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WO2025114614A1 - Anticorps anti-récepteurs alpha de l'interleukine-4 canins (il-4rα) et leurs utilisations - Google Patents

Anticorps anti-récepteurs alpha de l'interleukine-4 canins (il-4rα) et leurs utilisations Download PDF

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WO2025114614A1
WO2025114614A1 PCT/EP2024/084368 EP2024084368W WO2025114614A1 WO 2025114614 A1 WO2025114614 A1 WO 2025114614A1 EP 2024084368 W EP2024084368 W EP 2024084368W WO 2025114614 A1 WO2025114614 A1 WO 2025114614A1
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canine
antibody
seq
antibodies
antigen
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Olivier Leger
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Vetoquinol SA
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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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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 is in the field of therapeutic antibodies and especially anti- canine interleukine-4-receptor alpha (IL-4Ro) monoclonal antibody.
  • the present invention relates to anti-canine IL-4Ro monoclonal antibodies of high potency regarding inhibition of IL-4Ro signaling pathway.
  • the present invention thus also relates to the use of such antibodies for treating and/or preventing itch and/or inflammatory skin due to atopic dermatitis and allergies in dogs and in particular for treating canine atopic dermatitis.
  • atopic dermatitis The pathogenesis of atopic dermatitis is however quite complex. It is likely that a defective skin barrier allows microbial adherence, penetration of allergenic proteins, and initiation of abnormal inflammatory and allergic responses.
  • the immune response in dogs with atopic dermatitis, as in human is dominated by TH2 cells and involves cytokines such as IL-4, IL-5, IL-6, IL-13, and IL-31 (Marsella et al., 2012; Olivry et al., 2016), whereas development of chronic inflammation involves a mix of TH1 , TH2, TH17, and TH22-cell mediators (Olivry et al., 2016).
  • a caninized anti -canine IL-31 mAb has been developed to neutralize the effects of canine IL-31 for inducing pruritus in various ecies, including rodents, dogs, and non-human primates.
  • the anticanine IL-31 mAb has a limited anti-inflammatory effect on AD skin lesions and inflammation compared to existing therapeutic options like steroids, JAK-inhibitor or cyclosporin (Tamamoto-Mochizuki et al., 2019).
  • Dupilumab (corresponding to Dupi-H2L2 in W02017/102920) is a fully human monoclonal antibody directed against the human IL-4 receptor a subunit that blocks the signalling of IL-4 and IL-13, both key cytokines in Th2-mediated pathways, in humans.
  • dupilumab has shown a favourable safety profile with no dose- limiting toxicity.
  • the robust effects of dupilumab on skin inflammation and pruritus confirm the pathogenic role of IL-4 and IL-13 signalling in adult atopic dermatitis, and further support the application of Th2 cytokine antagonists in the treatment of this disease (Hamilton et al., 2015).
  • TH2-cell mediators development of chronic inflammation in canine atopic dermatitis, involves, like human counterpart, a mix of TH1 , TH2, TH17, and TH22-cell mediators (Olivry et al., 2016).
  • dupilumab directly down-regulates Th2-related markers, it also resulted in down-regulations of some Th17/Th22- related markers, proving its effects beyond narrow IL-4/IL-13 inhibition (Renert-Yuval and Guttman-Yassky, 2019).
  • Dupilumab is however not suitable for use in dogs in view of its fully human nature, besides it was generated against human IL-4Ro and does not cross-react well with canine IL-4Ro.
  • W02016/156588 is disclosing murine anti-canine interleukin-4 receptor alpha (IL-4Ro) antibodies that have a high binding affinity for canine IL-4Ro.
  • the 4 best antibodies for inhibiting binding of clL-4 to clL-4Ro expressed on CHO cells are the 4H3, 4D8, 2E2 and 11 H2 antibodies.
  • Such antibodies are announced to have the ability to inhibit the signaling from both canine IL-4 and IL-13. However, no data have been shown to demonstrate such ability.
  • W02021 /123089 and W02021 /123091 each disclose one murine anticanine interleukin-4 receptor alpha (IL-4Ro) antibody (146E2 in W02021 /123089 and 152H11 in W02021 /123091 ) as well as caninized versions thereof (c146E2-H3L3 and c152H11 -H3L3), and their comparison to a caninized version of the 4H3 antibody of W02016/156588 (c4H3) in their ability to inhibit IL-4 or IL-13 mediated STAT6 phosphorylation using an alphalisa assay.
  • IL-4Ro murine anticanine interleukin-4 receptor alpha
  • W02021 /188631 discloses several murine anti-canine interleukin-4 receptor alpha (IL-4Ro) antibodies (clone I, clone B, M3, M5, M8, and M9) and shown that they inhibit the binding of clL-4 to clL-4Ro (clone I, clone B) or inhibit STAT6 phosphorylation following binding of clL-4 to clL-4Ro (M3, M8 and M9 mouse antibodies).
  • IL-4Ro murine anti-canine interleukin-4 receptor alpha
  • IL-4Ro new anti-canine interleukine-4-receptor alpha
  • the anti-canine IL-4Ro monoclonal antibodies of the present invention have a significantly higher potency than those disclosed in prior art.
  • C146E2-H3L3 was found to be the more potent to inhibit the signaling from both canine IL-4 and IL-13.
  • the antibodies according to the invention were found to be about 5 times more potent than C146E2-H3L3.
  • the present invention thus relates to an anti-canine IL-4Ro antibody, in which heavy and light chains respectively comprises CDR-H and CDR-L with the following amino acid sequences:
  • CDR1 -H-27E1 a heavy chain comprising three CDR-H (heavy chain CDR) with the following amino acid sequences CDR1 -H-27E1 : SEQ ID NO: 1 (SYGMG), CDR2-H-27E1 : SEQ ID NO: 2 (VINPAVSGSRQGYAPAVKG), and CDR3-H-27E1 : SEQ ID NO: 3 (HASX 4 YWRGAGKIDA where X 4 is N or T), and
  • CDR1 -L-27E1 a light chain comprising three CDR-L (light chain CDR) with the following amino acid sequences CDR1 -L-27E1 : SEQ ID NO: 4 (SGGSGNDYG), CDR2-L-27E1 : SEQ ID NO: 5 (DNDKRPS), and CDR3-L-27E1 : SEQ ID NO: 6 (GGYDRHTYDA).
  • the present invention also relates to a vector comprising the nucleic acid(s) according to the invention.
  • Figure 3 represents concentration-dependent canine IL-4 STAT6 nluc reporter activity in MDCK clone #13.
  • Figure 6 represents titration curves from the MDCK cell-based assay with canine IL-4-induced STAT6-nluc reporter activation for antibody 27E1 .
  • Figure 7 represents titration curves from the MDCK cell-based assay with canine IL-13-induced STAT6-nluc reporter activation for antibody 27E1 .
  • Figure 8 represents titration curves from the MDCK cell-based assay with canine IL-4-induced STAT6-nluc reporter activation for Fab fragments of antibody 27E1 .
  • Figure 11 represents titration curves from the MDCK cell-based assay with canine IL-4-induced STAT6-Luc2P reporter activation for prior art antibodies 152H11 , 146E2, Clone B, 4H3, Clone I, M3, M8, M9 and Dupilumab.
  • Figure 12 represents titration curves from the MDCK cell-based assay with canine IL-13-induced STAT6-Luc2P reporter activation for prior art antibodies 152H11 , 146E2, Clone B, 4H3, Clone I, M3, M8, M9 and Dupilumab.
  • Figure 13 represents titration curves from the AlphaLISA p-STAT-6 Assay with canine IL-4 induction (2 ng/mL) for CAN-27E1 -338-VHN/329-VLA compared to the prior art antibodies 152H11 and 146E2.
  • Figure 15 represents histological staining of RCE stimulated with the pro- inflammatory cytokines (positive control).
  • Figure 16 represents histological staining of RCE-AD treated with the monoclonal antibody targeting the IL-4R CAN-27E1 -338-VHN/329-VLA.
  • Figure 17 represents histological staining of RCE-AD treated with the reference molecule oclacitinib.
  • Figure 18 represents ELISA quantification of IL-8 for evaluating inflammation in the RCE-AD model with increasing concentrations of the monoclonal antibody targeting the IL-4R CAN-27E1 -338-VHN/329-VL, compared to the reference molecule and controls.
  • Figure 19 represents titration curves from the MDCK cell-based assay with canine IL-4-induced STAT6-Luc2P reporter activation for 27E1 -mono-1 compared to CAN-27E1- 338-VHN/329-VLA.
  • Figure 20 represents titration curves from the MDCK cell-based assay with canine IL-4-induced STAT6-Luc2P reporter activation for 27E1 -mono-2 compared to CAN-27E1 - 338-VHN/329-VLA.
  • Figure 21 represents titration curves from the MDCK cell-based assay with canine IL-13-induced STAT6-Luc2P reporter activation for 27E1 -mono-1 compared to CAN-27E1 - 338-VHN/329-VLA.
  • Figure 22 represents titration curves from the MDCK cell-based assay with canine IL-13-induced STAT6-Luc2P reporter activation for 27E1 -mono-2 compared to CAN-27E1 - 338-VHN/329-VLA.
  • IL-4Ro new anti-canine interleukine-4-receptor alpha
  • the anti-canine IL-4Ro monoclonal antibodies of the present invention have a significantly higher potency than those disclosed in prior art.
  • C146E2-H3L3 was found to be the more potent to inhibit the signaling from both canine IL-4 and IL-13.
  • the antibodies according to the invention were found to be about 5 times more potent than C146E2-H3L3.
  • the present invention first relates to an anti-canine IL-4Ro antibody, in which heavy and light chains respectively comprises CDR-H and CDR-L with the following amino acid sequences:
  • CDR1 -H-27E1 a heavy chain comprising three CDR-H (heavy chain CDR) with the following amino acid sequences CDR1 -H-27E1 : SEQ ID NO: 1 (SYGMG), CDR2-H-27E1 : SEQ ID NO: 2 (VINPAVSGSRQGYAPAVKG), and CDR3- H-27E1 : SEQ ID NO: 3 (HASX 4 YWRGAGKIDA where X 4 is N or T), and
  • CDR1 -L-27E1 a light chain comprising three CDR-L (light chain CDR) with the following amino acid sequences CDR1 -L-27E1 : SEQ ID NO: 4 (SGGSGNDYG), CDR2-L-27E1 : SEQ ID NO: 5 (DNDKRPS), and CDR3-L-27E1 : SEQ ID NO: 6 (GGYDRHTYDA).
