 IL-4 receptor (Figure II). CCL17 expression was modestly increased by OX treatment, and this increase was also dependent on the type 2 IL-4R ( Figure 1J).
  • OX-induced dermatitis is mediated by IL-13 and IL-4 via the type 2 IL-4R
  • the type 2 IL-4 receptor mediates signals from both IL-4 and IL-13(5).
  • the present inventors sought to determine the cellular source for IL-4 and IL-13 in OX-challenged mice.
  • IL-4 and IL-13 reporter mice (4Get and Ill3 Smart , respectively) were treated with OX.
  • cells that have the capacity to secrete IL-4 are marked by eGFP(31).
  • Cells that express endogenous IL-13 are marked by the expression of the human CD4 surface marker (32).
  • IL-13 expression was not detected in skin homogenates by ELISA, -25% of all skin CD4 + T cells stained positive with anti-human CD4 ( Figures 11 A-B).
  • mice still displayed a thicker epidermis than OX-challenged IlI3r ⁇ 1 -/- mice, which were treated with either isotype control or anti-IL-4 antibody (Figure 2D).
  • TNF-a levels (as well as IL- ⁇ , data not shown) were decreased in OX-challenged H13ral ' A mice regardless of anti-IL-4 antibody treatment, and remained upregulated in OX-challenged WT mice despite anti-IL-4 treatment (Fig. 2E).
  • pro-inflammatory cytokine production in OX-induced dermatitis is exclusively mediated by IL-13 signaling via the type 2 IL-4R.
  • Immune cell infiltration in OX-induced dermatitis is synergistically mediated by the type 1 and type 2 IL-4Rs
  • Immunophenotyping of OX-challenged skin following anti-IL-4 antibody treatment revealed differential contribution of IL-4 and IL-13 via the type 1 and type 2 receptors to infiltrating cells into the skin as observed by assessment of total CD45 + cells (Figure 2L).
  • Infiltration of macrophages and T cells which were decreased in OX-challenged, isotype-treated IlI3r ⁇ 1 -/- mice, were not further reduced following anti-IL-4 treatment ( Figures 2M-N).
  • Neutrophil and monocyte accumulation which were decreased in isotype control-treated lll3ra-l ' /- mice following OX challenge, were further reduced following anti-IL-4 treatment ( Figures 20- P).
  • IL-13 is required for OX-induced dermatitis, TNF-a, CCL11, CCL17 ⁇ and neutrophilic infiltration into the skin
  • TNF-a levels were reduced in OX-challenged IlI3r ⁇ 1 -/- mice and were also reduced following neutralization of IL-13, the present inventors sought to define whether decreased neutrophilia was due to decreased TNF-a expression. To this end, TNF-a was neutralized in acetone- and OX-treated WT mice. Neutralization of TNF-a had no effect on ear thickness (Figure 3H), CXCL1 expression ( Figure 31), or infiltration of neutrophils into the skin (Figure 3J).
  • Oxazolone-induced dermatitis is mediated by the type 2 IL-4 receptor expressed on non- hematopoietic cells
  • IL-13R ⁇ 1 is expressed by hematopoietic and non-hematopoietic cells ( Figure 8).
  • the present inventors aimed to determine the relative contribution of these lineages to the type 2 IL- 4R-dependent response.
  • bone marrow chimeric mice were generated by transferring bone marrows that were obtained from either C57BL6 CD45.1 mice (herein: WT), or C57BL6 IlI3r ⁇ 1 -/- mice (expressing the CD45.2 variant), into WT or IlI3r ⁇ 1 -/- mice. Engraftment efficiency was determined in the peripheral blood by flow cytometry using CD45.1 and CD45.2 antibodies. All mice exhibited a nearly-complete leukocytes engraftment ( Figures 12A-D).
  • CXCL1 and CCL11 were dependent on the type 2 IL-4R, which is expressed by non-hematopoietic cells, as their expression was decreased in OX-challenged, IlI3r ⁇ 1 -/- recipient mice, regardless of genotype of the bone marrow donor ( Figures 4G-H).
  • mice were immunized and boosted with the extracellular domain (BCD) of mus musculus IL-13R ⁇ 1 in the presence of adjuvant. Thereafter, mice were bled and their antisera were tested for the ability to bind plate-immobilized IL-13R ⁇ 1 BCD (See method description online and, Figures 13A-B online).
  • BCD extracellular domain
  • MSS MSS Information regarding MSS is provided in Table 3, herein below.
  • MS 8 was capable of binding spleen monocytes (CD 11 b + Ly6G med Ly6C high ) from WT mice. Yet, it did not show any binding to spleen monocytes obtained from IlI3r ⁇ 1 -/- mice ( Figure 5E). To determine whether MSS can be used pharmacologically in vivo, its capacity to neutralize
  • IL-13 was tested in an acute model of IL-13 -induced eosinophilic lung inflammation where mice are intratracheally challenged with IL- 13(34). Consistent with its capability to neutralize IL-13 in vitro, IL- 13-challenged mice, which were intraperitoneally treated with MSS, exhibited reduced levels of CCL24 in their bronchoalveolar lavage fluid (HALF) compared with isotype control- treated mice ( Figure 5F). Decreased CCL24 expression was accompanied with a marked reduction in eosinophil HALF percentage and total cell counts (Figure 5G-H). These results demonstrate that MS 8 is a pharmacologically functional mouse IL-13R ⁇ 1 antagonist.
  • HALF bronchoalveolar lavage fluid
  • Antibody-mediated neutralization of the type 2 IL-4R protects mice from OX-induced dermatitis
  • mice were challenged with OX and treated with either isotype control or MS 8.
  • OX-challenged mice that were treated with MSS displayed a marked decrease in ear thickness ( Figure 6A). Histopathological assessment of the skin obtained from MS 8 -treated mice revealed decreased numbers of nuclei and decreased epidermal thickening ( Figures 6B-C).
  • mice treated with MSS displayed significantly decreased expression of TNF- ⁇ (Figure 6D).
  • Antibody-mediated neutralization of the type 2 IL-4R decreases OX-induced chemokine production and subsequent cellular migration
  • mice treated with MSS displayed decreased expression of CXCL1 and CCL11 in response to OX challenge ( Figures 6G-H). Although CCL24 expression was slightly reduced by MSS treatment, this did not reach statistical significance ( Figure 61). Subsequently, cellular infiltration of leukocytes following MSS treatment was also assessed. OX-challenged, MS8- treated mice displayed decreased total leukocyte infiltration ( Figure 6J). Decreased cellular infiltration in response to MSS treatment was evident in T cells, macrophages and neutrophils ( Figure 6J).
  • mice were immunized and boosted with the ECD of human IL-13R ⁇ 1 in the presence of adjuvant. Thereafter, mice were bled and their antisera were examined for their ability to bind plate-immobilized hIL-13R ⁇ 1 BCD ( Figures 15A-B). Thereafter, the ability of the antisera to neutralize hIL- 13R ⁇ 1 -dependent receptor activation was assessed using IL- 13-stimulated A549 cells ( Figure 15C). Serum from mouse #2 was capable of inhibiting IL-13-induced responses in vitro, suggesting the presence of neutralizing antibodies.
  • 2HA6 dose-dependently inhibited IL-13-induced CCL26 secretion ( Figure 7B) but not IL-4- or TNF-a-mediated secretion of CCL26 or IL-6, respectively.
  • 2HA6 was capable of specifically suppressing IL-13 through the human type 2 IL-4R.
  • Neutralizing antibodies towards receptors can block their activity by direct competition with their ligand on the ligand binding site, or by other means, such as preventing receptor dimerization.
  • a competitive ELISA testing the ability of IL-13 and 2HA6 to bind the ECD of hIL-13R ⁇ 1 was conducted ( Figure 7E). To this end, plates were coated with the ECD of hIL-13R ⁇ 1 ECD and IL-13 was added with no antibody treatment, with an isotype control antibody or with 2HA6.

Abstract

Isolated polypeptides are disclosed comprising a binding domain which binds specifically to human Interleukin 13 receptor, alpha 1 (IL-13Ralphal), wherein the polypeptide downregulates an activity of Interleukin 13 (IL- 13). Uses thereof are also disclosed.

Description

TARGETING IL-13 RECEPTOR ALPHA 1 IN ATOPIC DERMATITIS
AND ALLERGIC DISEASES
RELATED APPLICATIONS
This application claims the benefit of priority of US Provisional Patent Application No. 62/945,163 filed 8 December, 2019, the contents of which are incorporated herein by reference in their entirety.
SEQUENCE LISTING STATEMENT
The ASCII file, entitled 85343 SequenceListing.txt, created on 8 December 2020, comprising 37,973 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to agents which bind to IL- 13Rα1 receptor for the treatment of allergic diseases and, more particularly, but not exclusively, for the treatment of atopic dermatitis.
Atopic dermatitis (AD) is a common chronic inflammatory skin disease affecting approximately 230 million people worldwide. AD patients are classified into two endotypes, namely extrinsic and intrinsic AD. Extrinsic AD manifests with high serum IgE levels, whereas intrinsic AD has no IgE upregulation1. It is now acknowledged that AD is primarily a T cell-driven disease, as evident by clinically effective T cell-targeting drugs such as cyclosporine2, 3. Whilst T helper (Th)l, Thl7, and Th22 polarizations differ between AD endotypes, a strong Th2 axis is associated with both extrinsic and intrinsic AD4. In particular, IL-4 and IL-13 are produced at elevated levels in the lesional skin and are central regulators of many of the hallmark features of AD, including epidermal hyperplasia, skin barrier dysfunction, and production of eosinophil and T cell chemokines5, 6. The importance of IL-4, IL-13 and their associated receptors in AD is best exemplified by the ongoing pursuit to pharmacologically target these cytokines and/or their signaling components in AD5,7.
Background art includes Karo-Atar D, BioDrugs 2018; and Gandhi NA, et al. Expert Rev Clin Immunol 2017; 13:425-37.
Additional background art includes US Patent Application No. 20170340737. SUMMARY OF THE INVENTION
According to an aspect of the present invention there is provided an isolated polypeptide comprising a binding domain which binds specifically to human Interleukin 13 receptor, alpha 1 (IL-13Rα1), wherein the polypeptide downregulates an activity of Interleukin 13 (IL-13), the polypeptide comprising each of the following amino acid sequences:
(i) SEQ ID NO: 28 or SEQ ID NO: 34;
(ii) SEQ ID NO: 29 or SEQ ID NO: 35; and
(iii) SEQ ID NO: 30 or SEQ ID NO: 36.
According to an aspect of the present invention there is provided a pharmaceutical composition comprising the isolated polypeptide described herein as the active agent and a pharmaceutically acceptable carrier.
According to an aspect of the present invention there is provided a method of treating a disease mediated by IL-13 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated polypeptide described herein, thereby treating the disease.
According to embodiments of the present invention, the activity comprises induction of CCL26 secretion.
According to embodiments of the present invention, the polypeptide is an antibody. According to embodiments of the present invention, the polypeptide is an antibody having an antigen recognition domain which comprises the CDR sequences of an antibody having a heavy chain as set forth in SEQ ID NO: 55, wherein the CDR sequences are in the same orientation as the antibody having the heavy chain as set forth in SEQ ID NO: 55.
According to embodiments of the present invention, the isolate polypeptide further comprises a light chain.
According to embodiments of the present invention, the CDR1 of a heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 28, CDR2 of the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 29, and CDR3 of the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:
30.
According to embodiments of the present invention, the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 55.
According to embodiments of the present invention, the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 56. According to embodiments of the present invention, the polypeptide has an affinity between O.lnM -lOnM for the human IL-13Rα1 as measured by ELISA.
According to embodiments of the present invention, the antibody is selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE antibody.
According to embodiments of the present invention, the antibody is a bi-specific antibody. According to embodiments of the present invention, the first target of the bi-specific antibody is human IL-13Rα1, and the second target of the bi-specific antibody is human IL-4.
According to embodiments of the present invention, the isolated polypeptide described herein is for use in treating a disease mediated by IL-13.
According to embodiments of the present invention, the disease mediated by IL-13 is selected from the group consisting of atopic dermatitis, asthma, eosinophilia, urticaria, and allergic rhinitis fibrotic diseases, COPD and cancer.
