Anti-canine interleukine-4-receptor alpha (il-4rΑ) antibodies and the uses thereof

Abstract

The present invention relates to anti-canine IL-4Rα monoclonal antibodies of high potency regarding inhibition of IL-4Rα signaling pathway, and the use thereof 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.

Description

[0001] ANTI-CANINE INTERLEUKINE-4-RECEPT0R ALPHA (IL-4RA) ANTIBODIES AND THE USES THEREOF

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention is in the field of therapeutic antibodies and especially anti- canine interleukine-4-receptor alpha (IL-4Ro) monoclonal antibody. In particular, 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.

[0004] BACKGROUND ART

[0005] Atopic dermatitis (AD) in dogs is a common inherited chronic inflammatory and pruritic skin disease involving abnormalities in skin barrier function and cutaneous inflammation, secondary staphylococcal and Malassezia skin and ear infections, and hypersensitivity to environmental allergens, food allergens, or staphylococcal or Malassezia allergens (or both pathogens) (Nuttall et al., 2019). The estimated prevalence of AD in the dog is approximately 10-15% (Gedon and Mueller, 2018).

[0006] In dogs, clinical signs of an environmental allergy mainly develop between 6 months and 3 years of age. The most common clinical signs include generalized pruritus (seasonal, nonseasonal, or nonseasonal with seasonal worsening), erythema, papules, pustules, crusts, and excoriations. Head (perioral, periocular, and ears), flexor aspect of elbows, carpal and tarsal joints, paws (digits, claws, and interdigital aspects), ventral abdomen, perineum, and ventral tail are most commonly affected. Predilection sites differ from breed to breed (Gedon and Mueller, 2018) (Griffin and DeBoer, 2001 ) (Wilhem S,et al., 2011 ) (Santoro, 2018).

[0007] Clinical, immunological, histological and pathological features of atopic dermatitis in dogs are so similar to the human counterpart, that canine atopic dermatitis has been suggested as an animal model for human AD (Mineshige et al., 2018) (Marsella and Girolomoni, 2009) (Gedon and Mueller, 2018). The most important limitations of available treatments for canine atopic dermatitis are cost, side effects, compliance and lag phase. Because of their diversity in lag phases, some therapeutic options are more suitable for treating acute flares (eg, glucocorticoids, oclacitinib), whereas others are more indicated for maintenance and / or prevention of flares (eg, allergen-specific immunotherapy, cyclosporine) (Santoro, 2019).

[0008] However, for a medication required for many months / years it is always prudent to find alternative therapies when treatment is needed for extended periods of time (Marsella and De Benedetto, 2017). In addition, long-term use of glucocorticoids is associated with multiple adverse effects because glucocorticoid receptors are present in almost all cells. In addition to some adverse side effects, short-lived benefits of relief are provided by some treatments (oclacitinib), which sometimes are followed by a rapid return of clinical signs even at a higher level than before the initiation of therapy (rebound). Overall, the currently available treatment modalities cannot provide the much-needed convenient, safe, long-term solution, and alternative treatments are needed.

[0009] 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. Initially, 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).

[0010] Among treatments, the use of monoclonal antibodies was also disclosed. In dogs, 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. Despite effectively controlling pruritus in dogs with atopic dermatitis, 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).

[0011] In addition, because IL4 and IL13 and their specific signaling pathways are considered attractive targets for the treatment of allergy (Kelly-Welch, 2003), the signaling pathway was considered as an attractive target for atopic dermatitis.

[0012] 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. In all clinical studies to date, 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). Beside 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). Although 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.

[0013] W02016 / 156588 (Intervet) 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 (no signaling results from ligation of c-IL-4 to clL-4Ro in these 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.

[0014] W02021 / 123089 and W02021 / 123091 (Intervet) 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. Both caninized C146E2-H3L3 and C152H11 -H3L3 are more potent than c4H3. In addition, Figures 3 and 4 of their common priority application US62 / 951 ,793 compares C146E2-H3L3, C152H11 -H3L3, and c4H3 in the same alphalisa assay and shows that C146E2-H3L3 is more potent than C152H11 -H3L3.

[0015] W02021 / 188631 (Kindred) 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).

[0016] Nevertheless, there remains a need for further improved anti-clL-4Ro antibodies with increased ability to inhibiting IL-4Ro signaling pathway, in particular for caninized antibodies. SUMMARY OF THE INVENTION

[0017] In the context of the present invention, the inventors surprisingly found that new anti-canine interleukine-4-receptor alpha (IL-4Ro) monoclonal antibodies of particularly high potency regarding inhibition of IL-4Ro signaling pathway. Compared to anti-canine IL-4Ro monoclonal antibodies of the prior art, such potent antibodies could have the advantage of using lower doses for disease treatment and having a longer lasting effect, thereby allowing subjects to be treated less frequently. This will bring comfort to the subjects to be treated and lower the overall cost of the treatment.