  • canine may also be referred to as a “dog”.
  • Canines can be categorized as belonging to the subspecies with the trinomial name Canis lupus familiaris (Canis familiaris domesticus) or Canis lupus dingo.
  • Canines include any species of dog Canis sp. and includes both feral and pet varieties, the latter also being referred to as companion animals.
  • antibody or “immunoglobulin” means a glycoprotein that specifically binds to another molecule referred to as its “antigen”.
  • An antibody is generally composed of two types of glycopeptide chains called “heavy chain” and “light chain”, an antibody being made up of two heavy chains and two light chains, bound by disulfide bridges. Each chain is made up of a “variable region” and a “constant region”. The constant region of a particular isotype of heavy or light chain is normally identical from one antibody to another of the same isotype, excluding somatic mutations. In return, the variable region (referred to as “VH” for the heavy chain variable region and “VL” for the light chain variable region) varies from one antibody to another.
  • genes coding for antibody heavy chains and light chains are generated by recombination of, respectively, three and two segments of distinct genes called VH, DH and JH-CH for the heavy chain and VL and JL-CL for the light chain.
  • the CH and CL segments do not participate in recombination and form the constant regions of the heavy and light chains respectively.
  • Recombinations of the VH-DH-JH and VL-JL segments form the variable regions of heavy and light chains, respectively.
  • the VH and VL regions have three hypervariable zones or complementarity determining regions (also known as “complementarity determining region” or “CDR”) called CDR1 , CDR2 and CDR3, the CDR3 region being the most variable, since it is located at the recombination zone.
  • CDR complementarity determining region
  • CDR regions are found in the part of the antibody that will be in contact with the antigen and are therefore very important for antigen recognition.
  • antibodies maintaining the three CDR regions and each of the heavy and light chains of an antibody mostly keep the antigenic specificity of the original antibody.
  • an antibody only maintaining one of the CDRs, and particularly CDR3 also keeps the specificity of the original antibody.
  • the CDR1 , CDR2 and CDR3 regions are each preceded by FR1 , FR2 and FR3 regions, respectively, corresponding to “framework regions” (“FR”) which vary from one VH or VL segment to another.
  • FR framework regions
  • the CDR3 region is also followed by a framework region FR4.
  • the CDRs of an antibody are defined from the amino acid sequence of its heavy and light chains compared to criteria known to the skilled person.
  • Various methods for determining CDRs have been proposed, and the portion of the amino acid sequence from a heavy or light chain variable region of an antibody defined as a CDR varies depending on the method chosen.
  • the first determination method is the one proposed by Kabat et al. (Kabat et al. Sequences of proteins of immunological interest, 5 th Ed., U.S. Department of Health and Human Services, NIH, 1991 , and later editions). In this method, CDRs are defined based on sequence variability. Another method was proposed by Chothia et al., 1987. In this method, CDRs are defined based on the location of the structural loop regions.
  • This numbering provides a standardized definition of framework regions ((FR1 -IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and complementarity determining regions (CDR1 -IMGT: positions 27 to 38, CDR2-IMGT: positions 56 to 65 and CDR3-IMGT: positions 105 to 117).
  • framework regions (FR1 -IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and complementarity determining regions (CDR1 -IMGT: positions 27 to 38, CDR2-IMGT: positions 56 to 65 and CDR3-IMGT: positions 105 to 117).
  • CDR sequences are defined according to the Kabat nomenclature.
  • CDRs have been determined by using either IgBLAST, a sequence analysis tool for antibody variable domain sequences, developed by NCBI and freely accessible at https://www.ncbi.nlm.nih.gov/igblast or the program AbNum (antibody numbering) from professor’s Andrew C.R. Martin group at UCL website; http://www.bioinf.org.uk/abs/abnum/, which lead to exactly the same CDR sequences.
  • IL-4 receptor a (abbreviated as “IL-4Ra”) is a typical representative of the class I cytokine receptor (CKR) family, which consists of single-pass transmembrane proteins that are non-covalently bound by cytoplasmic Janus kinases (JAKs) that contribute the enzymatic activity for signal transduction (Boulay et al., 2003; Nelms et al., 1999). Most class I CKRs form heterodimers (Weidemann et al., 2007). Formation of the active CKR heterodimers occurs in a two-step process, whereby the cytokine ligand first binds to one of the subunits that exhibits a higher ligand affinity.
  • CKR cytoplasmic Janus kinases
  • the second receptor chain is recruited into the complex by the occupied subunit (Whitty and Riera, 2008). Formation of these heterodimeric CKR complexes then triggers crossactivation of the cytoplasmic JAKs that in turn transform the CKR tails into docking sites for various downstream signaling pathways including the STAT6 pathway and the insulin receptor substrate pathway (Leonard and O’Shea, 1998; Kelly-Welch et al., 2003).
  • the IL-4Ro chain can recruit the IL-2Ry chain (also called common y-chain), which is also used by the high-affinity receptors for IL-2, -7, -9, -15, and -21 , and is thus classified as a common y-chain-using receptor (Boulay et al., 2003).
  • This IL-4-induced heterodimer of IL-4Ro and IL-2Ry is referred to as type 1 IL-4R complex.
  • the IL- 4-bound IL-4Ro subunit can recruit the IL-13Ro1 chain to form a type 2 complex, which is also induced when IL-13Ro1 bound by IL-13 recruits IL-4Ro (Nelms et al., 1999).
  • type 1 IL-4R signaling is restricted to cells of hematopoietic origin, whereas type 2 signaling is more widely distributed (Murata et al., 1998).
  • the antibodies according to the invention bind to canine IL-4Ro, and do not bind with significant affinity to canine antigens other than canine IL-4Ro.
  • the antibodies according to the invention may however bind to some orthologs of canine IL-4Ro.
  • the antibodies according to the invention preferably do not bind with significant affinity to human IL-4Ro.
  • the amino acid sequence of human IL-4Ro is shown in NCBI reference sequence NM_000418.4, as follows (SEQ ID NO: 9):
  • binding refers to an interaction between molecules to form a complex which, under physiologic conditions, is relatively stable. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non- covalent bonds, interactions or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as IL-4Ro, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of association (k1 ) to dissociation (k-1 ) of an antibody to a monovalent antigen (k1 / k-1 ) is the association constant K, which is a measure of affinity.
  • K varies for different complexes of antibody and antigen and depends on both k1 and k-1 .
  • the association constant K for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art, including Surface Plasmon resonance (SPR) and biolayer interferometry (BLI) technologies.
  • SPR Surface Plasmon resonance
  • BLI biolayer interferometry
  • antibodies according to the invention bind to canine IL-4Ro with an affinity of at most 5x10E-11 M as measured using BLI technology (Octet K2 instrument).
  • Antibodies according to the invention also preferably do not show any measurable affinity to human IL-4Ro by using BLI technology (Octet K2 instrument).
  • Antibodies according to the invention have the ability to inhibit the signaling activated by both canine IL-4 and IL-13, preferably the STAT6-signaling activated by canine IL-4 and IL-13.
  • Binding of IL-4 to the IL-4Ro/IL-2Ryc heterodimer normally activates a signaling pathway dependent on STAT6 transcription factor, which homodimerizes and goes to the nucleus to activate M2 type macrophages gene expression.
  • Antibodies according to the invention have the ability to inhibit the STAT-6-signaling activated by the binding of canine IL-4 to canine IL-4Ro.
  • the ability to inhibit e STAT-6-signaling activated by the binding of canine IL-4 to canine IL-4Ro of the antibodies according to the invention is higher than that of the C146E2-H3L3 antibody disclosed in W02021 /123089 (with a light chain of amino acid sequence SEQ ID NO:39 of W02021 /123089 and a heavy chain of amino acid sequence SEQ ID NO:42 of W02021 /123089) in the same STAT6-signaling activation assay performed in the same conditions.
  • antibodies according to the invention further have the ability to inhibit the STAT-6-signaling activated by the binding of canine IL-13 to canine IL-13Ro1.
  • the ability to inhibit the STAT-6-signaling activated by the binding of canine IL-13 to canine IL-13Ro1 is at least equal to that of the C146E2-H3L3 antibody disclosed in W02021 /123089 in the same STAT6-signaling activation assay performed in the same conditions.
  • IL-13 receptor alpha 1 chain (abbreviated as “IL-13Ra1”) is a member of type- 1 cytokine receptors. IL-13Ro1 bound by IL-13 recruits IL-4Ro to form type-2 receptors.
  • the cDNA encoding canine IL-13Ro1 has been reported by Tang in 2001.
  • the canine IL- 13Ro1 cDNA has an open reading frame that encodes 405 amino acid residues (without signal peptide) shown in NCBI reference sequence XM_538150.5, which is as follows (with the natural signal peptide) (SEQ ID NO: 10):
  • Inhibition of signaling activated by canine IL-4 and IL-13, and in particular of STAT6-signaling activated by canine IL-4 and IL-13, may be measured by any method known in the art. Two particularly useful methods are: a) a cell-based assay using as reporter canine cells endogenously expressing signaling pathway components required for evaluation of STAT6-signaling after IL-4Ro/IL-2Ryc (type 1 ) or IL-4Ro/IL-13Ro1 (type 2) receptor activation.
  • the reporter cells are preferably canine cells (in particular MDCK cells available in ECACC and ATCC) expressing, preferably stably, a reporter system for measuring STAT6 phosphorylation and/or activation of STAT6 transcription factors.
  • the reporter system may notably be selected from a gene encoding a detectable molecule (e.g. a bioluminescent protein such as luciferase; a fluorescent protein such as GFP...) under the control of a STAT6-regulated promoter.
  • a detectable molecule e.g. a bioluminescent protein such as luciferase; a fluorescent protein such as GFP
  • one or more STAT6 binding consensus sequences may be inserted just upstream of a minimal promoter.