According to embodiments of the present invention, the disease is atopic dermatitis. According to embodiments of the present invention, the method further comprises administering to the subject an agent which downregulates the amount and / or activity of IL-4.
According to embodiments of the present invention, the agent is an antibody directed against the IL-4.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIGs. 1 A-K. The type 2 IL-4 receptor is required for oxazolone-induced atopic dermatitis. Wild type (WT) and IlI3rα1-/- mice were challenged with acetone or oxazolone for 3 weeks. Thereafter, ear thickness was measured (A). Quantitation of epidermal thickness (B) and representative photomicrographs of hematoxylin and eosin stained slides are shown (C). Skin protein levels of TNF-a (D), IL-4 (E), CXCL1 (G), CCL11 (H), CCL24 (I), CCL17 (J), as well as serum IgE (F) are shown. In addition, total numbers of skin T cells, neutrophils (Neuts), eosinophils (Eos) and macrophages (Macs) following acetone and oxazolone-challenge is shown (K). Data are shown as mean ± SEM; ns- non-significant, *p < 0.05, **p < 0.01, ***p <0.001. Each dot represents a single mouse from n=3 independent experiments.
FIGs.2A-Q. Differential roles for IL-4 and IL-13 via the type 2 IL-4R in oxazolone-driven atopic dermatitis. Wild type (WT) and IlI3rα1-/- mice were challenged with oxazolone and treated with anti-IL-4 or isotype control antibody (Isotype). Schematic representation of the differential signaling components which this experiment enables to dissect (A). In (C) ear thickness was measured and representative photomicrographs (B) and quantitation of epidermal thickness (D) of hematoxylin and eosin stained slides are shown. Skin protein levels of TNF-a (E), IL-4 (F), CXCL1 (H), CCL11 (I), CCL24 (J), CCL17 (K) as well as serum IgE (G) are shown. In addition, total numbers of skin infiltrating leukocytes (CD45+ cells) (L), macrophages (M), T cells (N), neutrophils (O), monocytes (P) and eosinophils (Q) were determined. Data are shown as mean ± SEM; ns- non-significant, *p < 0.05, **p < 0.01, ***p <0.001. Each dot represents a single mouse from n=3 independent experiments.
FIGs. 3A-J. Neutralization of IL-13 and TNF-a in oxazolone-treated wild type mice demonstrates a key role for IL-13 in oxazolone-induced dermatitis, CXCL1 expression and infiltration of neutrophils. Wild type (WT) mice were challenged with oxazolone and treated with isotype control or with neutralizing antibodies targeting IL-13 (A-G) or TNF-a (H-J). Subsequently, ear thickness (B, H) and epidermal thickening (C) were measured. Skin protein levels of soluble IL-13Ra2 (A), TNF-a (D), CCL11 (E), CCL17 (E), CCL24 (E) and CXCL1 (I) are shown. In addition, total numbers of skin infiltrating neutrophils (F, J) and eosinophils (G) were determined. Data are shown as mean ± SEM; ns- non-significant, *p < 0.05, **p < 0.01, ***p <0.001. Each dot represents a single mouse from n=3 independent experiments.
FIGs. 4A-N. Dissecting the contribution of the type 2 IL-4R expressed by hematopoietic and non-hematopoietic cells to oxazolone-induced allergic skin disease. Wild type (WT) and IlI3rα1-/- mice were irradiated. Thereafter, recipient mice received bone marrow cells from WT or IlI3rα1-/- mice. Ten weeks later, mice were challenged with oxazolone and ear thickness was measured (A). Representative photomicrographs (C) and quantitation of epidermal thickness (B) of hematoxylin and eosin stained slides are shown. The protein levels of skin TNF-a (D), IL-4 (E), serum IgE (F), CCL11 (G), CXCL1 (H), CCL24 (I). In addition, total numbers of skin infiltrating leukocytes (J), macrophages (K) neutrophils (L), monocytes (M) and eosinophils (N) were determined. Data are shown as mean ± SEM; ns- non-significant, *p < 0.05, **p < 0.01, ***p <0.001. Each dot represents a single mouse from n=3 independent experiments.
FIGs. 5A-H. Assessment of an anti-mouse IL-13Rα1 neutralizing antibody. Binding properties of MSS mAb to mouse (m) or human (h) IL-13Rα1 ECD, or streptavidin (A). ELISA- estimated affinity (Kd) to mIL-13Rα1 was calculated (A). MC38 cells were treated with lOng/ml of IL-13 (B), IL-4 (C), or TNF-a (D) in the presence of anti-IL-13Rα1 (MSS) or isotype control mAb at decreasing 2-fold concentrations. Thereafter, the levels of soluble (s) IL-13Ro2 (B-C) and IL-6 (D) in the culture supernatants were measured (A-D, n=3). Specificity of MSS binding to IL- 13Rα1 was determined by binding of wild type (WT) and IlI3rα1-/- splenic lymphocytes (Lymph), monocytes (Monos), neutrophils (Neuts), eosinophils (Eos), and dendritic cells from WT or IlI3rα1-/- mice (E) (n=3 mice). IL-13 was administered intratracheally to WT mice and MSS or isotype control were injected intraperitoneally. CCL24 levels (F) as well as eosinophil percentage
(G) and total cell count (H) were determined (n=8 mice). Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ***p <0.001.
FIGs. 6A-J. Pharmacological neutralization of the type 2 IL-4R alleviates oxazolone- induced atopic dermatitis. Wild type mice were challenged with oxazolone for 3 weeks and treated with isotype control or anti-IL-13Rα1 (MSS). Thereafter, ear thickness was measured (A). Representative photomicrographs (C) and quantitation of epidermal thickness (B) of hematoxylin and eosin stain slides are shown. Skin protein levels of TNF-a (D), IL-4 (E), CXCL1 (G), CCL11
(H), CCL24 (I) as well as serum IgE (F) are shown. In addition, total numbers of skin T cells, neutrophils (Neuts), eosinophils (Eos) and macrophages (Macs) following isotype control and MSS treatment were determined (J). Data are shown as mean ± SEM; ns- non-significant, *p < 0.05, **p < 0.01, ***p <0.001. Each dot represents a single mouse from n=3 independent experiments.
FIGs.7A-E. Generation and assessment of an anti-human IL-13Rα1 neutralizing antibody. Binding properties of 2HA6 mAb to mouse (m) or human (h) IL-13Rα1 ECD, or streptavidin (A). ELISA-estimated affinity (Kd) to hIL-13Rα1 was calculated (A). Data are shown as mean ± SD. A549 cells were treated with lOng/ml of IL-13 (B), IL-4 (C), or TNF-a (D) in the presence of anti- IL-13Rα1 (2HA6) or isotype control mAb. Thereafter, the levels of CCL26 (B-C) and IL-6 (D) in the culture supernatants were measured. The ability of anti-IL-13Rα1 to compete with hIL-13 on the binding site of IL-13Rα1 was measured by a competitive ELISA (E). Data are shown as mean ± SEM; *p < 0.05, **p < 0.01 from n=3. FIG. 8. Expression of Il-13Rα1 in the skin. Wild type mice were treated with acetone (Control) or challenged with oxazolone. Thereafter, the ears were enzymatically digested and the expression of IL-13Rα1 was determined by flow cytometry. Data are shown as mean ± SEM; * < p 0.05, **p < 0.01.
FIGs. 9A-B. IL-13Rα1 regulates oxazolone-induced dermatitis. Wild type (WT) C57BL/6 and III 3 ra1-/- mice were challenged with oxazolone and euthanized on Day 25. Representative ear photographs of mice from each group are shown.
FIGs. lOA-O. The type 2 IL-4 receptor is required for DFNB-induced atopic dermatitis. Wild type (WT) and IlI3rα1-/- mice were challenged with acetone or DNFB for 3 weeks. Thereafter, ear thickness was measured (A). Quantitation of epidermal thickness (B) and representative photomicrographs of hematoxylin and eosin stained slides of acetone- and oxazolone-challenged skin are shown (C). The levels of skin protein levels of TNF-a (D), IL-4 (E), CXCL1 (G), CCL11 (H), CCL24 (I), CCL17 (J), as well as serum IgE (F) are shown. In addition, total numbers of skin leukocytes (J), as well as macrophages (K), T cells (L), neutrophils (M), monocytes (N), and eosinophils (O) is shown (K). Data are shown as mean ± SEM; ns- nonsignificant, *p < 0.05, **p < 0.01, ***p <0.001 from at least n=3.
FIGs. 11A-B. Assessing the cellular source for IL-4 and IL-13 in oxazolone-induced dermatitis. IL-4 reporter (4Get) and IL-13 reporter ( IU3Smart ) mice were treated with oxazolone to induce experimental atopic dermatitis. Twenty for hours after the last oxazolone challenge, the ears were obtained, enzymatically digested and single cell suspensions stained to identify CD4+, CD8+, NKT and γδ T cells. The percentage of cells (out of the total gated population) that are positive for GFP or stained positive for human CD4 (hCD4), which represents IL-13 expressing cells in shown (A-B). Each dot represents a different mouse.
FIGs. 12A-D. Engraftment efficiency of following adoptive transfer of wild type and Ι113Γα1-/- bone marrow cells. Wild type (WT) C56BL/6 mice, WT B6 CD45.1 mice, and H13ral' /- mice were irradiated with 9 Gy total body irradiation (TBI). Twenty-four hours post-TBI, recipients received 5xl06 bone-marrow cells obtained from donor CD45.1 or IlI3rα1-/- mice. Two weeks later, bloods were drawn from representative mice (one from each group, A-D) and blood cells were stained with CD45. l-APC/eF780 and CD45.2-PE antibodies by flow cytometry. (A) Recipient - C56BL/6; Donor - B6 CD45.1. (B) Recipient - B6 CD45.1; Donor - II3ral'-/-. (C) Recipient - lll3ra-/- Donor - B6 CD45.1. (D) Recipient - lll3ral','\ Donor - IlI3rα1-/-. Shown % are gated on live cells.
FIGs. 13A-C. IL-13Rα1 antisera inhibit IL-13-induced IL-13Ra2 secretion by MC38 cells Wild type mice were immunized with the recombinant extracellular domain of mouse mouse (m) IL-13Rα1 and boosted fortnightly three times. Prior to immunization and following the 3rd boost, blood was drawn and sera were examined for binding mIL-13Rα1 (A), or streptavidin (control antigen) (B). Subsequently, MC38 cells were treated with a 1:100 dilution in saline of serum from pre-immunized mice (Pre Immunization) or antisera (Post Immunization) in presence of lOOng/ml recombinant mouse IL-13 (C). The ability of antisera to block IL-13-induced soluble (s) IL-13Ra2 levels in the cell supernatants were determined. Data are shown as mean ± SD; **p < 0.01.
FIGs. 14A-B. Schematic representation of the Yeast Display system used in this study to isolate IL-13Rα1-binding antibodies. (A) Saccharomyces cerevisiae cells displaying a single chain variable fragment (scFv) library originated from mouse V-genes, where one cell expresses one distinct scFv clone. During secretion, the fusion Alpha-agglutinin (Aga)2-scFv protein forms disulfide bonds with the membrane protein Agal, resulting in scFv display on the cell surface, thus allowing it to bind soluble antigens (e.g. biotinylated IL-13Rα1). Antigen binding ability is evaluated by detection of the scFv-bound biotinylated antigen by Allophycocyanin (APC)- conjugated streptavidin (SA). Furthermore, a c-myc tag located at the C-terminus of the Aga2- scFv fusion protein, allows to assess the efficiency of scFV display by detecting a complex comprised of mouse anti-myc and Alexa flour 488 (AF488)-conjugated anti-mouse IgG antibodies. The yeast library was analyzed by flow cytometry (Galios, Beckman Coulter) (B) and APC7AF488+ clones were enriched by repetitive sorting cycles (B). Thereafter, the output scFvs were sequenced. B , Representative enrichment procedure of clones binding mouse (m) IL- 13Ra 1.
FIGs. 15A-C. IL-13Rα1-specific antisera inhibit IL-13-induced CCL26 secretion by A549 cells. Wild type C57BL/6 mice were immunized with the recombinant extracellular domain of human (h) IL-13Rα1 and boosted fortnightly three times. Prior to immunization and nine days following the 3rd boost, blood was drawn and sera were examined for binding hIL-13Rα1 (A), or streptavidin (control antigen) (B). Subsequently, A549 cells were treated with a 1:100 dilution in saline of serum from pre-immunized mice (Pre Immunization) or antisera (Post Immunization) in presence of 100ng/ml recombinant mouse IL-13 (C). The ability of antisera to block IL-13 -induced CCL26 secretion levels in the culture supernatants were determined. Data are shown as mean ± SD; **p < 0.01. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to agents which bind to IL- 13Rα1 receptor for the treatment of allergic diseases and, more particularly, but not exclusively, for the treatment of atopic dermatitis. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
IL-13 and IL-4 are potent mediators of type 2-associated inflammation such as those found in atopic dermatitis (AD). IL-4 shares overlapping biological functions with IL-13, a finding that is mainly explained by their ability to signal via the type 2 IL-4 receptor (R), which is comprised of IL-4Ra in association with IL-13Rα1.
The present inventors sought to define the role of the type 2 IL-4R in AD. Two distinct models of experimental AD in IlI3rα1-/- mice, which lack the type 2 IL-4R were set up. The results revealed that dermatitis including ear and epidermal thickening were dependent on type 2 IL-4R signaling. Furthermore, expression of TNF-a, was dependent on the type 2 IL-4R, whereas, induction of IL-4, IgE, CCL24, and skin eosinophilia were dependent on the type 1 IL-4R. Neutralization of IL-4, IL-13 and TNF-a as well studies in bone marrow-chimeric mice revealed that dermatitis, TNF-a, CXCL1, and CCL11 expression were exclusively mediated by IL-13 signaling via the type 2 IL-4R expressed by non-hematopoietic cells. Conversely, induction of IL- 4, CCL24, and eosinophilia were dependent on IL-4 signaling via the type 1 IL-4R expressed by hematopoietic cells.
Whilst reducing the present invention to practice, the present inventors pharmacologically targeted IL-13Rα1 and established proof-of-concept for therapeutic targeting of this pathway in AD. As illustrated in Figures 6A-J, an antibody targeting mouse IL-13Rα1 of the type 2 IL-4R alleviated oxazolone-induced atopic dermatitis.
The present inventors also generated an antibody which recognizes human IL-13Rα1 (Figures 7A-E) and propose that this antibody can be used for the treatment of allergic diseases including AD. Furthermore, the antibody can serve as a basis for the generation of additional antibodies for the treatment of such diseases.
Thus, according to a first aspect of the present invention, there is provided an isolated polypeptide comprising a binding domain which binds specifically to human Interleukin 13 receptor, alpha 1 (IL-13Rα1), wherein the polypeptide downregulates an activity of IL-13, the polypeptide comprising each of the following amino acid sequences:
(i) SEQ ID NO: 28 or SEQ ID NO: 34;
(ii) SEQ ID NO: 29 or SEQ ID NO: 35; and
(iii) SEQ ID NO: 30 or SEQ ID NO: 36. The term "polypeptide" as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=0, 0=C-NH, CH2-0, CH2-CH2, S=C- NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drag Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
Peptide bonds (-CO-NH-) within the polypeptide may be substituted, for example, by N- methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-0-0-C(R)-N-), ketomethylen bonds (- CO-CH2-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-
CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic nonnatural acid such as Phenylglycine, Tic, naphtylalanine (Nal), phenylisoserine, threoninol, ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
In addition to the above, the polypeptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc.).
As used herein in the specification and in the claims section below the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids. Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used in the polypeptides of the present invention.
Table 1
Figure imgf000011_0001
Table 2
Figure imgf000011_0002
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
The term “IL-13Rα1” (also known as CD213A1) refers to a subunit of the IL-13 receptor which dimerizes with the IL-4Ra subunit. Together with IL-4Ra subunit, the IL-13Rα1 subunit mediates the biological effects of IL-13. An exemplary mRNA which encodes human is set forth in RefSeq No. NM_001560.3. An exemplary amino acid sequence of human IL-13Rα1 is set forth in SEQ ID NO: 65 (UniProtKB - P78552).
The polypeptides described herein bind specifically to human Interleukin 13 receptor, alpha 1 (IL-13Rα1). According to a specific embodiment, the polypeptides bind with at least 2 fold, 5 fold, 10 fold or even higher affinity to human IL-13Rα1 as compared to mouse IL-13Rα1, as measured by ELISA. According to a specific embodiment, the polypeptides bind with at least 2 fold, 5 fold, 10 fold or even higher affinity to human type 2 IL-4 receptor as compared to human type 1 IL-4 receptor, as measured by ELISA.
For example, the polypeptides described herein may have an apparent affinity between O.lnM -lOnM for the human IL-13Rα1 as measured by ELISA. In some embodiments the affinity is equivalent to the Kd. In another embodiment, the polypeptides may have an affinity (or Kd) between O.SnM-SnM for the human IL-13Rα1 as measured by ELISA. Additional contemplated affinities (Kds) are between 500 nM- 0.5 nM, 100 nM-1 nM, 50 nM-1 nM, 20 nM-1 nM, 10 nM-1 nM, as measured by ELISA.
As used herein the term “KD” refers to the equilibrium dissociation constant between the polypeptide and its respective target.
Furthermore, the polypeptides of the present invention are capable of inhibiting one or more functional activities of hIL-13Rα1. For example, in one embodiment, the polypeptides inhibit (or downregulate) CCL26 secretion from epithelial cells, as measured by ELISA. In another embodiment, the polypeptides may inhibit IL-13-induced eotaxin release in fibroblast cells. In yet another embodiment, the polypeptides may inhibit IL-13-induced STAT6 phosphorylation in fibroblast cells. In yet another embodiment, the polypeptides may inhibit IL-4-induced eotaxin release in fibroblast cells. In yet another embodiment, the polypeptides may inhibit IL-4-induced STAT6 phosphorylation in fibroblast cells. In specific embodiments, the polypeptides inhibit all of the above functional activities of hIL-13Rα1.
The polypeptides of the instant invention may or may not inhibit the binding of IL-13 to isolated IL-13Rα1 (i.e., IL-13Rα1 that is not part of a dimeric receptor with IL4Ra). In one embodiment, the polypeptides of the instant invention prevent dimerization of IL-13Rα1 with IL- 4Ra. In another embodiment, the polypeptides described herein compete for the binding of IL-13 with IL-13Rα1.
The polypeptide may be selected from the group consisting of TCR, CAR-T and an antibody.
According to a specific embodiment, the polypeptide is an antibody. Although the reference to antibodies is in more details as compared to other polypeptides having affinity binding entities, the description of this embodiment should not be construed as limiting and the present invention is equally related to binding entities as described herein especially in the sense of cell therapy as further described hereinbelow.
The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VL that are capable of binding to an epitope of an antigen in an MHC restricted manner. As a more general statement the term “antibody” aims to encompass any affinity binding entity which binds a cell surface presented molecule with an MHC restricted specificity. Thus, CDRs of the antibodies of some embodiments of the present invention may be implanted in artificial molecules such as T cell receptors or CARs as further described hereinbelow. Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.
As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3). Examples of heavy chain CDR sequences are provided by SEQ ID NOs: 28, 29, 30, 34, 35 and 36. Examples of light chain CDR sequences are provided by SEQ ID NOs: 40, 41, 42, 46, 47 or 48.
The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by IMGT (see Giudicelli V, Chaume D, Bodmer J, Miiller W, Busin C, Marsh S, et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206-11), Kabat et al. (See, e.g., Rabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Rabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www(dot)bioinf-org(dot)uk/abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996), the "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156- 1166, 2008) and IMGT [Lefranc MP, et al. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 27: 55-77].
As used herein, the “variable regions” and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches. According to a specific embodiment, the CDRs are determined according to Rabat et al. (supra).
In one embodiment, the antibodies provided herein have an antigen recognition domain which comprises the CDR sequences of an antibody having a heavy chain as set forth in SEQ ID NO: 55, wherein the CDR sequences are in the same orientation as the antibody having the heavy chain as set forth in SEQ ID NO: 55. Thus, for example, an antibody comprising SEQ ID NO: 28 (as CDR1 of the heavy chain), SEQ ID NO: 29 (as CDR2 of the heavy chain) and SEQ ID NO: 30 (as CDR3) of the heavy chain is contemplated, wherein the CDRs are determined according to the IGMT method. An antibody comprising SEQ ID NO: 34 (as CDR1 of the heavy chain), SEQ ID NO: 35 (as CDR2 of the heavy chain) and SEQ ID NO: 36 (as CDR3) of the heavy chain is contemplated, wherein the CDRs are determined according to the Rabat method.
In one embodiment, the antibodies provided herein have an antigen recognition domain which comprises the CDR sequences of an antibody having a light chain as set forth in SEQ ID NO: 56, wherein the CDR sequences are in the same orientation as the antibody having the heavy chain as set forth in SEQ ID NO: 56. Thus, for example, an antibody comprising SEQ ID NO: 40 (as CDR1 of the light chain), SEQ ID NO: 41 (as CDR2 of the light chain) and SEQ ID NO: 42 (as CDR3) of the light chain is contemplated, wherein the CDRs are determined according to the IGMT method. An antibody comprising SEQ ID NO: 46 (as CDR1 of the light chain), SEQ ID NO: 47 (as CDR2 of the light chain) and SEQ ID NO: 48 (as CDR3) of the light chain is contemplated, wherein the CDRs are determined according to the Rabat method.
According to a specific embodiment, the light chain comprises the CDR sequences SEQ ID NO: 40, SEQ ID NO: 47 and SEQ ID NO: 42.
The variable region of the heavy chain of the antibodies of this aspect of the present invention may have an amino acid sequence at least 90 % identical, 91 % identical, 92 % identical, 93 % identical, 94 % identical, 95 % identical, 96 % identical, 97 % identical, 98 % identical, 99 % identical to SEQ ID NO: 55, wherein the CDR sequences remain as defined herein above.
Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:
(i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;
(ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule;
(iii) disulfide-stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond;
(iv) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CHI domains thereof;
(v) Fab’, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);
(vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and
(vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementaritydetermining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856- 859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845- 51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
In an embodiment in which the antibody is a full length antibody, the heavy and light chains of an antibody of the invention may be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, or two, complete light chains) or may include an antigen-binding portion (a Fab, F(ab').sub.2, Fv or a single chain Fv fragment ("scFv")). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2,
IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. In some embodiments, the immunoglobulin isotype is selected from IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl (e.g., human IgGl) or IgG4 (e.g., human IgG4). The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit. According to a particular embodiment the antibody is an IgGl antibody having a heavy chain constant domain as set forth in SEQ ID NO: 62.
According to a particular embodiment the antibody is an IgGl antibody having a light chain constant domain as set forth in SEQ ID NO: 64.
Bispecific configurations of antibodies are also contemplated herein. A bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen. According to a specific embodiment the BsMAb is engineered to simultaneously bind to IL-13Rα1 and IL-4.
As used herein the phrase “chimeric antigen receptor (CAR)” refers to a recombinant or synthetic molecule which combines antibody-based specificity for a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits cellular immune activity to the specific antigen.
As used herein the phrase “T Cell Receptor" or “TCR” refers to soluble and non-soluble forms of recombinant T cell receptor.
It will be appreciated that the polypeptides (e.g. antibodies) of the present invention may be conjugated to a functional moiety (also referred to as an “immunoconjugate”) such as a detectable or a therapeutic moiety. The immunoconjugate molecule can be an isolated molecule such as a soluble and/or a synthetic molecule.
Various types of detectable or reporter moieties may be conjugated to the antibody of the invention. These include, but not are limited to, a radioactive isotope (such as [125]iodine), a phosphorescent chemical, a chemiluminescent chemical, a fluorescent chemical (fluorophore), an enzyme, a fluorescent polypeptide, an affinity tag, and molecules (contrast agents) detectable by Positron Emission Tomagraphy (PET) or Magnetic Resonance Imaging (MRI).