  • a sandwich immunoassay for quantitative detection of phospho-STAT6 (phosphorylated on Tyr641 ) in cellular lysates referred to as an alphaLISA assay) using:
  • Acceptor beads coated with an agent that binds to the tag of and immobilizes the tagged anti-phospho-STAT6 antibody wherein the acceptor beads contain a fluorophore that emits light when excited by energy transferred by singlet oxygen (such as an Europium chelate, which emits light in a narrow peak at 615 nm; or rubrene, which emits light between 520 and 620 nm, preferably an Europium chelate).
  • a fluorophore that emits light when excited by energy transferred by singlet oxygen (such as an Europium chelate, which emits light in a narrow peak at 615 nm; or rubrene, which emits light between 520 and 620 nm, preferably an Europium chelate).
  • donor beads contain phthalocyanine, which converts ambient oxygen to singlet oxygen upon illumination at 680 nm, as a photosensitizer and acceptor beads contain an Europium chelate, which emits light in a narrow peak at 615 nm, as a fluorophore emitting light when excited by energy transferred by singlet oxygen.
  • the anti -canine IL-4Ra antibodies according to the invention have been shown to have a huge potency for blocking signaling mediated by IL-4 and IL-13.
  • Several caninized variants of an initial chicken antibody exhibit these advantageous properties, and also show ability to improve symptoms associated with IL-4 and/or IL-13 mediated disorders and diseases, especially those associated with atopic dermatitis in dogs.
  • the anti-canine IL-4Ro antibodies according to the invention have heavy and light chains respectively comprising CDR-H and CDR-L with the following amino acid sequences:
  • CDR1 -H-27E1 a heavy chain comprising three CDR-H (heavy chain CDR) with the following amino acid sequences CDR1 -H-27E1 : SEQ ID NO: 1 (SYGMG), CDR2-H-27E1 : SEQ ID NO: 2 (VINPAVSGSRQGYAPAVKG), and CDR3- H-27E1 : SEQ ID NO: 3 (HASX 4 YWRGAGKIDA where X 4 is N or T), and
  • CDR1 -L-27E1 a light chain comprising three CDR-L (light chain CDR) with the following amino acid sequences CDR1 -L-27E1 : SEQ ID NO: 4 (SGGSGNDYG), CDR2-L-27E1 : SEQ ID NO: 5 (DNDKRPS), and CDR3-L-27E1 : SEQ ID NO: 6 (GGYDRHTYDA).
  • caninized anti-canine IL-4Ro antibodies with these 6 CDRs have been shown by the inventors to have particularly potent ability to inhibit the STAT6-signaling activated by canine IL-4 and IL-13.
  • anti-canine IL-4Ro antibodies have heavy and light chains respectively comprising CDR-H and CDR-L with the following amino acid sequences:
  • CDR1 -H-27E1 a heavy chain comprising three CDR-H (heavy chain CDR) with the following amino acid sequences CDR1 -H-27E1 : SEQ ID NO: 1 (SYGMG), CDR2-H-27E1 : SEQ ID NO: 2 (VINPAVSGSRQGYAPAVKG), and CDR3- H-27E1 : SEQ ID NO: 11 (HASTYWRGAGKIDA), and
  • CDR1 -L-27E1 a light chain comprising three CDR-L (light chain CDR) with the following amino acid sequences CDR1 -L-27E1 : SEQ ID NO: 4 (SGGSGNDYG), CDR2-L-27E1 : SEQ ID NO: 5 (DNDKRPS), and CDR3-L-27E1 : SEQ ID NO: 6 (GGYDRHTYDA).
  • IL-4Ro antibodies have heavy and light chains respectively comprising CDR-H and CDR-L with the following amino acid sequences:
  • CDR1 -H-27E1 a heavy chain comprising three CDR-H (heavy chain CDR) with the following amino acid sequences CDR1 -H-27E1 : SEQ ID NO: 1 (SYGMG), CDR2-H-27E1 : SEQ ID NO: 2 (VINPAVSGSRQGYAPAVKG), and CDR3-H-27E1 : SEQ ID NO: 12 (HASNYWRGAGKIDA), and - a light chain comprising three CDR-L (light chain CDR) with the following amino acid sequences CDR1 -L-27E1 : SEQ ID NO: 4 (SGGSGNDYG), CDR2-L-27E1 : SEQ ID NO: 5 (DNDKRPS), and CDR3-L-27E1 : SEQ ID NO: 6 (GGYDRHTYDA).
  • VH heavy chain variable regions
  • the CDR-H sequences mentioned above are preferably integrated in the following VH framework regions:
  • VH FR1 EVQLVESGGDLVKPGGTLRLSCVASGFTFS (SEQ ID NO: 13);
  • VH FR2 WVRQSPGKGLQWVA (SEQ ID NO: 14);
  • VH FR3 RATISRDDAKNTLYLQLNSLRAEDTAVYYCAK (SEQ ID NO: 15);
  • VH FR4 WGQGTLVTVSS (SEQ ID NO: 16).
  • Anti-canine IL-4Ro antibodies according to the invention preferably have a heavy chain comprising a variable region of sequence: EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASX104YWRGAGKIDAWGQGTLVTVSS, where X104 is N or T (SEQ ID NO: 17).
  • anti-canine IL-4Ro antibodies have a heavy chain comprising a variable region with of sequence: EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSS (SEQ ID NO:
  • IL-4Ro antibodies have a heavy chain comprising a variable region with of sequence: EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASNYWRGAGKIDAWGQGTLVTVSS (SEQ ID NO:
  • VL light chain variable regions
  • the CDR-L sequences mentioned above are preferably integrated in the following VL framework regions:
  • VL FR1 SSVLTQPPSVSVSLGQTATITC (SEQ ID NO: 20);
  • VL FR2 WX2QQKSPGQAPMTVIY (SEQ ID NO: 21 ), where X 2 is F (WFQQKSPGQAPMTVIY (SEQ ID NO: 22) or where X 2 is is Y (WYQQKSPGQAPMTVIY (SEQ ID NO: 23), and preferably X 2 is F;
  • VL FR3 GIPDRFSGSX10SGSTX15TLTISGAQAEDEAEYFC (SEQ ID NO: 24), where X10 is G or S and X15 is S or H, thus VL FR3 may be of sequence where X10 is S and X15 is H (GIPDRFSGSSSGSTHTLTISGAQAEDEAEYFC (SEQ ID NO: 25)), but preferably VL FR3 is where X10 is G and Xi 5 is S (GIPDRFSGSGSGSTSTLTISGAQAEDEAEYFC (SEQ ID NO: 26)); and VL FR4: FGSGTQLTVL
  • the CDR-L sequences mentioned above are integrated in the VL framework regions FR1 , FR2, FR3 and FR4 with SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 27, respectively.
  • Anti-canine IL-4Ro antibodies according to the invention preferably have a light chain comprises a variable region of sequence:
  • anti-canine IL-4Ro antibodies according to the invention have a light chain comprises a variable region of sequence:
  • IL-4Ro antibodies have a light chain comprises a variable region of sequence:
  • VH and VL are Preferred combinations of heavy and light chains variable regions
  • anti-canine IL-4Ro antibodies comprise:
  • a heavy chain comprising a variable region with a sequence (SEQ ID NO: 19) EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASNYWRGAGKIDAWGQGTLVTVSS and a light chain comprising a variable region with a sequence (SEQ ID NO: 29) SSVLTQPPSVSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL, or
  • a heavy chain comprising a variable region with a sequence (SEQ ID NO: 19) EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASNYWRGAGKIDAWGQGTLVTVSS and a light chain comprising a variable region with a sequence (SEQ ID NO: 30) SSVLTQPPSVSVSVSLGQTATITCSGGSGNDYGWYQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSSSGSTH TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL, or
  • a heavy chain comprising a variable region with a sequence (SEQ ID NO: 18) EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSS, and a light chain comprising a variable region with a sequence (SEQ ID NO: 29) SSVLTQPPSVSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL, or
  • a heavy chain comprising a variable region with a sequence (SEQ ID NO: 18) EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSS, and a light chain comprising a variable region with a sequence (SEQ ID NO: 30) SSVLTQPPSVSVSVSLGQTATITCSGGSGNDYGWYQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSSSGSTH TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL.
  • anti-canine IL-4Ro antibodies comprise:
  • a heavy chain comprising a variable region with a sequence (SEQ ID NO: 18) EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSS, and
  • the anti-canine IL-4Ro antibodies of the present invention are preferably caninized antibodies.
  • the above-described VH and VL amino acid sequences are caninized sequences.
  • a “caninized” antibody means an antibody that contains CDRs derived from an antibody of non-canine origin, the other parts of the antibody molecule being derived from one (or more) canine antibodies.
  • Caninized antibodies may be prepared using a similar approach as the well-known techniques described for humanization and offer the advantage of reduced immunogenicity when administered as therapeutics to dogs. Procedures for the production of humanized monoclonal antibodies include those described in Riechmann et al., 1988, Liu et al., 1987, Larrick et al., 1989, and Winter and Harris, 1993.
  • Caninized antibodies according to the invention may be prepared from techniques known to the skilled person.
  • Antibodies were caninized by grafting the three CDRs, as defined by the Kabat nomenclature, from the light chain variable region (VL) into a canine germline VL with a sequence as-homologous-as-possible to the one of the parental antibody VL.
  • the three CDRs from the heavy chain variable region (VH) were grafted into a canine germline VH with a sequence as-homologous-as-possible to the parental antibody VH.
  • germline sequence refers to a sequence of unrearranged immunoglobulin DNA sequences.
  • the source of unrearranged immunoglobulin sequences used for the invention is the IMGT database (Giudicelli et al Nucl. Acids Res., 2005; http://www.imgt.org).
  • a few amino acid residues in the canine framework regions of the selected canine germline variable regions may be changed to the amino acid residues that were present in the parental variable regions (so called back-mutations intended to maintain high affinity to the antigen).
  • canine framework refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the CDR residues as defined by the Kabat nomenclature.
  • the added-value of combining a structural model with pure sequence analysis is the potential to discriminate between paratope-facing and non-paratopic residues in the CDR regions.
  • the purpose of the structural model is to permit expanding the limits of the caninization process, taking it beyond mere CDR-grafting.
  • the structural models permit making more intelligent choices regarding back-mutations in light of the particular germlines involved.
  • the Kabat CDR definitions are not as strictly structural as those of other systems; thus, for some germlines the Kabat definitions are too broad.