Examples of suitable fluorophores include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FTTC), Cy-chrome, ihodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red, PE-Cy5, and the like. For additional guidance regarding fluorophore selection, methods of linking fluorophores to various types of molecules see Richard P. Haugland, “Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992- 1994”, 5th ed., Molecular Probes, Inc. (1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.;
Hermanson, “Bioconjugate Techniques”, Academic Press New York, N.Y. (1995); Kay M. et al, 1995. Biochemistry 34:293; Stubbs et al, 1996. Biochemistry 35:937; Gakamsky D. et al, “Evaluating Receptor Stoichiometry by Fluorescence Resonance Energy Transfer,” in “Receptors: A Practical Approach,” 2nd ed., Stanford C. and Horton R. (eds.), Oxford University Press, UK. (2001); U.S. Pat. No. 6,350,466 to Targesome, Inc.]. Fluorescence detection methods which can be used to detect the antibody when conjugated to a fluorescent detectable moiety include, for example, fluorescence activated flow cytometry (FACS), immunofluorescence confocal microscopy, fluorescence in-situ hybridization (FISH) and fluorescence resonance energy transfer (FRET).
Numerous types of enzymes may be attached to the antibody of the invention [e.g., horseradish peroxidase (HPR), beta-galactosidase, and alkaline phosphatase (AP)] and detection of enzyme-conjugated antibodies can be performed using ELISA (e.g., in solution), enzyme-linked immunohistochemical assay (e.g., in a fixed tissue), enzyme-linked chemiluminescence assay (e.g., in an electrophoretically separated protein mixture) or other methods known in the art [see e.g., Khatkhatay MI. and Desai M., 1999. J Immunoassay 20: 151-83; Wisdom GB., 1994. Methods Mol Biol. 32:433-40; Ishikawa E. et ai, 1983. J Immunoassay 4:209-327; Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208; Schuurs AH. and van Weemen BK., 1980. J Immunoassay 1:229-49).
The affinity tag (or a member of a binding pair) can be an antigen identifiable by a corresponding antibody [e.g., digoxigenin (DIG) which is identified by an anti-DIG antibody) or a molecule having a high affinity towards the tag [e.g., streptavidin and biotin]. The antibody or the molecule which binds the affinity tag can be fluorescently labeled or conjugated to enzyme as described above.
Various methods, widely practiced in the art, may be employed to attach a streptavidin or biotin molecule to the antibody of the invention. For example, a biotin molecule may be attached to the antibody of the invention via the recognition sequence of a biotin protein ligase (e.g., BirA) as described in the Examples section which follows and in Denkberg, G. et al, 2000. Eur. J. Immunol. 30:3522-3532. Alternatively, a streptavidin molecule may be attached to an antibody fragment, such as a single chain Fv, essentially as described in Cloutier SM. et al, 2000. Molecular Immunology 37:1067-1077; Dubel S. et al, 1995. J Immunol Methods 178:201; Huston JS. et al, 1991. Methods in Enzymology 203:46; Kipriyanov SM. etal., 1995. Hum Antibodies Hybridomas 6:93; Kipriyanov SM. et ai, 1996. Protein Engineering 9:203; Pearce LA. et ai, 1997. Biochem Molec Biol Inti 42:1179-1188).
Functional moieties, such as fluorophores, conjugated to streptavidin are commercially available from essentially all major suppliers of immunofluorescence flow cytometry reagents (for example, Pharmingen or Becton-Dickinson). According to some embodiments of the invention, biotin conjugated antibodies are bound to a streptavidin molecule to form a multivalent composition (e.g., a dimer or tetramer form of the antibody).
As mentioned, the antibody may be conjugated to a therapeutic moiety. The therapeutic moiety can be, for example, a cytotoxic moiety, a toxic moiety, a cytokine moiety and a second antibody moiety comprising a different specificity to the antibodies of the invention.
The functional moiety (the detectable or therapeutic moiety of the invention) may be attached or conjugated to the antibody of the invention in various ways, depending on the context, application and purpose.
When the functional moiety is a polypeptide, the immunoconjugate may be produced by recombinant means. For example, the nucleic acid sequence encoding a toxin (e.g., PE38KDEL) or a fluorescent protein [e.g., green fluorescent protein (GFP), red fluorescent protein (REP) or yellow fluorescent protein (YfP)] may be ligated in-frame with the nucleic acid sequence encoding the antibody of the invention and be expressed in a host cell to produce a recombinant conjugated antibody. Alternatively, the functional moiety may be chemically synthesized by, for example, the stepwise addition of one or more amino acid residues in defined order such as solid phase peptide synthetic techniques.
A functional moiety may also be attached to the antibody of the invention using standard chemical synthesis techniques widely practiced in the art [see e.g., hypertexttransferprotocol://worldwideweb (dot) chemistry (dot) org/portal/Chemistry)], such as using any suitable chemical linkage, direct or indirect, as via a peptide bond (when the functional moiety is a polypeptide), or via covalent bonding to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer. Chimeric peptides may be linked via bonding at the carboxy (C) or amino (N) termini of the peptides, or via bonding to internal chemical groups such as straight, branched or cyclic side chains, internal carbon or nitrogen atoms, and the like. Description of fluorescent labeling of antibodies is provided in details in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110.
As illustrated in Figures 6A-C, the present inventors have shown that the antibodies of the present invention may be used to treat diseases mediated by IL-13.
Examples of such diseases include, but are not limited to asthma, allergy, allergic rhinitis, chronic sinusitis, hay fever, atopic dermatitis, chronic obstructive pulmonary disease ("COPD"), pulmonary fibrosis, esophageal eosinophilia, scleroderma, psoriasis, psoriatic arthritis, fibrosis, inflammatory bowel disease (particularly, ulcerative colitis), anaphylaxis, and cancer (particularly, Hodgkin's lymphoma, glioma, and renal carcinoma), and general Th2-mediated disorders/conditions .
Additional contemplated cancers that can be treated with the polypeptides described herein include but are not limited to adrenocortical carcinoma, hereditary; bladder cancer; breast cancer, breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptibility to; breast cancer, type 4; breast cancer, type 4; breast cancer- 1; breast cancer-3; breast-ovarian cancer, Buridtt’s lymphoma; cervical carcinoma; colorectal adenoma; colorectal cancer, colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type 3; colorectal cancer, hereditary nonpolyposis, type 6; colorectal cancer, hereditary nonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrial carcinoma; esophageal cancer; gastric cancer, fibrosarcoma, glioblastoma multiforme; glomus tumors, multiple; hepatoblastoma; hepatocellular cancer, hepatocellular carcinoma; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, acute myeloid, with eosinophilia; leukemia, acute nonlymphocytic; leukemia, chronic myeloid; Li-Fraumeni syndrome; liposarcoma, lung cancer, lung cancer, small cell; lymphoma, non-Hodgkin’s; lynch cancer family syndrome Π; male germ cell tumor; mast cell leukemia; medullary thyroid; medulloblastoma; melanoma, meningioma; multiple endocrine neoplasia; myeloid malignancy, predisposition to; myxosarcoma, neuroblastoma; osteosarcoma; ovarian cancer, ovarian cancer, serous; ovarian carcinoma; ovarian sex cord tumors; pancreatic cancer, pancreatic endocrine tumors; paraganglioma, familial nonchromaffin; pilomatricoma; pituitary tumor, invasive; prostate adenocarcinoma; prostate cancer; renal cell carcinoma, papillary, familial and sporadic; retinoblastoma; rhabdoid predisposition syndrome, familial; rhabdoid tumors; rhabdomyosarcoma; small-cell cancer of lung; soft tissue sarcoma, squamous cell carcinoma, head and neck; T-cell acute lymphoblastic leukemia; Turcot syndrome with glioblastoma; tylosis with esophageal cancer; uterine cervix carcinoma, Wilms’ tumor, type 2; and Wilms’ tumor, type 1, etc.
According to a specific embodiment, the disease is atopic dermatitis.
The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
As used herein, the term “subject” includes mammals, preferably human beings at any age which suffer from the pathology. The antibodies of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term "active ingredient" refers to the antibody accountable for the biological effect.
Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drags may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Conventional approaches for drag delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB ; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
The term “tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (TCRLrantibody) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., dermatitis) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-1)· Dosage amount and interval may be adjusted individually to provide antibody levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit (diagnostic or therapeutic), which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S . Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
The present invention further provides for the administration of the disclosed anti-hlL- 13Rα1 antibody molecules for purposes of gene therapy. In such a method, the cells of a subject would be transformed with nucleic acid encoding the antibody molecules of the invention. Subjects comprising the nucleic acids will then produce the antibody molecules endogenously. Previously, Alvarez, et al, Clinical Cancer Research 6:3081-3087, 2000, introduced single-chain anti-ErbB2 antibodies to subjects using a gene therapy approach.
Nucleic acids encoding any polypeptide or antibody molecule of the invention may be introduced to a subject. In specific embodiments, the antibody molecule is a human, single-chain antibody. The nucleic acids may be introduced to the cells of a subject by any means known in the art. In specific embodiments, the nucleic acids are introduced as part of a viral vector. Examples of particular viruses from which the vectors may be derived include lentiviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphavirus, influenza virus, and other recombinant viruses with desirable cellular tropism.
Various companies produce viral vectors commercially, including, but by no means limited to, AVIGEN, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, and lentiviral vectors), CLONTECH (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), GENVEC (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).
Methods for constructing and using viral vectors are known in the art (see, e.g., Miller, et al, BioTechniques 7:980-990, 1992). In specific embodiments, the viral vectors are replication defective, that is, they are unable to replicate autonomously, and thus are not infectious, in the target cell. The replication defective virus may be a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsulating the genome to produce viral particles. Defective viruses which entirely or almost entirely lack viral genes may be used as well. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted.
Examples of vectors comprising attenuated or defective DNA virus sequences include, but are not limited to, a defective herpes virus vector (Kanno et al, Cancer Gen. Ther. 6: 147-154, 1999; Kaplitt et al, J. Neurosci. Meth. 71:125-132, 1997 and Kaplitt et al, J. Neuro One. 19:137-147, 1994).
Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Attenuated adenovirus vectors, such as the vector described by Strafford-Perricaudet et al, J. Clin. Invest. 90:626-630, 1992 are desirable in some instances. Various replication defective adenovirus and minimum adenovirus vectors have been described (see, e.g., W094/26914, W094/28938, W094/28152, W094/12649, WO95/02697 and W096/22378). The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al, Gene 101:195, 1991; EP 185573; Graham, EMBO J. 3:2917, 1984; Graham et al, J. Gen. Virol. 36:59, 1977). The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The use of vectors derived from the AAV s for transferring genes in vitro and in vivo has been described
(see Daly, et al, Gene Ther. 8:1343-1346, 2001, Larson et al, Adv. Exp. Med. Bio. 489:45-57, 2001; WO 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368 and 5,139,941 and EP 488528B1).
In another embodiment, the gene can be introduced in a retroviral vector, e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289, and 5,124,263; Mann et al, Cell 33:153, 1983; Markowitz et al, J. Virol., 62:1120, 1988; EP 453242 and EP178220. The retroviruses are integrating viruses which infect dividing cells.
Lentiviral vectors can be used as agents for the direct delivery and sustained expression of nucleic acids encoding an antibody molecule of the invention in several tissue types, including brain, retina, muscle, liver and blood. The vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the antibody molecule. For a review, see Zufferey et al, J. Virol. 72:9873-80, 1998 and Kafri et al, Curr. Opin. Mol. Ther. 3:316-326, 2001. Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for gene therapy. An example is a tetracycline-inducible VS V-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 10.sup.6 IU/ml for at least 3 to 4 days; see Kafri et al, J. Virol. 73:576-584, 1999. The vector produced by the inducible cell line can be concentrated as needed for efficiently transducing nondividing cells in vitro and in vivo.
Sindbis virus is a member of the alphavirus genus and has been studied extensively since its discovery in various parts of the world beginning in 1953. Gene transduction based on alphavirus, particularly Sindbis virus, has been well-studied in vitro (see Straus et al, Microbiol. Rev., 58:491-562, 1994; Bredenbeek et al, J. Virol., 67:6439-6446, 1993; Ijima et al, Int. J. Cancer 80:110-118, 1999 and Sawai et al, Biochim. Biophyr. Res. Comm. 248:315-323, 1998. Many properties of alphavirus vectors make them a desirable alternative to other virus-derived vector systems being developed, including rapid engineering of expression constructs, production of high-titered stocks of infectious particles, infection of nondividing cells, and high levels of expression (Strauss et al, 1994 supra). Use of Sindbis virus for gene therapy has been described. (Wahlfors et al, Gene. Ther. 7:472-480, 2000 and Lundstrom, J. Recep. Sig. Transduct. Res. 19(1- 4):673-686, 1999. In another embodiment, a vector can be introduced to cells by lipofection or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo and in vitro transfection of a gene encoding a marker (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987 and Wang et al, Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987). Useful lipid compounds and compositions for transfer of nucleic acids are described in WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127.
It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al, J. Biol. Chem. 267:963-967, 1992; Williams et al, Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). Receptor- mediated DNA delivery approaches can also be used (Wu et al, J. Biol. Chem. 263:14621-14624, 1988). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Vilquin et al, Gene Ther. 8:1097, 2001; Payen et al, Exp. Hematol. 29:295-300, 2001; Mir, Bioelectrochemistry 53:1-10, 2001; WO 99/01157, WO 99/01158 and WO 99/01175).
Pharmaceutical compositions suitable for such gene therapy approaches and comprising nucleic acids encoding an anti-hIL-13Ralphal antibody molecule of the present invention are included within the scope of the present invention.
In another aspect, the present invention provides a method for identifying, isolating, quantifying or antagonizing IL-13Ralphal in a sample of interest using an antibody molecule of the present invention. The antibody molecules may be utilized as a research tool in immunochemical assays, such as western blots, ELISAs, radioimmunoassay, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art (see, e.g., Immunological Techniques Laboratory Manual, ed. Goers, J. 1993, Academic Press) or various purification protocols. The antibody molecules may have a label to facilitate ready identification or measurement of the activities associated therewith. One skilled in the art is readily familiar with the various types of detectable labels (e.g., enzymes, dyes, or other suitable molecules which are either readily detectable or cause some activity/result that is readily detectable) useful in the above protocols.
The polypeptides of the present invention (e.g. antibodies) may be co-administered with additional agents to increase its therapeutic effect. For example, the present inventors contemplate administration of agents that down- regulate (or inhibit) IL-4. Such agents include antibodies, small molecule inhibitors, polynucleotide agents directed against genes or RNA that encode IL-4. For example, the present invention contemplates co-administration (or coformulation) with Tofacitinib or Ruxolitinib, which blocks IL-4 signaling.
It is expected that during the life of a patent maturing from this application many relevant IL-4 inhibitors will be developed and the scope of the term IL-4 inhibitor is intended to include all such new technologies a priori.
As used herein the term “about” refers to ± 10 % .
The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".
The term “consisting of’ means “including and limited to”.
The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes Ι-ΙΠ Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes Ι-ΙΠ Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes Ι-ΙΠ Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed.
(1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
MATERIALS AND METHODS
Mice : Male and female IlI3rα1-/- mice (backcrossed >F9 to C57BL/6) were generated as previously described19. C57BL/6 wild-type mice were obtained from Harlan Laboratories (Rehovot, Israel). All experiments were reviewed and approved by the Animal Care Committee of Tel Aviv University and were performed in accordance with its regulations and guidelines regarding the care and use of animals for experimental procedures. All of the experiments were conducted in the specific pathogen free facilities of the Tel Aviv University. In all experiments, age-, weight-, and sex-matched mice were used. Oxazolone-induced dermatitis: Mice (6- to 8-week-old, male and female) were sensitized on day 0 by the application of 15 μΐ of 1% oxazolone (OX) (Sigma- Aldrich, Rehovot, Israel) in acetone on both flanks of both ears (60 μΐ per mouse). Starting on day 7, mice were challenged in the same manner with 0.5% oxazolone, three times per week, for up to 10 challenges. Control mice were similarly treated with acetone. Twenty-four hours after the last challenge, the mice were bled for obtaining sera and euthanized. Ear thickness was measured by a digital caliper, and total ear tissues were taken for further analyses. In the IL-4 neutralization experiments, mice were treated intraperitoneally twice a week with rat IgGl anti-IL-4 (120 pg/mouse, clone 11B11, BioXCell, Lebanon, NH) or rat IgGl isotype control (clone TNP6A7, BioXCell, Lebanon, NH). In the IL- 13Rα1 -neutralization experiments, mice were treated intraperitoneally three times a week with anti-IL-13Rα1 (clone MSS, produced in house, 200 pg/mouse) or mouse IgGl isotype control (clone MOPC-21, BioXCell, Lebanon, NH).
Histology: Slides of paraffin-embedded sections (5 pm) were processed and stained with hematoxylin and eosin (H&E) by Patho-Lab Diagnostics, Israel. To measure epidermal thickness, 3 independent images were captured at x20 magnification from each slide. Using Image! software
(NIH), 10 epidermal thickness measurements of each slide were made and average thickness was calculated per image. The end value per mouse represents an average of 3 independent measurements.
Ear lysate preparation: Ears were placed in ice-cold IP Lysis Buffer (750 pi, Pierce #87787, Thermo Fisher Scientific, Waltham, MA) supplemented with a protease inhibitors cocktail (Sigma, Rehovot, Israel). Thereafter, the ears were homogenized, centrifuged (14,000g, 10 minutes, 4°C), and supernatants were collected, aliquoted, and stored at -20°C.
Enzyme-linked immunosorbent assay (ELISA): Cytokines and IgE levels were measured by ELISA according to manufacturer's instructions. The following kits for mouse proteins were used: IL-4 (BioLegend, CA, USA); IL-6, CCL17, CCL24, IL-13Ra2 (R&D systems, Minneapolis, MN, USA); TNF-o, CCL2, CCL11, CXCL1 (Peprotech, Rehovot, IL); IgE (BD Biosciences, San Jose, CA, USA). The following kits for human proteins were used: CCL26 (R&D systems, Minneapolis, MN, USA); IL-6 (Peprotech, Rehovot, IL). In vitro binding affinity and specificity assessments of MSS and 2HA6 monoclonal antibodies were carried out by an in-house developed ELISA. Briefly, 96-well plates were coated with the recombinant extracellular domains (ECDs) of human or mouse IL-13Rα1 proteins (ACROBiosystems, Newark, DE, USA), or with control antigens. HRP-conjugated secondary goat anti-human antibodies or goat anti-mouse IgG (H+L) (Jackson Immunoresearch, West Grove, PA, USA) were used to detect 2HA6 or MSS, respectively. The signal was developed with the chromogenic substrate TMB (Thermo Fisher Scientific, Waltham, MA) and the reaction was stopped by an equivalent volume of 1M H2SO4 and read at 450 nm.
Flow cytometry: Single-cell suspensions of enzymatically-digested ears were stained using the following antibodies: CD45-APC, CDllb-PerCP/Cy5.5, CD1 lc-FITC, CD8a-PE (eBioscience, San Diego, CA, USA); SiglecF-PE (BD Biosciences, San Jose, CA, USA); Ly6C- PE/Cy7, F4/80-AF700, CD4-AF488, CD3e-PE/Cy7 (BioLegend, San Diego, CA, USA); Ly6G- APC/Cy7, B220-PerCP/Cy5.5 (Biogems, Westlake Village, CA, USA). DAPI (Sigma-Aldrich, Rehovot, IL) was used as a marker for cell death. Cell counts were measured using Flow-Count Fluorospheres (Beckman Coulter, Brea, CA) according to the manufacturer’s instructions. Events were acquired using Gallios Flow Cytometer, and data were analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA).
Isolation and production of anti-IL-13Ral monoclonal antibodies: Female C57BL/6 or IlI3rα1-/- mice were immunized subcutaneously with human or mouse IL-13Rα1 ECD (ACROBiosystems, Newark, DE, USA) in complete Freund's adjuvant (Sigma-Aldrich, Rehovot, IL) (30 μg/mouse). Three additional boosts were made fortnightly with human or mouse IL- 13Rα1
ECD (30 pg/mouse) in incomplete Freund's adjuvant (Sigma-Aldrich, Rehovot, IL). Nine days after the last boost, mice were euthanized and antigen-specific B cells and plasma cells from spleens and bone marrows were sorted using FACSAria Fusion (BD Biosciences, San Jose, CA, USA) using a biotinylated antigen and the following antibodies: B220-AF488 (eBioscience, San Diego, CA, USA); CD19-PE (BioLegend, San Diego, CA, USA); CD138-PE (BD Biosciences, San Jose, CA, USA). cDNA libraries were made from the sorted cells and mouse V-genes were amplified using the appropriate primers.
Variable heavy (VH) and light (VL) chains were assembled into single chain Fv (scFv) antibodies by cloning into a pETcon2-based yeast display vector. Clones were selected for binding biotinylated human or mouse IL-13Rα1 ECD using Yeast Display (method described in22). Finally, scFv clones were converted to full-size antibody heavy and light chains and expressed as human or mouse IgGls in Expi293F mammalian expression systems (Thermo Fisher, Waltham, MA, USA). IgGs were purified using affinity chromatography on protein A or protein G columns (GE Healthcare, Chicago, IL, USA).
MC38 or A549 cells were seeded (4x1ο4 cells/well in 96-well plates and l.SxlO5 cells/well in 24-well plates, respectively) and left to adhere. Once adherent, medium (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and lx Penicillin/Streptomycin) was removed and renewed with 100-200 μΐ of medium containing one of the following treatments: human IL-4, human IL-13, or human TNF-a, which were mixed with either the anti-human IL-13Rα1 antibody 2HA6 (expressed as a human IgGl) or with an isotype control (bevacizumab, human IgGl anti- VEGF-A, Roche, Israel) for A549 cells, or mouse IL-4, mouse IL-13, or mouse TNF-a, with the anti-mouse IL-13Rα1 antibody MSS (expressed as a mouse IgGl) and its respective isotype control (MOPC-21, mouse IgGl, BioXCell, Lebanon, NH) for MC38 cells. After 48 hours of incubation, the cell culture media were collected, centrifuged (3000 rpm, 2 min, 4 °C) and supernatants were collected. Thereafter, the levels of CCL26 (for A549 cells), IL-13Ra2 (for MC38 cells) and IL-6 (for both cell lines) were measured by ELISA.
IL-13- induced lung inflammation: MSS (anti-mouse IL-13Rα1) or MOPC-21 (mouse IgGl isotype control) were administered to C57BL/6 mice (intraperitoneal, 200 pg/mouse, days 0 and 2). IL-13 (1 μg/mouse, Peprotech, Rehovot, IL) or saline were administered intratracheally 2 hours after the injection of MSS. On day 4, the mice were euthanized and bronchoalveolar lavage fluid (BALF) was obtained. BALF CCL24 levels (measured by ELISA) and eosinophil counts (measured by flow cytometry) were assessed as readouts for IL-13-induced pathology.
RESULTS
IL-13Rα1 is differentially expressed by hematopoietic and non-hematopoietic cells in the skin
To determine the role of the type 2 IL-4 receptor in atopic dermatitis (AD), a chronic model of repetitive skin challenges with oxazolone (OX) was used. This model has been shown to induce a Th2-dominated inflammatory response that is similar to human AD (24, 25). First, the cellular source for IL-13Rα1 expression in the skin was determined. Under baseline conditions, among the hematopoietic lineage, IL-13Rα1 was highly expressed by monocytes and neutrophils and to lesser extent by eosinophils and lymphocytes. The expression of IL-13Rα1 was observed on the surface of non-hematopoietic cells as well, with nearly no expression by fibroblasts (defined as CD45" /PDGFRa* cells), which may express the type 2 IL-4R(26). Assessment of IL-13Rα1 expression in OX-challenged mice revealed that the expression of IL-13Rα1 was stable on most cells. Nonetheless, a marked reduction was observed on the surface of monocytes (Figure 8), whereas IL-13Rα1 was increased on the surface of macrophages (Figure 8).
OX- and DNFB-induced dermatitis are mediated via the type 2 IL-4 receptor: To define the role of the type 2 IL-4 receptor in atopic skin disease, WT and Ill3ral-/- mice were challenged with OX. OX-challenged WT mice displayed increased ear thickening (Fig. 1A and Figure 9). In contrast, ear thickness in OX-challenged H13ral'-/- mice was comparable to acetone-treated mice (Figure 1A). Histopathological assessment of the skin obtained from OX-challenged WT mice revealed hyperkeratosis, increased numbers of nuclei and epidermal thickening (Figures 1B-C). OX-challenged lll3ral'-/ mice displayed decreased numbers of nuclei and decreased epidermal thickening (Figures 1B-C). To further establish the role of the type 2 IL-4 receptor in dermatitis, WT and IlI3rα1-/- mice were treated with 2,4-dinitrofluorobenzene (DNFB)(27). DNFB- challenged IlI3rα1-/- mice displayed decreased dermatitis as observed by decreased ear and epidermal thickening in comparison with DNFB -treated WT mice (Figures lOA-O).