  • the light chain CDR3 is usually well-described with high probability.
  • the difficult case is invariably CDR3 of the heavy chain, but the inventors have shown that antibodies with the CDR3 of the heavy chain defined above have particularly high potency.
  • the constant regions of antibodies according to the invention are preferably canine constant regions.
  • the anti-canine IL-4Ro antibodies of the present invention may be of several canine isotypes, according to the nature of their constant region and which correspond to the canine immunoglobulins IgG, IgA, IgM, IgE and IgD.
  • the anti-canine IL-4Ro antibody according to the present invention is of canine isotype IgG, and more preferably of canine isotype IgGB.
  • canine there are four IgG heavy chains referred to as A, B, C and D. These heavy chains represent four different subclasses of dog IgG, which are referred to as IgGA, IgGB, IgGC and IgGD.
  • Each IgG heavy chain consist of one variable domain (VH) and three constant domains referred to as CH1 , CH2 and CH3.
  • the CH1 domain is connected to the CH2 domain via an amino acid sequence referred to as the “hinge” or alternatively as the “hinge region”.
  • the DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. 2001 .
  • the amino acid and DNA sequences for these heavy chains are also available from the GenBank data bases.
  • the amino acid sequence of IgGA heavy chain has the gene accession number AF354264.1
  • IgGB has accession number AF354265.1
  • IgGC has accession number AF354266.1
  • IgGD has accession number AF354267.1 .
  • Canine antibodies also contain two types of light chains, kappa and lambda. The amino acid sequence of these light chains can be obtained from UniProtKB or IMGT databases.
  • the heavy chain of the anti -canine IL-4Ro antibody comprises a wild type canine IgGB constant region (SEQ ID NO: 31 ): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG
  • SEQ ID NO: 31 ASTTAPSVFPLAPSCGSTSGST
  • its heavy chain’s constant region may particularly comprise mutations for reducing its affinity for the Fey receptor(s) or complement protein(s) to which its isotype binds.
  • any antibody IgG isotype can be used in which the Fc portion is modified (e.g., by introducing 1 , 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to Fc receptors (see, e.g., WO 2003/101485, the disclosure of which is herein incorporated by reference).
  • Assays such as cell-based assays, to assess Fc receptor binding are well known in the art, and are described in, e.g. WO 2003/101485.
  • Fc region of canine IgGB region may influence binding to Fey receptor(s) (e.g., any one or more of CD16A, CD16B, CD32A, CD32B and/or CD64) or complement protein(s) (e.g., C1q) and result in “Fc silent” antibodies that have minimal interaction with effector cells or complement protein(s).
  • Fey receptor(s) e.g., any one or more of CD16A, CD16B, CD32A, CD32B and/or CD64
  • complement protein(s) e.g., C1q
  • the preferred mutants are in the CH2 domain of canine IgGB (SEQ ID NO: 31) heavy chain’s constant regions and comprise at least one of the following mutations (numbering of the positions being according to Eu numbering nomenclature (Edelman et al. 1969):
  • N297G or N297A which destroys the N -glycosylation site NST in the Fc, resulting in a non-glycosylated (aglycosylated) antibody with reduced ADCP and ADCC activities, or - any combination thereof.
  • preferred mutant canine IgGB heavy chain’s constant regions may contain:
  • K228P-M234A-L235A-P329G in order to improve manufacturability (K228P), reduce binding to Fc gamma receptors and avoid ADCP and ADCC (mutations M234A-L235A) and reduce binding of C1q complement protein (P329G), such as the sequence (SEQ ID NO: 34): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPPCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQ
  • N297G-P329G for having a non-glycosylated (aglycosylated) Fc with reduced ADCP and ADCC activities (N297G) and reduce binding of C1q complement protein (P329G), such as sequence (SEQ ID NO: 36): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFGGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDK
  • N297G-P329A for having a non-glycosylated (aglycosylated) Fc with reduced ADCP and ADCC activities (N297G) and reduce binding of C1q complement protein (P329A), such as sequence (SEQ ID NO: 37): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFGGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALASPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSR
  • N297A-P329G for having a non-glycosylated (aglycosylated) Fc with reduced ADCP and ADCC activities (N297A) and reduce binding of C1q complement protein (P329G), such as sequence (SEQ ID NO: 38): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDK
  • N297A-P329A for having a non-glycosylated (aglycosylated) Fc with reduced ADCP and ADCC activities (N297A) and reduce binding of C1q complement protein (P329A), such as sequence (SEQ ID NO: 39): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALASPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSR
  • the constant region of the light chain of the anti-canine IL-4Ro antibody according the invention is a lambda type of sequence: GQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPD KWKSHSSFSCLVTHECSTVEKKVAPAECS (SEQ ID NO: 46), or a kappa type of sequence: RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTE YLSHELYSCEITHKSLPSTLIKSFQRSEC (SEQ ID NO: 47), and preferably a lambda type.
  • one even more advantageous embodiment concerns a chimeric or caninized isotype IgGB anti-canine IL-4Ro antibody, comprising a lambda type light chain constant region (SEQ ID NO: 46) associated with a heavy chain constant region of canine IgGB type with the four mutations K228P-M234A-L235A-P329G (SEQ ID NO: 34) or three mutations M234A-L235A-P329G (SEQ ID NO: 32), and preferably three mutations M234A- L235A-P329G (SEQ ID NO: 32) .
  • SEQ ID NO: 46 lambda type light chain constant region associated with a heavy chain constant region of canine IgGB type with the four mutations K228P-M234A-L235A-P329G (SEQ ID NO: 34) or three mutations M234A-L235A-P329G (SEQ ID NO: 32), and preferably three mutations M234A- L235A-
  • the glycosylation of the antibody heavy chain constant region may be altered in order to reduce its affinity for the Fey receptor(s) or C1 q complement protein(s) to which normally binds.
  • Antigen-binding fragments or antigen-binding derivatives are provided.
  • the present invention also relates to an antigen-binding fragment or an antigenbinding derivative of the antibody according to the invention.
  • antigen-binding fragment is meant an antibody fragment retaining the antigen-binding domain and thus having the same antigen specificity as the original antibody as well as similar potency for inhibiting the canine IL-4Ro signaling pathway.
  • An antigen-binding fragment according to the invention is advantageously selected from the group consisting of a Fab fragment, a Fab’ fragment, a Fab’-SH fragment, a F(ab’)2 fragment, an Fv fragment.
  • a “Fab fragment” refers to a fragment of an antibody of interest (the antibody comprising two heavy chains and two light chains) formed by the assembly of the part of the heavy chain located upstream of the papain cleavage site (the CH1 domain is included, but not the hinge region or the other CH domains) and the whole light chain of an antibody of interest comprising two heavy chains and two light chains.
  • the 2 chains are linked by a disulfide bridge, as in whole Ig.
  • Fab fragments are monomers of around 50 kDa.
  • a ”Fab' fragment refers to a fragment of an antibody of interest (the antibody comprising two heavy chains and two light chains) formed by the assembly of the part of the heavy chain located upstream of the pepsin cleavage site (the CH1 domain and the hinge region are included, but not the other CH domains) and the whole light chain of the antibody of interest.
  • a “Fab'-SH fragment” refers to a Fab’ fragment containing a free sulfhydryl group.
  • F(ab')2 fragment refers to a fragment of an antibody of interest (the antibody comprising two heavy chains and two light chains) formed by the pairing of 2 Fab' fragments by disulfide bridges at the cysteines of the hinge region.
  • a “Fv fragment” or “variable fragment” refers to a fragment of an antibody of interest (the antibody comprising two heavy chains and two light chains) formed by non- covalently associated VH and VL domains of the antibody.
  • antigen-binding derivative of an antibody is meant to comprise at least one antibody fragment according to the invention as defined above linked to at least one peptide or polypeptide or other polymers.
  • Such derivatives are notable selected in the group consisting of a single-chain variable fragment (scFv), a disulfide-stabilized Fv (dsFv), a minibody, a diabody, a tribody, a single chain Fab (scFab) and a ScFabAC.
  • scFv single-chain variable fragment or “single-chain Fv”
  • linker consisting of a reduced number of amino acids (generally 15 to 20), either in the VH-linker-VL format or in the VL-linker-VH format.
  • Amino acids in the linker are most often chosen from glycine, serine, threonine, asparagine, alanine and proline, with glycine and serine most often used in the majority.
  • a scFv generally has a molar mass of around 25 kDa.
  • dsFv or “disulfide-stabilized Fv”, it is referred to a Fv fragment derivative formed by the fusion of the VH domain to the VL domain of an antibody via an intramolecular disulfide bond.
  • a “minibody” or “miniantibody” refers to a derivative formed by the fusion of a scFv to a domain that tends to dimerize, in particular a CH3 domain.
  • a minibody generally has a molar mass of around 75 kDa.
  • a “diabody” refers to a dimer composed of two scFv fragments. The two scFv fragments may be identical (in which case the diabody has a single antigenic specificity) or different (in which case the diabody may be bispecific if the two scFvs recognize different antigens).
  • a diabody generally has a molar mass of around 50 kDa (2 times that of a scFv).
  • a ‘tribody’ or ‘triabody’ refers to a trimer composed of three scFv fragments.
  • the three scFv fragments may be identical (in which case the tribody has a single antigenic specificity) or different (in which case the tribody may be bi- or tri-specific, depending on the antigenic specificity of the three scFvs).
  • a tribody generally has a molar mass of around 75 kDa (3 times that of a scFv).
  • scFab single chain Fab
  • CL constant part of the light chain
  • VH variable part of the heavy chain
  • a « ScFabAC » is referring to a ScFab derivative without cysteins for connecting the constant part 1 of the heavy chain (CH1 ) and the constant part of the light chain (CL).
  • antigen-binding derivatives may be designed for extending half-life such as by fusion with a canine Fc fragment (preferably a canine IgGB fragment, optionally mutated as disclosed above in the section regarding preferred constant regions), canine serum albumin, VHH anti-canine serum albumin or by grafting an alternative scaffold directed against canine serum albumin or other chemical polymers known to extend in vivo half-life such as polyethyleneglycol (PEG), or polypeptides such as PAS polypeptides comprising repetitive sequences of proline, alanine and/or serine or such as unstructured hydrophilic, biodegradable protein polymers named “XTEN”.