In order to determine the role of the type 2 IL-4 receptor in the regulation of proinflammatory cytokines such as TNF-a, which has been shown to be involved in AD(28), skin expression of TNF-a was analyzed following OX- and DNFB-challenge, (Figure ID and Figures lOA-O).
IL-4 is a key cytokine mediating the induction of IgE, which serves as an important biomarker and effector of Th2 immunity and atopy(29). Assessment of skin IL-4 levels revealed that although IL-4 was decreased in OX-challenged IlI3rα1-/- mice (Figure IE), an IL-13Rα1- independent pathway exists for induction of IL-4. Serum IgE levels in WT mice were elevated following OX treatment (Figure IF). OX-challenged IlI3rα1-/- mice exhibited slightly higher serum IgE levels in comparison with OX-challenged WT mice (Figure IF). Interestingly, IL-4 was induced to a similar extent in DNFB-treated WT and H13ral'-/- mice (Figures lOA-O). Serum IgE was slightly (but statistically significantly) decreased in DNFB-treated IlI3rα1-/- mice (Figures lOA-O).
Skin chemokine production and subsequent cellular migration is differentially regulated by the type 2 IL-4 receptor
The inflammatory milieu in AD is characterized by the induction of various chemokines promoting cellular migration(iO). Therefore, the present inventors set about to determine whether the type 2 IL-4 receptor regulates chemokine production and subsequent cellular influx into the skin. OX-challenged WT mice displayed increased expression of CXCL1, CCL11, and CCL24 (Figures 1G-I). Induction of CXCL1 and CCL11 in response to OX challenge was dependent on the type 2 IL-4 receptor (Figures 1G-H), whereas OX-induced CCL24 expression was independent of the type 2 IL-4 receptor (Figure II). CCL17 expression was modestly increased by OX treatment, and this increase was also dependent on the type 2 IL-4R (Figure 1J). Consistent with these findings, cellular infiltration of leukocytes was differentially regulated by the type 2 IL-4 receptor. OX-challenged WT mice displayed increased accumulation of various CD45+ cells predominantly consisting of neutrophils (defined as: CD 1 lb+/Ly6Ghi/Ly6Cmed cells), eosinophils (defined as: CDllb+/Ly6G-/Ly6C-/SiglecF+ cells), as well as T cells (defined as: SSClow/FSClow/CDl lb-/CD3+ cells) and macrophages (defined as: CDllb+/Ly6G /Ly6C-/SiglecF /CD lie* cells) (Figure IK). While neutrophil accumulation was dependent on the type 2 IL-4 receptor, accumulation of T cells and macrophages was regulated to a lesser extent and eosinophil accumulation was independent of the type 2 IL-4 receptor (Figure IK).
Similar to the findings in the OX model, the expression of CXCL1 and CCL11 (but not CCL24) were decreased in the ears of DNFB -treated IlI3rα1-/- mice in comparison with WT mice (Figures lOA-O). Consistently, infiltration of neutrophils and T cells into the skin of DNFB-treated mice was reduced in the absence of IU3ral, whereas infiltration of eosinophils was not affected by the lack of Ill3ral (Figures lOA-O).
Taken together, these data demonstrate that the type 2 IL-4 receptor differentially mediates accumulation of immune cells in the skin, and highlight a type 2 IL-4R-independent mechanism for eosinophil recruitment into the skin in AD.
OX-induced dermatitis is mediated by IL-13 and IL-4 via the type 2 IL-4R
The type 2 IL-4 receptor mediates signals from both IL-4 and IL-13(5). Thus, the present inventors sought to determine the cellular source for IL-4 and IL-13 in OX-challenged mice. To this end, IL-4 and IL-13 reporter mice (4Get and Ill3Smart, respectively) were treated with OX. In this experimental setting, cells that have the capacity to secrete IL-4 are marked by eGFP(31). Cells that express endogenous IL-13 are marked by the expression of the human CD4 surface marker (32). Although IL-13 expression was not detected in skin homogenates by ELISA, -25% of all skin CD4+ T cells stained positive with anti-human CD4 (Figures 11 A-B). Human CD4 was also detected on the surface of 1.47% of γδ T cells and 2.07% of NKT cells. CDS* T cells were negative for human CD4 expression (Fig. S4 online). Assessment of eGFP* T cells in the skin of IL-4 reporter mice revealed that the cellular source for IL-4 expression following OX-challenge were NKT cells (-12.5%), CD4+ T cells (-8.5%) and a small proportion of CDS* T cells (-3.5%) (Figures 11 A-B).
In order to examine the relative contribution of IL-4 and IL- 13 via the type 2 IL-4 receptor to OX-induced pathology, IL-4 was neutralized using an anti-IL-4 antibody (33) in OX-challenged WT and IlI3rα1-/- mice. In this experimental setting, comparison between WT and IlI3rα1-/- mice in the isotype control-treated mice reflects the contribution of the type 2 IL-4 receptor to disease pathology similar to the experimental setting that was shown in Figure 1 (Figure 2A). Comparison between WT mice treated with isotype control and WT mice treated with anti-IL-4 should reveal the relative contribution of IL-4 to OX-induced pathology since these mice can still mediate IL- 13 signaling via the type 2 IL-4R (Figure 2A). Comparison between IlI3rα1-/- mice treated with isotype control and IlI3rα1-/- mice treated with anti-IL-4 should reveal the relative contribution of IL-4 via the type 1 IL-4 receptor to OX-induced pathology (Figure 2A). Finally, comparing OX- induced responses in WT and IlI3rα1-/- mice that have been treated with anti-IL-4 should reveal the relative contribution of IL-13 via the type 2 IL-4 receptor to OX-induced pathology (Figure
2A).
Consistent with the results shown in Figure 1, isotype control-treated IlI3rα1-/- mice displayed decreased OX-induced dermatitis as observed by ear and epidermal thickness measurements (Figures 2B-D). Neutralization of IL-4 did not further protect OX-challenged IlI3rα1-/- mice from ear thickening (Figure 2C). Histopathological analysis of the skin demonstrated a statistically-significant, but minor effect for IL-4 neutralization on OX-induced epidermal thickening in WT mice (Figure 2B, 2D). Nonetheless, these mice still displayed a thicker epidermis than OX-challenged IlI3rα1-/- mice, which were treated with either isotype control or anti-IL-4 antibody (Figure 2D). The finding that anti-IL-4 treatment did not reduce epidermal thickness in OX-challenged IlI3rα1-/- mice, demonstrates that epidermal thickening is not mediated by the type 1 IL-4R and is probably driven by IL-4 and IL-13 signaling via the type 2 IL-4R.
TNF-a levels (as well as IL-Ιβ, data not shown) were decreased in OX-challenged H13ral' A mice regardless of anti-IL-4 antibody treatment, and remained upregulated in OX-challenged WT mice despite anti-IL-4 treatment (Fig. 2E). Thus, pro-inflammatory cytokine production in OX-induced dermatitis is exclusively mediated by IL-13 signaling via the type 2 IL-4R.
Of note, IL-4 levels in the skin were abolished in mice treated with the IL-4 neutralizing mAb, thus validating the efficiency of the anti-IL-4 treatment (Figure 2F). Reduced expression of IL-4 was not due to competition of the neutralizing antibody with the ELISA kits(2i). Consistent with previous observations (Figure IF), IgE levels in isotype-treated, OX-challenged IlI3rα1-/- mice, were slightly higher (albeit not statistically significant) than those of isotype control-treated, OX-challenged WT mice (Figure 2G). Neutralizing IL-4 in OX-challenged WT and IlI3rα1-/- mice caused a dramatic reduction in serum IgE levels (Figure 2G). Thus, IgE production following OX- challenge is exclusively mediated by IL-4 signaling through the type 1 IL-4R.
Immune cell infiltration in OX-induced dermatitis is synergistically mediated by the type 1 and type 2 IL-4Rs
To further pursue the roles of IL-4, IL-13 and the type 2 IL-4R in OX-induced dermatitis, the expression of various chemokines was assessed in OX-challenged WT and IlI3rα1-/- mice that were treated with an IL-4 neutralizing mAb.
Similar to previous finding (Figure 1), OX-challenged IlI3rα1-/- mice that were treated with isotype control, displayed significant reduction in CXCL1, CCL11 and CCL17, but not CCL24 (Figure 2H-K). Expression of CXCL1, CCL11, and CCL17 in OX-challenged IlI3rα1-/- mice were dependent on IL-13 signaling via the type 2 IL-4 receptor since no further reduction was observed in the levels of these chemokines in anti-IL-4-treated, OX-challenged WT or IlI3rα1-/- mice (Figure 2H-I, 2K). Anti-IL-4-treated, OX-challenged WT and IlI3rα1-/- mice displayed a marked reduction in CCL24 levels, which was reduced to the same extent in these mice following anti-IL-4 treatment (Fig. 2J). Thus, OX-induced CCL24 expression is primarily dependent on IL-4 signaling via the type 1 IL-4 receptor.
Immunophenotyping of OX-challenged skin following anti-IL-4 antibody treatment revealed differential contribution of IL-4 and IL-13 via the type 1 and type 2 receptors to infiltrating cells into the skin as observed by assessment of total CD45+ cells (Figure 2L). Infiltration of macrophages and T cells, which were decreased in OX-challenged, isotype-treated IlI3rα1-/- mice, were not further reduced following anti-IL-4 treatment (Figures 2M-N). Neutrophil and monocyte accumulation, which were decreased in isotype control-treated lll3ra-l' /- mice following OX challenge, were further reduced following anti-IL-4 treatment (Figures 20- P). Eosinophilic infiltration was not altered in OX-challenged, isotype control-treated IlI3rα1-/- mice but was reduced following anti-IL-4 treatment (Figure 2Q). Collectively, these data demonstrate that in OX challenged mice, IL-13 signaling via the type 2 IL-4R regulates T cell and macrophage accumulation, whereas neutrophil and monocyte accumulation is dependent both on IL-13 signaling via the type 2 IL-4R as well as IL-4 interacting with the type 1 IL-4R (Figures 2M-P). Eosinophilic infiltration was dependent on IL-4 signaling via the type 1 IL-4R (Figure 2Q).
IL-13 is required for OX-induced dermatitis, TNF-a, CCL11, CCL17 \ and neutrophilic infiltration into the skin
To better establish the respective roles of IL-13 and IL-4, a pharmacological approach was taken, where IL-13 was neutralized in OX-treated WT mice using a commercial rat anti-mouse IL-13 IgGl antibody. Neutralization of IL-13 was confirmed by complete reduction in soluble IL- 13 Ra.2 (sIL-13Ra2) expression in anti-IL- 13 -treated mice (Figure 3 A). Similar to the findings in IlI3rα1-/- mice (Figure 1), neutralization of IL-13 resulted in a significant reduction in ear thickness, epidermal thickness, and TNF-a expression in the skin (Figures 3B-D). Furthermore, neutralization of IL-13 caused a marked reduction in the expression of CCL11 and CCL17 but had no effect on CCL24 expression (Figure 3E). Consistent with the findings in OX-challenged IlI3rα1-/- mice (Figure 1), neutralization of IL-13 resulted in decreased infiltration of neutrophils to the skin whereas eosinophil levels were not affected (Figures 3F-G).
Since TNF-a levels were reduced in OX-challenged IlI3rα1-/- mice and were also reduced following neutralization of IL-13, the present inventors sought to define whether decreased neutrophilia was due to decreased TNF-a expression. To this end, TNF-a was neutralized in acetone- and OX-treated WT mice. Neutralization of TNF-a had no effect on ear thickness (Figure 3H), CXCL1 expression (Figure 31), or infiltration of neutrophils into the skin (Figure 3J).
Oxazolone-induced dermatitis is mediated by the type 2 IL-4 receptor expressed on non- hematopoietic cells
IL-13Rα1 is expressed by hematopoietic and non-hematopoietic cells (Figure 8). Thus, the present inventors aimed to determine the relative contribution of these lineages to the type 2 IL- 4R-dependent response. To this end, bone marrow chimeric mice were generated by transferring bone marrows that were obtained from either C57BL6 CD45.1 mice (herein: WT), or C57BL6 IlI3rα1-/- mice (expressing the CD45.2 variant), into WT or IlI3rα1-/- mice. Engraftment efficiency was determined in the peripheral blood by flow cytometry using CD45.1 and CD45.2 antibodies. All mice exhibited a nearly-complete leukocytes engraftment (Figures 12A-D).
In comparison with WT recipient mice, and irrespectively of the donor mice, IlI3rα1-/- recipient mice displayed decreased OX-induced ear thickness (Figure 4A) and decreased epidermal thickness (Figures 4B-C). In fact, IlI3rα1-/- mice that received WT bone marrow cells were protected from OX-induced ear thickening exactly to the same extent as the control lll3ral' A mice (Fig. 4 A, p = 0.9999). These data demonstrate that expression of the type 2 IL-4R on non- hematopoietic cells is necessary for the development of OX-induced dermatitis. Similarly, skin TNF-a levels were decreased in IlI3rα1-/- recipient mice, regardless of the genotype of the graft bone marrow (Figure 4D).
Skin IL-4 levels were increased in OX-challenged, IlI3rα1-/- recipient mice irrespectively of the donor bone marrow (Figure 4E). Consistent with this finding, serum IgE levels in OX- challenged, IlI3rα1-/- recipient mice were slightly higher than those of the OX-challenged WT mice (Figure 4F). This finding suggests a suppressive pathway for IgE production, which is mediated by the type 2 IL-4R that is expressed by non-hematopoietic cells.
Expression of CXCL1 and CCL11 was dependent on the type 2 IL-4R, which is expressed by non-hematopoietic cells, as their expression was decreased in OX-challenged, IlI3rα1-/- recipient mice, regardless of genotype of the bone marrow donor (Figures 4G-H).
While CCL24 levels were comparable among WT mice and III 3 ral^' mice, which received IlI3rα1-/- bone marrow cells (Figure 41), CCL24 was dramatically increased in IlI3rα1-/- mice that received bone marrow cells from WT mice (Figure 41).
Immunophenotyping of the cellular infiltrate in OX-challenged mice following bone marrow transfer revealed that accumulation of neutrophils and macrophages (Figure 4J-L) in OX- challenged skin was dramatically suppressed in recipient IlI3rα1-/- mice irrespective of bone marrow donor. Monocyte and eosinophil numbers in the skin were comparable in all groups (Figure 4N). These data demonstrate that expression of CXCL1 and accumulation of neutrophils following OX challenges is mediated by non-hematopoietic cells via the type 2 IL-4R.
Generation of a mouse IL-13Raα1-neutralizing mAh
The data suggests that neutralization of the type 2 IL-4R, i.e. by blocking IL-13Rα1, may be a beneficial approach for treating AD. Thus, an IL-13Rα1 -neutralizing mAb was generated. To this end, IlI3rα1-/- mice were immunized and boosted with the extracellular domain (BCD) of mus musculus IL-13Rα1 in the presence of adjuvant. Thereafter, mice were bled and their antisera were tested for the ability to bind plate-immobilized IL-13Rα1 BCD (See method description online and, Figures 13A-B online). Subsequently, antisera were examined for receptor neutralizing activity using IL-13-stimulated MC38 cells (Figure 13C). Serum from mouse #6, which also displayed the highest antibody titer (Figure 13A), was capable of inhibiting IL-13-induced responses in vitro, suggesting the presence of neutralizing antibodies. Subsequently, bone marrow cells were obtained from mouse #6, plasma cells were sorted (CD138+), and then antibody genes coding for variable heavy (VH) and variable light (VL) fragments were cloned as scFvs into a yeast display vector. Yeast display was applied to enrich mIL-13Rα1 BCD-specific clones (see illustration in Figure 14A). Following several enrichment steps, a mIL-13Rα1 BCD-specific scFv was isolated, which was then re-formatted to a mouse IgGl anti-IL-13Rα1 mAb termed 'MS8'.
Information regarding MSS is provided in Table 3, herein below.
Table 3
Figure imgf000045_0001
Figure imgf000046_0001
In vitro, MSS was capable of binding mIL-13Rα1 (ECso = 1.46nM, by ELISA) but not hIL-13Rα1 or streptavidin, which was used as a control antigen (Figure 5A). Furthermore, MS 8 dose-dependently inhibited IL-13-induced secretion of sIL-13R(x2 by MC38 cells (Figure 5B). MSS was capable of partially inhibiting IL-4-induced effects in MC38 cells as well (Figure 5C). Importantly, MSS did not impair TNF-ct-induced IL-6 production by MC38 cells (Fig. 5D), demonstrating a specific inhibitory effect on IL-13 and IL-4 through the type 2 IL-4R. Furthermore, MS 8 was capable of binding spleen monocytes (CD 11 b+Ly6GmedLy6Chigh) from WT mice. Yet, it did not show any binding to spleen monocytes obtained from IlI3rα1-/- mice (Figure 5E). To determine whether MSS can be used pharmacologically in vivo, its capacity to neutralize
IL-13 was tested in an acute model of IL-13 -induced eosinophilic lung inflammation where mice are intratracheally challenged with IL- 13(34). Consistent with its capability to neutralize IL-13 in vitro, IL- 13-challenged mice, which were intraperitoneally treated with MSS, exhibited reduced levels of CCL24 in their bronchoalveolar lavage fluid (HALF) compared with isotype control- treated mice (Figure 5F). Decreased CCL24 expression was accompanied with a marked reduction in eosinophil HALF percentage and total cell counts (Figure 5G-H). These results demonstrate that MS 8 is a pharmacologically functional mouse IL-13Rα1 antagonist.
Antibody-mediated neutralization of the type 2 IL-4R protects mice from OX-induced dermatitis
Next, the present inventors sought to determine whether in vivo neutralization of the type 2 IL-4R via targeting IL-13Rα1 may protect mice from OX-induced dermatitis. Accordingly, WT mice were challenged with OX and treated with either isotype control or MS 8. OX-challenged mice that were treated with MSS displayed a marked decrease in ear thickness (Figure 6A). Histopathological assessment of the skin obtained from MS 8 -treated mice revealed decreased numbers of nuclei and decreased epidermal thickening (Figures 6B-C).
Furthermore, mice treated with MSS displayed significantly decreased expression of TNF- α (Figure 6D). Neutralization of IL-13Rα1 resulted in slightly increased expression of IL-4 (Figure 6E). Nonetheless, no differences in serum IgE were observed between MSS and isotype- treated mice (Figure 6F, p = 0.065).
Antibody-mediated neutralization of the type 2 IL-4R decreases OX-induced chemokine production and subsequent cellular migration
Similar to the results which were obtained for OX- and DNFB-challenged IlI3rα1-/- mice (Figure 1), mice treated with MSS displayed decreased expression of CXCL1 and CCL11 in response to OX challenge (Figures 6G-H). Although CCL24 expression was slightly reduced by MSS treatment, this did not reach statistical significance (Figure 61). Subsequently, cellular infiltration of leukocytes following MSS treatment was also assessed. OX-challenged, MS8- treated mice displayed decreased total leukocyte infiltration (Figure 6J). Decreased cellular infiltration in response to MSS treatment was evident in T cells, macrophages and neutrophils (Figure 6J). Interestingly, and despite the finding that eosinophilic infiltration following OX- challenge was independent of IL-13Rα1, MSS treatment resulted in significantly decreased eosinophilia as well (Figure 6J). Collectively, these data suggest that targeting IL-13Rα1 in atopic dermatitis may have beneficial therapeutic value.
Generation of a human IL-13Rα1-neutralizing mAb IlI3rα1-/- mice were immunized and boosted with the ECD of human IL-13Rα1 in the presence of adjuvant. Thereafter, mice were bled and their antisera were examined for their ability to bind plate-immobilized hIL-13Rα1 BCD (Figures 15A-B). Thereafter, the ability of the antisera to neutralize hIL- 13Rα1 -dependent receptor activation was assessed using IL- 13-stimulated A549 cells (Figure 15C). Serum from mouse #2 was capable of inhibiting IL-13-induced responses in vitro, suggesting the presence of neutralizing antibodies. Subsequently, bone marrow cells were obtained from mouse #2, antigen-specific B cells were sorted, and then genes of VH and VL antibody fragments were cloned as scFvs into a yeast display vector. Yeast display was applied to enrich hIL-13Rα1 BCD-specific clones. Following several enrichment steps, a chimeric antibody (i.e. human IgGl domains fused to mouse V-genes) that neutralizes hIL-13Rα1 termed '2HA6' was generated. 2HA6 was capable of binding hIL- 13Rα1 (apparent affinity = 0.67nM, by ELISA) but not mIL-13Rα1 or streptavidin (Figure 7A). 2HA6 dose-dependently inhibited IL-13-induced CCL26 secretion (Figure 7B) but not IL-4- or TNF-a-mediated secretion of CCL26 or IL-6, respectively. Thus, 2HA6 was capable of specifically suppressing IL-13 through the human type 2 IL-4R. Neutralizing antibodies towards receptors can block their activity by direct competition with their ligand on the ligand binding site, or by other means, such as preventing receptor dimerization. To determine whether 2HA6 directly competes with IL- 13 on its respective binding site, a competitive ELISA testing the ability of IL-13 and 2HA6 to bind the ECD of hIL-13Rα1 was conducted (Figure 7E). To this end, plates were coated with the ECD of hIL-13Rα1 ECD and IL-13 was added with no antibody treatment, with an isotype control antibody or with 2HA6.
Thereafter, an HRP-conjugated anti-IL-13 detection antibody was added followed by TMB and optical density absorbance was determined. As expected, in the absence of any competing antibody or in the presence of an isotype control Ab, IL-13 was capable of binding the hIL-13Rα1 ECD (Figure 7E). IL-13 was capable of binding the ECD of hIL-13Rα1 even in the presence of neutralizing concentrations of 2HA6 (Figure 7E). Collectively, these data suggest that 2HA6 neutralized IL-13 activity by a mechanism that does not involve direct competition with IL-13.
Information regarding the antibody sequence is summarized in Table 4, herein below.
Table 4
Figure imgf000048_0001
Figure imgf000049_0001
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
In addition, any priority documents) of this application is/are hereby incorporated herein by reference in its/their entirety.
REFERENCES
1. T. Bieber et al., Clinical phenotypes and endophenotypes of atopic dermatitis: Where are we, and where should we go? Journal of Allergy and Clinical Immunology 139, S58-S64 (2017).
2. M. Sudrez-Farinas et al, Intrinsic atopic dermatitis shows similar ΊΉ2 and higher TH17 immune activation compared with extrinsic atopic dermatitis. Journal of Allergy and Clinical Immunology 132, 361-370 (2013).
3. D. Karo-Atar, A. Bitton, I. Benhar, A. Munitz, Therapeutic Targeting of the Interleukin- 4/Interleukin-13 Signaling Pathway: In Allergy and Beyond. BioDrugs, (2018).
4. J. D. Hamilton, B. Ungar, E. Guttman-Yassky, Drug evaluation review: dupilumab in atopic dermatitis. Immunotherapy 7, 1043-1058 (2015).
5. A. Wollenberg et al., Treatment of atopic dermatitis with tralokinumab, an anti-IL-13 mAb. J Allergy Clin Immunol 143, 135-141 (2019).
6. E. L. Simpson et al., Two Phase 3 Trials of Dupilumab versus Placebo in Atopic Dermatitis. N Engl J Med 375, 2335-2348 (2016).
7. M. Shirley, Dupilumab: First Global Approval. Drugs 77, 1115-1121 (2017).
8. S. M. Zurawski et al., The primary binding subunit of the human interleukin-4 receptor is also a component of the interleukin- 13 receptor. The Journal of biological chemistry 270, 13869-13878 (1995).
9. T. A. Wynn, IL-13 effector functions. Annu Rev Immunol 21, 425-456 (2003).
10. J. A. Elias, C. G. Lee, T. Zheng, Y. Shim, Z. Zhu, Interleukin- 13 and leukotrienes: an intersection of pathogenetic schema. AmJ Respir Cell Mol Biol 28, 401-404 (2003).
11. A. N. McKenzie, G. Zurawski, Interleukin- 13: characterization and biologic properties. Cancer Treat Res 80, 367-378 (1995).
12. G. K. Hershey, IL-13 receptors and signaling pathways: an evolving web. J Allergy Clin Immunol 111, 677-690; quiz 691 (2003).