  • PEG polyethyleneglycol
  • PAS polypeptides comprising repetitive sequences of proline, alanine and/or serine or such as unstructured hydrophilic, biodegradable protein polymers named “XTEN”.
  • Antigen -binding derivatives of particular interest are:
  • Fab-Fc fusions in which the C-terminal end of the Fab VH domain is fused to the N-terminal end of a canine Fc fragment (preferably a canine IgGB fragment, optionally mutated as disclosed above in the section regarding preferred constant regions).
  • a canine Fc fragment preferably a canine IgGB fragment, optionally mutated as disclosed above in the section regarding preferred constant regions.
  • the monovalent Fab-Fc can be obtained using the Knobs-into-holes’ technology originally described by Ridgway et al. in 1996.
  • the Knob-into-Hole (KiH) technology uses complementary mutations into each CH3 domain of the antibody Fc fragment of the two heavy chains, which results in asymetric molecule which has been further stabilized by implementing an artificial disulfide bridge (Carter, 2001 ).
  • One heavy chain may notably consists in Fab-Fc with a CH3 KiH “hole” variant having mutations T366S-L368A-Y407V and introduction of the Y349C mutation for engineering a disulfide bridge with the corresponding «knob » heavy chain.
  • the other heavy chain consists in hinge- Fc with KiH « knob » variant having the mutation T366W and introduction of S354C mutation for engineering a disulfide bridge with the corresponding « hole » heavy chain.
  • scFab-Fc monovalent single chain Fab-Fc fusions, in which the C-terminal end of a single chain Fab fragment (scFab) is fused to the N-terminal end of a canine Fc fragment (preferably a canine IgGB fragment, optionally mutated as disclosed above in the section regarding preferred constant regions).
  • the monovalent scFab-Fc can be obtained using the Knobs-into-holes’ technology originally described by Ridgway et al. in 1996 as disclosed above.
  • the scFab can be designed by fusing the light chain to the heavy chain fused with the canine constant domain (CH1 -hinge-CH2-CH3)) using a peptide linker, such as the an 50-long peptide linker having the following sequence: GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG (SEQ ID NO: 48).
  • the antigen-binding derivatives of the present invention retain its ability to recognize canine IL-4Ro and to inhibit the signaling pathway activated by the binding of canine IL-4 to canine IL-4Ro with equivalent or the same level of that of the original antibody.
  • antigen-binding fragments and antigen-binding derivatives may be used to produce bispecific antibodies, which also represent an aspect of the present invention.
  • the present invention is thus related to a bispecific antibody comprising an antigen-binding fragment or an antigen-binding derivative as described above, and an antigen-binding fragment or an antigen-binding derivative directed to one other target relevant for treating atopic dermatitis.
  • the skilled person well knows how to product bi- or multi -specific antibodies and especially as described in Fan et al.2015.
  • the antibody, antigen-binding fragment or antigen-binding derivative thereof according to the invention, as well as bi- or multi -specific antibody as described above, may be produced from any host cell, any transgenic non-human animal or transgenic plant described in the present description, and notably below in the section concerning the nucleic acids, vectors, host cells, transgenic non-human animals or transgenic plants according to the invention.
  • the present invention also relates to a nucleic acid (herein also called nucleic or nucleotide sequence) or a combination of two nucleic acids, encoding the antibodies, antigen-binding fragment or antigen-binding derivative thereof or encoding the bispecific antibody according to the invention, all as described above.
  • a nucleic acid herein also called nucleic or nucleotide sequence
  • a combination of two nucleic acids encoding the antibodies, antigen-binding fragment or antigen-binding derivative thereof or encoding the bispecific antibody according to the invention, all as described above.
  • sequence of a nucleic acid according to the invention may be optimized to promote the expression thereof in a host cell, a transgenic non-human animal of interest.
  • a host cell a transgenic non-human animal of interest.
  • synonymous codons there are in general several three- nucleotide combinations encoding the same amino acid (except for methionine and tryptophan), called synonymous codons.
  • some of these combinations are in general used preferentially by a cell or a given organism (this is referred to as genetic code usage bias).
  • a nucleic acid according to the invention advantageously comprises at least one of SEQ ID NOs: 53 to 56, as described in Table 3 below, which encode the amino acid sequences of the VH and VL regions of the antibodies according to the invention.
  • the present invention also relates to a nucleic acid or a combination of two nucleic acids encoding the heavy and/or light chain constant regions of an antibody, antigenbinding fragment or antigen-binding derivative thereof, or of a bispecific antibody according to the invention as described above.
  • the present invention also relates to a nucleic acid or a combination of two nucleic acids encoding the entire heavy and/or light chains of an antibody, antigen-binding fragment or antigen-binding derivative thereof, or of a bispecific antibody according to the invention as described above.
  • Vectors
  • the present invention also relates to a vector comprising a nucleic acid or combination of nucleic acids according to the invention.
  • a vector comprises the elements necessary for the expression of said nucleic sequence(s), and notably a promoter, a transcription initiation codon, termination sequences, and suitable transcription regulatory sequences. These elements vary according to the host used for the expression and are easily selected by persons skilled in the art based on their general knowledge.
  • the vector advantageously comprises a Kozak consensus sequence, i.e.
  • the vector can notably be a plasmid or viral vector. It is used to clone or express the nucleic acids according to the invention.
  • the present invention also relates to a host cell, a transgenic non-human animal or a transgenic plant comprising at least one nucleic acid or combination of nucleic acids according to the invention or a vector according to the invention.
  • the host cell may be of prokaryotic or eukaryotic origin, and may in particular be selected from bacterial, insect, plant, fungus, yeast or mammalian cells.
  • the antibody, antigen-binding fragment or derivative thereof according to the invention may then be produced by culturing the host cell under suitable conditions.
  • a host cell according to the invention can notably be obtained by transforming a cell line by the expression vector(s) for the heavy and light chains of an antibody, antigenbinding fragment or antigen-binding derivative thereof according to the invention, and separating the various cell clones obtained.
  • the transformed cell line is preferably of eukaryotic origin, and may in particular be selected from insects, plants, yeast, or mammalian cells.
  • Suitable cell lines available for antibody production notably include lines selected from Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) fibroblasts, murine lymphoid cell lines (NSO and Sp2/0), Human embryonic kidney (HEK293) cells and Human embryonic retinal (PER.C6) cells.
  • a transgenic non-human animal according to the invention may be obtained by directly injecting the gene(s) of interest (here, the sequences encoding the heavy and light chains of the antibody) into a fertilized egg (Gordon et al. -1980).
  • a transgenic non- human animal may also be obtained by introducing the gene(s) of interest (here, the sequences encoding the heavy and light chains of the antibody) into an embryonic stem cell and preparing the animal by a chimera aggregation method or a chimera injection method (see Manipulating the Mouse Embryo, A Laboratory Manual, Second edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993)).
  • a transgenic non-human animal may also be obtained by a cloning technique in which a nucleus, into which the gene(s) of interest (here, the sequences encoding the heavy and light chains of the antibody) has/have been introduced, is transplanted into an enucleated egg (Ryan et al., 1997;Cibelli et al., 1998, WOOO/26357).
  • a transgenic non-human animal producing an antibody of interest can be prepared by the methods above. The antibody may then be accumulated in the transgenic animal and harvested, notably from the animal’s milk or eggs.
  • transgenic non-human animals For producing antibodies in the milk of transgenic non-human animals, preparation methods are notably described in W090/04036, W095/17085, W001 /26455, W02004/050847, W02005/033281 , W02007/048077. Methods for purifying proteins of interest from milk are also known (see W001 /26455, W02007/ 106078).
  • the transgenic non-human animals of interest notably include mice, rabbits, rats, goats, bovines (notably cows), and poultry (notably chicken).
  • a transgenic plant according to the invention may be selected from any plant allowing antibody production. Numerous antibodies have already been produced in transgenic plants and the technologies required for obtaining a transgenic plant expressing an antibody of interest and for recovering the antibody are well-known to a person skilled in the art (see Stoger et al., 2002, Fisher et al., 2003 Schillberg et al., 2005). It is also possible to influence the glycosylation obtained in the plants or any other necessary addition or modification in order to be similar to that of natural canine antibodies.
  • the present invention also relates to an antibody, antigen-binding fragment or antigen-binding derivative thereof, or a bispecific antibody according to the invention, for use as a medicinal product.
  • the antibody, functional fragment or derivative thereof, or bispecific antibody according to the invention is preferably used in the treatment or prevention of the following diseases, for which a role of IL-4 has been established: atopic dermatitis, contact dermatitis, psoriasis, allergic asthma, inflammatory bowel disease, neurodegeneration, chronic rhinosinusitis, and eosinophilic diseases, preferably in the treatment or prevention of canine atopic dermatitis.
  • the antibody, antigen-binding fragment or antigen-binding derivative thereof, or bispecific antibody according to the invention is advantageously used in the treatment or prevention of itch and/or inflammatory skin due to atopic dermatitis in dogs.
  • the antibody, antigen-binding fragment or antigenbinding derivative thereof, or bispecific antibody according to the invention is advantageously used in the treatment or prevention of itch and/or inflammatory skin due to allergies in dogs.
  • the present invention also concerns the use of an antibody, antigen-binding fragment or antigen-binding derivative thereof, or bispecific antibody according to the invention for preparing a medicinal product for treating or preventing of itch and/or inflammatory skin due to atopic dermatitis and/or allergies in dogs.
  • the present invention also concerns the use of an antibody, antigen-binding fragment or antigen-binding derivative thereof, or a bispecific antibody according to the invention in the treatment or prevention of itch and/or inflammatory skin due to atopic dermatitis and/or allergies in dogs.
  • the present invention also concerns a method for treating or preventing itch and/or inflammatory skin due to atopic dermatitis and/or allergies in dogs, comprising administering to dogs an effective amount of an antibody, antigen-binding fragment or antigen-binding derivative thereof, or a bispecific antibody according to the invention.
  • the present invention also concerns a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody, antigen -binding fragment or antigen-binding derivative thereof, or a bispecific antibody according to the invention for use in the treatment or prevention of itch and/or inflammatory skin due to atopic dermatitis and/or allergies in dogs.