13. J. Mattes et al., IL-13 induces airways hyperreactivity independently of the IL-4R alpha chain in the allergic lung. J Immunol 167, 1683-1692 (2001).
14. M. O. Daines et al., Level of expression of IL-13R alpha 2 impacts receptor distribution and IL-13 signaling. J Immunol 176, 7495-7501 (2006).
15. M. M. Mentink-Kane et al., IL-13 receptor alpha 2 down-modulates granulomatous inflammation and prolongs host survival in schistosomiasis. Proc Natl Acad Sci USA 101, 586-590 (2004).
16. M. M. Mentink-Kane, T. A. Wynn, Opposing roles for IL-13 and IL-13 receptor alpha 2 in health and disease. Immunol Rev 202, 191-202 (2004).
17. S. Fichtner-Feigl et al., Induction of IL-13 triggers TGF-betal-dependent tissue fibrosis in chronic 2,4,6-trinitrobenzene sulfonic acid colitis. J Immunol 178, 5859-5870 (2007).
18. S. Fichtner-Feigl, W. Strober, K. Kawakami, R. K. Puri, A. Kitani, IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-betal production and fibrosis. Nat Med 12, 99-106 (2006).
19. C. A. Herrick, L. Xu, A. N. McKenzie, R. E. Tigelaar, K. Bottomly, IL-13 is necessary, not simply sufficient, for epicutaneously induced Th2 responses to soluble protein antigen. J Immunol 170, 2488-2495 (2003).
20. T. Zheng et al., Transgenic expression of interleukin- 13 in the skin induces a pruritic dermatitis and skin remodeling. J Invest Dermatol 129, 742-751 (2009).
21. A. Munitz, E. B. Brandt, M. Mingler, F. D. Finkelman, M. E. Rothenberg, Distinct roles for IL-13 and IL-4 via IL-13 receptor alphal and the type Π IL-4 receptor in asthma pathogenesis. Proc Natl Acad Sci USA 105, 7240-7245 (2008). 22. T. R. Ramalingam et al., Unique functions of the type Π interleukin 4 receptor identified in mice lacking the interleukin 13 receptor alphal chain. Nat Immunol 9, 25-33 (2008).
23. M. E. Rothenberg et al., IL-13 Receptor { alpha} 1 Differentially Regulates Aeroallergen- Induced Lung Responses. J Immunol 187, 4873-4880 (2011).
24. M. Q. Man et al., Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges. J Invest Dermatol 128, 79-86 (2008).
25. A. Wollenberg et al., Treatment of atopic dermatitis with tralokinumab, an anti-IL-13 mAb. J Allergy Clin Immunol, (2018).
26. T. Murata, S. R. Husain, H. Mohri, R. K. Puri, Two different IL-13 receptor chains are expressed in normal human skin fibroblasts, and IL-4 and IL-13 mediate signal transduction through a common pathway. Int Immunol 10, 1103-1110 (1998).
27. X. Y. Yuan et al., Topical application of aloperine improves 2,4-dinitrofiuorobenzene- induced atopic dermatitis-like skin lesions in NC/Nga mice. Eur J Pharmacol 658, 263- 269 (2011).
28. D. Y. Leung, M. Boguniewicz, M. D. Howell, I. Nomura, Q. A. Hamid, New insights into atopic dermatitis. J Clin Invest 113, 651-657 (2004).
29. S. T. Holgate et al., Asthma. Nat Rev Dis Primers 1, 15025 (2015).
30. E. Gros, C. Bussmann, T. Bieber, I. Forster, N. Novak, Expression of chemokines and chemokine receptors in lesional and nonlesional upper skin of patients with atopic dermatitis. J Allergy Clin Immunol 124, 753-760 e751 (2009).
31. M. Mohrs, K. Shinkai, K. Mohrs, R. M. Locksley, Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter. Immunity 15, 303-311 (2001).
32. H. E. Liang et al., Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity. Nat Immunol 13, 58-66 (2011).
33. J. Ohara, W. E. Paul, Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor- 1. Nature 315, 333-336 (1985).
34. A. Munitz, M. L. McBride, J. S. Bernstein, M. E. Rothenberg, A dual activation and inhibition role for the paired immunoglobulin-like receptor B in eosinophils. Blood 111, 5694-5703 (2008).
35. N. Gour, M. Wills-Karp, IL-4 and IL-13 signaling in allergic airway disease. Cytokine 75, 68-78 (2015).
36. C. Perkins, M. Wills-Karp, F. D. Finkelman, IL-4 induces IL- 13-independent allergic airway inflammation. J Allergy Clin Immunol 118, 410-419 (2006).
37. A. D. Keegan et al., Similarities and differences in signal transduction by interleukin 4 and interleukin 13: analysis of Janus kinase activation. Proc Natl Acad Sci USA 92, 7681-7685 (1995).
38. G. Grunig et al., Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282, 2261-2263 (1998).
39. W. Chen et al., IL-13 receptor alpha2 contributes to development of experimental allergic asthma. J Allergy Clin Immunol 132, 951-958 e951-956 (2013).
40. C. H. He et al., Chitinase 3-like 1 regulates cellular and tissue responses via IL-13 receptor alpha2. Cell Rep 4, 830-841 (2013).
41. J. C. Hoving et al., Interleukin 13-mediated colitis in the absence of IL-4Ralpha signalling. Gut 66, 2037-2039 (2017).
42. S. Fichtner-Feigl et al., IL-13 signaling via IL-13R alpha2 induces major downstream fibrogenic factors mediating fibrosis in chronic TNBS colitis. Gastroenterology 135, 2003-2013, 2013 e2001-2007 (2008).
43. D. Karo-Atar et al., A protective role for IL-13 receptor alpha 1 in bleomycin-induced pulmonary injury and repair. Mucosal Immunol 9, 240-253 (2016). 44. E. L. Simpson etal., Efficacy and safety of lebrikizumab (an anti-IL-13 monoclonal antibody) in adults with moderate-to-severe atopic dermatitis inadequately controlled by topical corticosteroids: A randomized, placebo-controlled phase Π trial (TREBLE). J Am Acad Dermatol78, 863-871 e811 (2018).
45. S. M. Pope et al, Identification of a cooperative mechanism involving interleukin- 13 and eotaxin-2 in experimental allergic lung inflammation. JBiol Chem 280, 13952-13961 (2005).
46. I. S. Junttila et al, Tuning sensitivity to IL-4 and IL-13: differential expression of IL- 4Ralpha, IL-13Ralphal, and gammac regulates relative cytokine sensitivity. J Exp Med 205, 2595-2608 (2008).
47. T. Katakura, M. Miyazaki, M. Kobayashi, D. N. Herndon, F. Suzuki, CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. J Immunol 172, 1407-1413 (2004).
48. N. Makita, Y. Hizukuri, K. Yamashiro, M. Murakawa, Y. Hayashi, IL-10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration. Int Immunol 27, 131-141 (2015).
49. J. E. Frampton, H. A. Blair, Dupilumab: A Review in Moderate-to-Severe Atopic Dermatitis. Am J Clin Dermatol 19, 617-624 (2018).
50. S. Horie et al, Interleukin- 13 but not interleukin-4 prolongs eosinophil survival and induces eosinophil chemotaxis. Intern Med 36, 179-185 (1997).
51. W. Luttmann et al, Activation of human eosinophils by IL-13. Induction of CD69 surface antigen, its relationship to messenger RNA expression, and promotion of cellular viability. J Immunol 157, 1678-1683 (1996).

Claims

WHAT IS CLAIMED IS:
1. An isolated polypeptide comprising a binding domain which binds specifically to human Interleukin 13 receptor, alpha 1 (IL-13Rα1), wherein the polypeptide downregulates an activity of Interleukin 13 (IL-13), said polypeptide comprising each of the following amino acid sequences:
(i) SEQ ID NO: 28 or SEQ ID NO: 34;
(ii) SEQ ID NO: 29 or SEQ ID NO: 35; and
(iii) SEQ ID NO: 30 or SEQ ID NO: 36.
2. The isolated polypeptide of claim 1, wherein said activity comprises induction of
CCL26 secretion.
3. The isolated polypeptide of claims 1 or 2, being an antibody.
4. The isolated polypeptide of claim 3, being an antibody having an antigen recognition domain which comprises the CDR sequences of an antibody having a heavy chain as set forth in SEQ ID NO: 55, wherein said CDR sequences are in the same orientation as the antibody having said heavy chain as set forth in SEQ ID NO: 55.
5. The isolate polypeptide of claim 4, further comprising a light chain.
6. The isolated polypeptide of claim 4, wherein CDR1 of a heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 28, CDR2 of said heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 29, and CDR3 of said heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 30.
7. The isolated polypeptide of claim 4, wherein the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 55.
8. The isolated polypeptide of claims 5 or 6, wherein the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 56.
9. The isolated polypeptide of any one of claims 1-8, having an affinity between 0. InM -lOnM for said human IL-13Rα1 as measured by ELISA.
10. The isolated polypeptide of claim 3, being an antibody, wherein said antibody is selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE antibody.
11. The isolated polypeptide of claim 3, being an antibody, wherein said antibody is a bi-specific antibody.
12. The isolated polypeptide of claim 11, wherein the first target of said bi-specific antibody is human IL-13Rα1, and the second target of said bi-specific antibody is human IL-4.
13. A pharmaceutical composition comprising the isolated polypeptide of any one of claims 1-12 as the active agent and a pharmaceutically acceptable carrier.
14. A method of treating a disease mediated by IL-13 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated polypeptide of any one of claims 1-12, thereby treating the disease.
15. The isolated polypeptide of any one of claims 1-12, for use in treating a disease mediated by IL-13.
16. The method or isolated polypeptide of claims 14 or 15, wherein said disease mediated by IL-13 is selected from the group consisting of atopic dermatitis, asthma, eosinophilia, urticaria, and allergic rhinitis fibrotic diseases, COPD and cancer.
17. The method or isolated polypeptide of claim 16, wherein said disease is atopic dermatitis.
18. The method of claim 14, further comprising administering to the subject an agent which downregulates the amount and/or activity of IL-4.
19. The method of claim 18, wherein said agent is an antibody directed against said IL-
4.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003080675A2 (en) * 2002-03-22 2003-10-02 Amrad Operations Pty Ltd MONOCLONAL ANTIBODY AGAINST INTERLEUKIN-13 RECEPTOR ALPHA 1 (IL-13Rα1)
WO2008060813A2 (en) * 2006-10-19 2008-05-22 Merck & Co., Inc. High affinity antibody antagonists of interleukin-13 receptor alpha 1
WO2017191627A1 (en) * 2016-05-02 2017-11-09 Ramot At Tel-Aviv University Ltd. Antibodies to interleukin 13 receptor alpha-1 (il-13r alpha 1) and uses thereof
WO2018011405A1 (en) * 2016-07-15 2018-01-18 Universität Zürich Il-13ralpha1 antibodies for use in treatment of atopic inflammation, sepsis and neutropenia

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003080675A2 (en) * 2002-03-22 2003-10-02 Amrad Operations Pty Ltd MONOCLONAL ANTIBODY AGAINST INTERLEUKIN-13 RECEPTOR ALPHA 1 (IL-13Rα1)
WO2008060813A2 (en) * 2006-10-19 2008-05-22 Merck & Co., Inc. High affinity antibody antagonists of interleukin-13 receptor alpha 1
WO2017191627A1 (en) * 2016-05-02 2017-11-09 Ramot At Tel-Aviv University Ltd. Antibodies to interleukin 13 receptor alpha-1 (il-13r alpha 1) and uses thereof
WO2018011405A1 (en) * 2016-07-15 2018-01-18 Universität Zürich Il-13ralpha1 antibodies for use in treatment of atopic inflammation, sepsis and neutropenia

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BITTON ALMOG, AVLAS SHMUEL, REICHMAN HADAR, ITAN MICHAL, KARO-ATAR DANIELLE, AZOUZ NURIT P., ROZENBERG PERRI, DIESENDRUCK YAEL, NA: "A key role for IL -13 signaling via the type 2 IL -4 receptor in experimental atopic dermatitis", SCIENCE IMMUNOLOGY, vol. 5, no. 44, 14 February 2020 (2020-02-14), pages eaaw2938, XP055834011, DOI: 10.1126/sciimmunol.aaw2938 *

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