  • treatment is meant an improvement, observed at the clinical or biochemical level, of the subject’s disease.
  • prevention is meant the fact of preventing or delaying the onset of, or of decreasing the intensity of, the clinical or biochemical manifestations associated with the disease.
  • a clinical parameter of interest may be the gravity of the skin lesions measured by evaluating for example erythema, excoriations, and lichenification, compiled on the composite CADESI score (Olivry T et al., 2014), or quantification of itch.
  • Example 1 Generation of MDCK nLuc Stat6 cell-based luciferase assay to measure IL4- or IL13-induced STAT6 transcription factor activation
  • a dog kidney cell line - MDCK (Madin-Darby Canine Kidney) cells was used, that endogenously expresses Type I and Type II IL4 receptors and functional downstream signaling pathway components.
  • a nano-luciferase reporter construct that is regulated by activation of STAT6 transcription factor, the main intracellular signal transducer of IL-4 and IL- 13 -activated IL-4 receptors, was designed and generated.
  • Stable MDCK cells carrying STAT6-nanoluc reporter cassette were established that could respond in a concentration-dependent manner to canine IL-4 or canine IL-13 induction by activation of the nano-luciferase reporter.
  • This reporter cell line was used for evaluating the functionality of the chimeric and caninized versions of anti-IL-4Ralpha antibodies of the invention.
  • MDCK cells were purchased from ATCC (parental MDCK cell line CCL-34TM). At first the cells were tested for recombinant canine IL-4 and IL-13 responsiveness by measuring STAT6 phosphorylation, using immunoblot analysis. The analysis of immunoblot results demonstrates, that both, canine IL-4 and IL-13, can induce STAT6 Y-641 phosphorylation in MDCK cells.
  • a modified MDCK cell line was generated that expresses nano-luciferase (nluc) reporter gene under STAT6-regulated minimal promoter.
  • Synthetic DNA with 4 consensus STAT6 binding sites and minimal TATA-box promoter was designed, ordered and cloned 5’ to nluc ORF (see Figure 2).
  • the plasmid DNA construct contained also hygromycin resistance cassette to enable antibiotic selection of transgene carrying cells. Spel-Nhel linearized vector DNA fragment was extracted from agarose gel and purified; DNA was eluted into water.
  • Hygromycin- resistant MDCK cell clones were cultivated as duplicates on 48-well plates. The cells were starved in serum free media for 8h and induced with recombinant canine IL-4 (8 ng/ml) or BSA (0.1%) for 24h. The medium was discarded and the cells were lysed in passive lysis buffer (Promega) for 30 min at room temperature. Nano-Gio Luciferase Assay buffer and substrate mixture (N1120, Promega) was mixed 1 :1 with cell lysates and luminescence was recorded on Glomax luminometer (Promega).
  • a stable nano- luciferase reporter cell line can be used to measure canine I L-4- or IL-13-induced activation of IL4Ralpha receptor-mediated activation of STAT6 transcription factor in canine MDCK cells.
  • This STAT6-nluc reporter cell line was used to analyze receptor-blocking function of anti-IL-4Ralpha antibodies.
  • EXAMPLE 2 PRODUCTION AND PURIFICATION OF CANINE EXTRA CELLULAR DOMAIN IL-4 RECEPTOR ALPHA (c-ECD-IL-4Ra)
  • cECD-IL-4Ro Recombinant extra cellular domain of canine IL-4 receptor alpha
  • a 6 Histidine tag was added at the C-terminal of c-ECD-IL4Ra to allow purification by metal ion affinity chromatography (IMAC). Briefly, the cDNA coding for the cECD-IL- 4Ra (SEQ ID NO: 8) was cloned into pQMCF expression vectors (QMCF technology from Icosagen). Endotoxin free plasmid was transfected into CHOEBNAL T85 1 E9 CHO cell line and the established pool of cells was used to produce the recombinant protein in the CHO medium. Transient production was done at 900 mL final volume.
  • Recombinant cECD-IL-4Ra was then purified by IMAC using HisTrapTM Excel columns (GE Healthcare) followed by preparative gel filtration with Superdex 200 Increase 10/300 (GE Healthcare).
  • Monoclonal recombinant blocking antibodies against canine IL4Ro were developed from immunized chickens and rabbits using HybriFree Technology (Kivi et al. 2016).
  • Test animals were immunized 3-4 times, with 0.2 mg antigen per injection. Chickens were immunized after every 2-2.5 weeks (intramuscular), rabbits after every 3- 3.5 weeks (subscapular). The immunizations were in Freund's complete adjuvant or in Freund 's incomplete adjuvant or with the antigen in PBS. For boosting, half of the antigen amount was injected intramuscularly or suscapsularly in Freund 's incomplete adjuvant and another half intravenously in PBS. All test animals were boosted with canine IL4Ro- ECD (extra-cellular domain).
  • Immune response of each individual was measured by ELISA approximately 10 days after 3rd immunization from the blood serum of rabbit or from PEG6000 precipitated total IgY fraction of chicken egg yolk.
  • the specific antibody titer was measured by ELISA, coating the plates with recombinant canine IL4RaECD. Spleens of test animals were homogenized, splenocytes frozen and stored in liquid nitrogen.
  • Immuno modules (Thermo ScientificTM) or Pierce Streptavidin coated 96-well plates (Thermo Scientific, code 15121 ) were coated with canine IL4Ro-ECD-His or biotinylated canine IL4Ro-ECD-His antigen.
  • Spleen cells from chicken were used for panning and a number of panning reactions were performed. After 45 min. incubation, wells were washed with PBS to remove the unbound cells.
  • RNA was isolated and cDNA was synthesized using SuperScript® IV First-Strand Synthesis System for RT-PCR (Invitrogen) and used for VH and VL amplification using VH and VL primers specific for chicken and rabbit antibody sequences.
  • VH and VL were purified and ligation independent cloning) reactions were performed for hlgG1 -lambda (chicken) and hlgG1 -Kappa (rabbit) expression vectors library (“pools”) construction.
  • E. coli DH5o was transformed by hlgG1 expression vector pools, grown o/n in liquid medium on shaker at 37 °C. Plasmid DNA was purified and transfected into CHOEBNALT85-1 E9 cells for hlgG1 pools production. Supernatants of antibody pools were analyzed by ELISA (48 to 72 hours after transfection) to identify antigen specific reaction.
  • Ten lead antibodies, 8 from rabbits and 2 from chicken were chosen for cloning into canine IgGB scaffold as chimeric antibodies, to be produced, characterized and their potency, in suppressing canine IL4- or canine IL13-induced STAT6 activation, tested in the MDCK nluc stat6 cell-based-assay.
  • Synthetic codon optimized DNA encoding chicken- or rabbit-derived antibody VH and VL sequences were designed, ordered and DNA was cloned using LIC method into the appropriate canine IgGB, kappa or lambda pQCMF expression vectors (Icosagen).
  • CHOEBNAL T85 1 E9 cells were cotransfected with 5pg of canine light chain and 5pg of canine heavy chain vector DNAs using Reagent 007 for transient antibody production.
  • the cells were cultivated in 35ml volume of Xcell CHO-TF medium, for initial 72h at 37°C, for the production phase, the temperature was shifted to 30° C and the culture was additionally fed. The duration of the production phase was 9 days.
  • the cells were removed from expression culture supernatants by centrifugation. Then, clarified supernatants were filtered through the glass fiber prefilter and 0.45 pm filter.
  • Antibodies were purified by MabSelect SuRe affinity chromatography, eluted with 0.1 M Na-citrate pH 3.3 and neutralized with 1.5 M Tris pH 8.8. Collected MabSelect chromatography IgG fractions were concentrated using Amicon Ultra centrifuge filters (Merck Millipore) and gel filtrated with Superdex 200 Increase column into PBS pH 7.4.
  • Purified antibodies were sterile filtered, concentration was measured with NanoDrop 2000 (Thermo Scientific), aliquoted and stored at -75 °C. Purified antibodies were analyzed under non-reduced (-DTT) and reduced (+DTT) conditions by Coomassie staining on SDS-PAAG. Purity of purified antibodies was analyzed by size-exclusion chromatography (SEC) with Superdex 200 Increase column.
  • recombinant canine IL-4 (754-CL-025 from R&D Systems) or recombinant canine IL-13 (5894-CL-025 from R&D Systems) was used for induction on serum-starved (7-8h) 50-80% confluent MDCK reporter cells, grown in 96-well plates in serum free culture medium. Media volume was 100ul per well.
  • Purified chimeric IgGB antibodies were serially diluted (3-fold dilution; 8 steps) in serum free DMEM culture medium and were applied in triplicate to reporter cells, 10 minutes before addition of recombinant canine IL-4 or IL- 13.
  • Streptavidin sensors were equilibrated off-line in kinetic buffer (PBS, 0,1%BSA, 0.02% Tween20, 0.1% Proclin 300) for 10 min and then monitored on-line in kinetic buffer for 60 seconds for baseline establishment.
  • kinetic buffer PBS, 0,1%BSA, 0.02% Tween20, 0.1% Proclin 300
  • sensors were loaded with biotinylated canine-ECD-IL-4R alpha (5 pg/ml in kinetic buffer) for 30 sec.
  • sensors were transferred to antibody solution (-5 pg/ml kinetic buffer) for association for 300 sec.
  • a reference sample (kinetic buffer) was included to allow subtraction of background signal and assay drift.
  • dupilumab has a high affinity for recombinant human IL-4Ra (below 1 pM) and a much lower affinity for canine IL-4Ro (around 500 pM).
  • dupilumab shows a moderate inhibition of signalling induced by canine IL-4 (around 40 nM) and no apparent inhibition of signalling induced by canine IL-13.
  • the IC50 concentration of the chimeric antibodies tested for inhibition of canine IL-4-induced STAT6-nluc reporter activation is between 0.08-1.8 nM. Also, inhibition of canine IL-13-induced STAT6-nluc reporter activation is between 0.18-7.9 nM.
  • the most potent chimeric antibody from chicken 27E1 suppresses canine IL-4- induced STAT6-nluc reporter activation in cell-based assay at sub-nanomolar concentration.
  • a number of chicken and rabbit recombinant antibodies were obtained which show high inhibitory activity for both canine IL-4 and IL-13 in a MDCK cell-based STAT6-nluc reporter activation assay.
  • the chicken antibody 27E1 were found to be the most potent lead candidate.
  • Recombinant Fab fragments of 27E1 were produced with canine constant regions of IgGB isotype.
  • a 6 Histidine tag was added at the C-terminal of the heavy chain CH1 constant domain to allow purification by metal ion affinity chromatography (IMAC).
  • IMAC metal ion affinity chromatography
  • the cDNA coding for the Fab heavy (Fab-HC) and light (Fab-LC) chains sequences of 27E1 (27E1 Fab-HC and Fab-LC encoding sequences shown in SEQ ID NOS: 83 and 85, respectively) were cloned into pQMCF expression vectors (QMCF technology from Icosagen).
  • the obtained plasmid was co-transfected into CHOEBNAL T85 1 E9 CHO cell line and the established pool of cells was used to produce the antibody in the CHO medium.
  • the Fab fragments were then purified by IMAC using HisTrapTM Excel columns (GE Healthcare) followed by preparative gel filtration with Superdex 200 Increase 10/300 (GE Healthcare).
  • Fab fragment of 27E1 still have a subnanomolar IC50 which makes them very powerful antagonist molecules.
  • Example 6 preparation and characterization of caninized versions of 27E1
  • Antibody caninization was performed using standard CDR grafting method followed by construction and screening of caninized antibody variants containing rationally designed back mutations.
  • Biological activity of the caninized antibodies was determined in cell-based IL4Ralpha dependent STAT6-nluc reporter assays.
  • the ability to inhibit canine IL-4- and/or IL-13-induced, IL4R alpha mediated STAT6-nluc reporter activation in MDCK cells was determined by serially-diluting each protein-A purified individual antibody and calculating their IC50 concentrations.
  • an homology molecular model of the parental 27E1 antibody has been constructed.
  • residues from the canine framework are at so-called ‘vernier’ or ‘canonical’ positions.
  • ‘Vernier’ residues are structurally adjacent to the CDRs, and are known to affect CDR conformation and fine-tuning of antigen recognition.
  • ‘Canonical’ residues are positions whose adoption of specific sequence are signatures of cataloged three dimensional conformations of CDR sequences; i.e. an analysis of CDRs in resolved antibody structures permits the classification of these CDR structures on the basis of conformation, and the subsequent association of these conformations with specific sequence signatures at particular sequence locations.
  • the overall process of designing caninized versions of anti-canine IL-4 receptor alpha antibody 27E1 involved the following steps i) build a homology molecular model of antibody 27E1 in order to guide the caninization process ii) identify the amino acid sequence of the VH and VL CDRs of antibody 27E1 as defined by the Kabat nomenclature iii) identify a suitable canine V germline gene for both the VH and the VL that will be used as acceptor sequence iv) Identify the amino acid sequence of the CDRs as defined by the Kabat nomenclature of the canine V germline genes above v) replace the CDRs in the canine V germline genes with the corresponding VH and VL CDRs of antibody 27E1 vi) Replace some canine framework residues with antibody 27E1 framework residues that have been identified as critical by inspection of the molecular model vii) Synthesize the DNA encoding the caninized versions from step (vi), clone it into a
  • VL CDR1 which is unusually short, it was possible to identify a VL CDR1 template from the solved antibody structures with a Lambda chain VL CDR1 of IMGT length 4, the human antibody structure 3TV3. Only Lambda light chain is expressed in avians and one striking difference with mammalian lambda light chain is that the canonical structure of the chicken VLA CDR1 is significantly shorter.
  • VH CDR2 since there were no solved galline antibody structures having VH CDR2 with sufficiently homologous sequence, a search over human antibody structures from the closest matching human germline having VH CDR2 of identical length was conducted; the single resulting structure was 1AD0.
  • D(H73) An example of a vernier location is the heavy chain residue D(H73) (Kabat numbering).
  • D(H73) is occupied by asparagine (N) as opposed to the 27E1 aspartic acid (D).
  • D(H73) is surface-exposed, and packed behind other vernier residues that directly contact VH CDR2.
  • the sidechain contacts the vernier residues F(H29), S(H30) and R(H71 ).
  • D(H73/H82) may serve to stabilize the presentation of VH CDR2.
  • the asparagine substitution is isosteric and would generally result in the preservation of these conformationally stabilizing contacts. Therefore, in the proposed heavy chain germline there is a high probability that position (H73) need not be a candidate for back-mutation to the parental galline germline residue, aspartic acid.
  • G(L64) Kabat numbering
  • (L64) is occupied by serine (S) as opposed to the 27E1 glycine (G).
  • S serine
  • G 27E1 glycine
  • the G(L64) alpha carbon makes no vernier or CDR contacts, although the mainchain contacts the Vernier residue l(L48) and VL CDR2 at residues N(L51 ) and D(L52).
  • G(L64) appears to be crucial to the adoption of a specific three-dimensional conformation by VL CDR2.
  • Parental antibody 27E1 having VH and VL amino acid sequences of SEQ ID NOs: 58 (AVTLDESGGGLQTPGGALSLVCKASGFTFSSYGMGWMRQAPGKGLEWVAVINPAVSGSRQGYAPAVKGRATISR DDGQSTLRLQLNNLRTEDTGTYYCAKHASNYWNGAGRIDAWGHGTEVIVSS) and 59
  • ALTQPSSVSANPGETVKITCSGGSGNDYGWFQQKSPGSAPVTVIYDNDKRPSNIPSRFSGSGSGSTSTLTITGVQA EDEAVYFCGGYDRHTYDAFGAGTTLTVL were caninized by the introduction of the CDRs as defined by the Kabat nomenclature into the VH of heavy chain frameworks (FR1 , FR2, FR3) from the canine germline subgroup IGHV3-38*01 together with canine IGHJ4*01 (FR4), and the introduction into the VL of light chain frameworks (FR1 , FR2, FR3) from the canine germline subgroup IGLV3-29*01 , together with IGLJ4*01 (FR4).
  • IGHV3-38*01 EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYDMSWVRQSPGKGLQWVAVIWNDGSSTYYADAVKGRFTISRDNA KNTLYLQMNSLRAEDTAVYYCAK
  • IGHJ4*01 NFDYWGQGTLVTVSS
  • IGLJ4*01 YVFGSGTQLTVL
  • SEQ ID NO: 62 IGHV3-38*01 : EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYDMSWVRQSPGKGLQWVAVIWNDGSSTYYADAVKGRFTISRDNA KNTLYLQMNSLRAEDTAVYYCAK
  • the sequence of the canine germline IGLV3-29*01 is: SSVLTQPPSVSVSLGQTATITCSGESLSRYYAQWYQQKPGQAPMTVIYGDRERPSGIPDRFSSSSSENTHTLTISGAQ AEDEAEYYCEIWDASADD (SEQ ID NO: 63) .
  • caninized 27E1 VH and VL versions were produced and purified as described in Example 4 for the chimeric versions, and different combinations of these versions were produced. All the purified caninized versions were evaluated for their inhibitory potency for both canine IL-4 and IL-13 stimulation in the MDCK nLuc STAT6 cell-based luciferase assay following the same protocol as described in Example 4 for the chimeric versions.
  • variable VH and VL sequences from chicken origin 4 caninized versions with a significant IC50 inhibitory activity for both canine IL-4 and IL-13 in a MDCK cell-based STAT6-nluc reporter activation assay were successfully obtained.
  • An acute canine atopic dermatitis (cAD) house dust mite (HDM) -sensitized dogs model was used.
  • dogs were treated with the chimeric canine IgGB version of the chicken antibody 27E1 containing the mutations K228P-M234A-L235A-P329G in the Fc domain (silencing of effector functions of the Fc).
  • the monoclonal chimeric antibody anti-IL4Ralpha mentioned above (Test item) was used, formulated as a solution for injection stored at -75 °C ( ⁇ 15 °C) at a concentration of 10 mg/mL or 20 mg/ml, and was administered in a single subcutaneous injection.
  • Dermipred® Reference item; prednisolone
  • Dermatophagoides farinae Allergic element
  • the canine species was selected because it is the target animal species for the future clinical use as a treatment of canine atopic dermatitis.
  • the number of animals included in the study was 16 all had been previously successfully sensitised D. farinae. Four groups were created:
  • Group 1 received a single subcutaneous injection of anti-IL4R at a dose of 5mg/kg seven days before the first application of D. farinae
  • Group 2 received a single subcutaneous injection of anti-IL4R at a dose of 5mg/kg three days before the first application of D. farinae
  • Group 3 treated once daily with Dermipred at a dose of 1 mg/kg, on 7 consecutive days starting one day before the first application of D. farinae
  • the animals were housed in groups of 2 or 3 in kennels with wood shavings on the floor and toys were available.
  • the food was Royal Canin special Beagle kibble.
  • the ration was adapted to the weight of the animal. Water was provided ad libitum.
  • Test Item The administration of the Test Item to the first group of dogs (group 1 ), was designated as Day 0. Subsequent study days were counted forward (Day 1 , day 2,..) and preceding days referred to as negative numbers (Day -1 , day -2,..).
  • Dogs in groups 1 and 2 received a single subcutaneous administration of Test Item at a dose of 5 mg/kg on day 0 for group 1 and on day 4 for group 2.
  • Dogs in group 3 received the Reference item administered once a day per os from D6 to D 12 at a dose of 1 mg/kg.
  • a dermatological examination took place once a day from study days D7 to D14.
  • the schedule of events is described in Table 5 below and in Figure 9.
  • anti-IL4Ralpha 27E1 antibody as described herein is at least as much effective for treating atopic dermatitis in dogs as glucocorticoids (Dermipred®).
  • MDCK cell-reporter gene assay (MDCK 5x STAT6 Luc2P)
  • MDCK 5x STAT6 Luc2P was generated to measure IL4-induced STAT6 transcription factor activation.
  • Canine kidney MDCK NBL-2 cell line (ATCC CCL-34) was choose since naturally expressing IL4/13 signaling pathway (receptors and effectors).
  • the plasmid pGL4 (Luc2P_STAT6-RE_Hygro) was purchased at Promega.
  • the MDCK 5x STAT6 Luc2P was generated to express luciferase reporter gene under STAT6-regulated minimal promoter.
  • MDCK cells was stably transfected with the pGL4 (Luc2P_STAT6-RE_Hygro) mixed with chemical lipofection agent. 48h post-transfection antibiotic selection was initiated and a stable polyclonal pool of cells was established after 20 days.
  • the polyclonal cells were seeded at 10 cells/mL in multiple 96-well plates by adding 100pL per well. After 24h we noted the wells that have only one cell (clone). Each clone was expended for 3-4 weeks and conserved for further screening. Molecular screening was done on each clone with PCR to verify the presence of the reporter gene (luciferase). 32 clones were positives for the presence of the reporter gene. A second molecular screening (RT-qPCR) was done on the 32 clones to determine the expression of IL4Ra and IL13Ra.
  • Non essential amino acids 1X (Gibco; 11140035)
  • Non essential amino acids 1X (Gibco; 11140035)
  • inhibitory activity for canine IL-4 or IL13-stimulation of the following different prior art anti-IL4R antibody candidates was measured in the MDCK 5x STAT6 Luc2P cell line, so as to rank the prior art antibodies potency:
  • the MDCK 5xSTAT6 Luc2P cells were collected and seeded into a white 96-well assay plate at 0,5x106 cells/mL in 10Opl bioassay medium per well, then incubate at 37°C with 5% CO2 for 5h.
  • the antibodies (control and candidates) were serially diluted in a ratio of 1 :3 in bioassay medium with the starting concentration of 100pg/mL in a another 96-well plate. The liquid in to each well in the plate was discarded and 25pl of diluted antibodies were added to the cells for 1 h at 37° C with 5% CO2.
  • the four-parameter model was used to fit the dose response curve that correlates RLU and value of antibodies concentration.
  • the inhibitory activity (potency) of CAN- 27E1 -338-VHN/329-VLA according to the invention was measured for canine IL-4 stimulation in the parental MDCK cell line CCL-34TM and compared the one of the prior art anti-IL4 pathway caninized candidates 152H11 (c152H11 -H3L3) and 146E2 (c146E2-H3L3) described in applications W02021/123089 and W02021/123091 .
  • 96-well microplate was seeded with 8 x 104 MDCK cells per well (in 200 pL) and incubated at 37° C overnight. 2. Medium was removed, and wells were washed with 200pl of cell growth medium without serum.
  • Antibodies were pre-diluted at 12.5 pg/mL and then 3-fold serial diluted in cell growth media without serum. 40 pL of the serial dilutions were transferred to each well. All was done in triplicate.
  • Canine IL-4 was diluted to 10 ng/mL and then 10 pL were added to wells containing antibodies. All was done in triplicate. The plate was incubated for 15 min at 37°C.
  • canine IL-4 was diluted to 25 ng/mL and then 3-fold serial diluted in cell growth media without serum. 50 pL/well of dilutions were transferred to 96-well containing cells. The plate was incubated for 15 min at 37° C.
  • the Acceptor Mix was prepared from the AlphaLISA p-STAT6 Assay Kit and 5 pL per well was added to 10 pL of the cell lysates in 384-well Proxiplate. Plate was sealed, covered with foil, and then incubated for 2 hours at room temperature.
  • the Donor Mix was prepared from the AlphaLISA p-STAT6 Assay kit under subdued laboratory lighting and 5 pL was added per well. The plate was sealed, covered with foil, and then incubated for 2 hours at room temperature.
  • the plate was read using the AlphaScreen settings on the BMG Clariostar.
  • CAN-27E1 -338-VHN/329-VLA has an IC50 at least 5-fold lower than a monoclonal anti-clL-4R antibody C146E2-H3L3 which is the best anti-clL4R candidate in the prior art. It should also be noted that CAN-27E1 -338-VHN/329-VLA is the only anti-clL-4R antibody that fully inhibits clL-4 induced STAT6 phosphorylation.
  • Example 10 Evaluation of the effect of a monoclonal antibody of the invention targeting the canine IL4R on an Atopic Reconstructed Canine Epidermis model (RCE- AD)
  • the reference molecule used is oclacitinib and was tested at 10 pM concentration.
  • the efficacy of the monoclonal antibody and the reference molecule are evaluated and compared to negative control (RCE not stimulated with the pro-inflammatory cytokines) and positive control (RCE stimulated with the pro-inflammatory cytokines) by: histological morphological analysis (hemalun eosin staining), and evaluation of inflammation (ELISA quantification of IL-8).
  • the inflammation was evaluated by ELISA quantification of IL-8 in the medium.
  • the assay reveals a strong increase of IL-8 secretion in the positive control (RCE- AD) and a decrease of this inflammation with the mAb targeting the IL-4R and the reference molecules.
  • the mAb targeting the IL-4R according to the invention triggers and stimulates the recovering of the skin barrier function by lowering the epidermal inflammation (hyperplasia, spongiosisi and acanthosis) and by increasing the number of keratohyaline granules (profillagrin precursors).
  • Monovalent Fab Fc format i.e. IgG-like format but with only one arm
  • caninized 27E1 anti-canine IL4Ralpha antibody version VHN / VLA
  • canine IgGB Fc containing the mutations M234A, L235A and P329G to reduce Fc related effector functions.
  • the monovalent Fab-Fc was obtained using the Knobs-into-holes’ technology originally described by Ridgway et al. in 1996.
  • the Knob-into-Hole (KiH) technology uses complementary mutations into each CH3 domain of the antibody Fc fragment of the two heavy chains, which results in asymetric molecule which has been further stabilized by implementing an artificial disulfide bridge (Carter, 2001 ).
  • One heavy chain consists in Fab-Fc with a CH3 KiH “hole” variant having mutations T366S-L368A-Y407V and introduction of the Y349C mutation for engineering a disulfide bridge with the corresponding «knob » heavy chain.
  • the other heavy chain consists in hinge-Fc with KiH « knob » variant having the mutation T366W and introduction of S354C mutation for engineering a disulfide bridge with the corresponding « hole » heavy chain.
  • the monovalent Fab-Fc molecule was obtained by co-transfection of three genes in CHO cells; “hole” heavy chain, “knob” heavy chain and 27E1 light chain.
  • the resulting molecule was purified from the supernatant by affinity purification on protein A followed by a preparative size exclusion chromatography.
  • scFab Fc format i.e. IgG-like format but with only one arm
  • caninized 27E1 anti-canine IL4Ralpha antibody version VHN / VLA
  • the monovalent scFab-Fc was obtained using the Knobs-into-holes’ technology originally described by Ridgway et al. in 1996.
  • the Knob-into-Hole (KiH) technology uses complementary mutations into each CH3 domain of the antibody Fc fragment of the two heavy chains, which results in asymetric molecule which has been further stabilized by implementing an artificial disulfide bridge (Carter, 2001 ).
  • One heavy chain consists in scFab-Fc with a CH3 KiH “hole” variant having mutations T366S-L368A- Y407V and introduction of the Y349C mutation for engineering a disulfide bridge with the corresponding «knob » heavy chain.
  • the other heavy chain consists in hinge-Fc with KiH « knob » variant having the mutation T366W and introduction of S354C mutation for engineering a disulfide bridge with the corresponding « hole » heavy chain.
  • the scFab was designed by fusing the light chain (variable light chain domain of canonized 27E1 VL version A (VLA) fused with the canine constant Kappa light chain domain (CK)) to the heavy chain (variable heavy chain domain of canonized 27E1 VH version N (VHN) fused with the canine constant IgGB domains (CH1 -hinge-CH2-CH3)) using an 50-long peptide linker having the following sequence: GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGG (SEQ ID NO: 48).
  • the monovalent scFab-Fc was obtained by co-transfetion of two genes, “hole” heavy chain and “knob” heavy chain.
  • the resulting molecule was purified from the supernatant by affinity purification on protein A followed by a preparative size exclusion chromatography.
  • amino acid sequences are the following: scFab27E1 -VHN-canlgGB-MALAPG-Y349C-T366S-L368A-Y407V
  • the inhibitory potency for canine IL-4 stimulation of CAN-27E1 -338-VHN/329-VLA and its antigen-binding derivatives candidates 27E1 -mono-1 and 27E1 -mono-2 as described in Example 11 was tested and compared in the MDCK cell-based STAT6-Luc2P reporter activation assay, using the same protocol as described in Example 8.
  • IC50 obtained for 27E1 -mono-1 and for 27E1 -mono-2 compared to 27E1 are depicted in Figures 19 and 20, respectively, and shown in Table 8 below with IC50 ratio versus 27E1 .
  • Example 13 INHIBITORY POTENCY OF ANTIGEN-BINDING DERIVATIVES FOR IL13 STIMULATION
  • the inhibitory potency for canine IL-13 stimulation of CAN-27E1 -338-VHN/329-VLA and its antigen-binding derivatives candidates 27E1 -mono-1 and 27E1 -mono-2 as described in Example 11 was tested and compared in the MDCK cell-based STAT6-Luc2P reporter activation assay, using the same protocol as described in Example 4 for the chimeric versions.
  • IC50 obtained for 27E1 -mono-1 and for 27E1 -mono-2 compared to 27E1 are depicted in Figures 21 and 22, respectively, and shown in Table 9 below with IC50 ratio versus 27E1 .
  • Cibelli JB Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM.Cibelli JB, et al. 1998 Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science. 280:1256-8.
  • IMGT/GENE-DB a comprehensive database for human and mouse immunoglobulin and T cell receptor genes Nucl. Acids Res., 33(S1 ):D256-D261.
  • Lefranc MP Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin- Contet V, Lefranc G. 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(1 ):55-77.
  • Interleukin-13 receptor alpha but not alpha chain: a functional component of interleukin-4 receptors. Blood 91 , 3884-3891 .

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Abstract

La présente invention concerne des anticorps monoclonaux anti-IL-4Rα canins ayant un pouvoir élevé concernant l'inhibition de la voie de signalisation IL-4Rα, et leur utilisation pour traiter et/ou prévenir les démangeaisons et/ou les inflammations de la peau dues à la dermatite atopique et aux allergies chez les chiens, et en particulier pour traiter la dermatite atopique canine.
PCT/EP2024/084368 2023-11-30 2024-12-02 Anticorps anti-récepteurs alpha de l'interleukine-4 canins (il-4rα) et leurs utilisations Pending WO2025114614A1 (fr)

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