[0018] The anti-canine IL-4Ro monoclonal antibodies of the present invention have a significantly higher potency than those disclosed in prior art. In particular, of all tested prior art antibodies, C146E2-H3L3 was found to be the more potent to inhibit the signaling from both canine IL-4 and IL-13. However, the antibodies according to the invention were found to be about 5 times more potent than C146E2-H3L3.

[0019] In this context, 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:

[0020] - 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 (HASX4YWRGAGKIDA where X4is N or T), and

[0021] - 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).

[0022] The present invention also relates to an antigen-binding fragment or antigenbinding derivative of the antibody according to the invention, preferably an antigenbinding fragment selected in the group consisting of a Fab fragment, a Fab’ fragment, a Fab’-SH fragment, a F(ab’)2 fragment, an Fv fragment; or an antigen-binding derivative selected in the group consisting of synthetic or semisynthetic antibody-derived molecule selected from a scFv, a dsFv, a minibody, a diabody, a tribody, a scFab and a ScFabAC; optionally designed for extending half-life such as by fusion with a canine Fc fragment, canine serum albumin, VHH anti-canine serum albumin or by grafting an alternative scaffold directed against canine serum albumin or chemical polymers such as polyethyleneglycol (PEG) or polypeptides such as PAS or XTEN polypeptides.

[0023] The present invention also relates to a bispecific antibody comprising an antigenbinding fragment or derivative according to the invention and an antigen-binding fragment or derivative directed to one other target relevant for treating atopic dermatitis.

[0024] The present invention also relates to a nucleic acid or combination of two nucleic acids encoding the antibody according to the invention or the antigen-binding fragment or the antigen-binding derivative thereof according to the invention or the bispecific antibody according to the invention.

[0025] The present invention also relates to a vector comprising the nucleic acid(s) according to the invention.

[0026] The present invention also relates to a host cell comprising the nucleic acid(s) according to the invention or the vector according to the invention.

[0027] The present invention also relates to the anti-canine IL-4o antibody according to the invention or the antigen-binding fragment or derivative thereof according to the invention, or the bispecific antibody according to the invention, for use as a medicinal product, in particular for use in the treatment and / or prevention of itch and / or inflammatory skin due to atopic dermatitis and / or allergies in dogs, preferably in the treatment of canine atopic dermatitis.

[0028] DESCRIPTION OF THE FIGURES

[0029] Figure 1 represents IL-4 signaling pathways, with type 1 (IL-4Ro / yc) induced by binding of IL-4 to receptor IL-4Ro and with type 2 (IL-4Ro / IL-13Ro1 ) induced by both binding of IL-4 and IL-13 to their respective receptors.

[0030] Figure 2 represents the structure of STAT6-regulated nluc reporter cassette. 4 STAT6 transcription factor consensus binding sites (S6bs) locate just 5’ of the minimal TATA-box promoter (minP) to maximally affect the expression of nano-luciferase reporter. After genome integration of Spel-Nhel transgene cassette, 5’ synthetic polyA signal and transcriptional pause site should insulate the STAT6-nluc reporter cassette from potential transcriptional interference of nearby chromatin-bound factors.

[0031] Figure 3 represents concentration-dependent canine IL-4 STAT6 nluc reporter activity in MDCK clone #13.

[0032] Figure 4 represents concentration-dependent canine IL-13 STAT6 nluc reporter activity in MDCK clone #13.

[0033] Figure 5 represents analysis of purified cECD-IL-4Ra on Coomassie stained 4-15% SDS-PAGE gel under non-reducing conditions, with loading of 1.3 pg of purified product.

[0034] 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 .

[0035] 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 .

[0036] Figure 9 represents schedule of events for in vivo experiments in acute canine atopic dermatitis model.

[0037] Figure 10 represents dermatologic score on average, measured after in vivo experiments in the four groups of dogs, wherein Group 1 received a single subcutaneous injection of anti-IL4R at a dose of 5mg / kg seven days before the first application of D. farina, Group 2 received a single subcutaneous injection of anti-IL4R at a dose of 5mg / kg three days before the first application of D. farina, Group 3 were treated once daily with Dermipred® at a dose of 1mg / kg, on 7 consecutive days starting one day before the first application of D. farina, and Group 4 were untreated control.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Figure 14 represents histological staining of RCE not stimulated with the pro- inflammatory cytokines (negative control).

[0042] Figure 15 represents histological staining of RCE stimulated with the pro- inflammatory cytokines (positive control).

[0043] Figure 16 represents histological staining of RCE-AD treated with the monoclonal antibody targeting the IL-4R CAN-27E1 -338-VHN / 329-VLA.

[0044] Figure 17 represents histological staining of RCE-AD treated with the reference molecule oclacitinib.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] In the context of the present invention, the inventors surprisingly found that new anti-canine interleukine-4-receptor alpha (IL-4Ro) monoclonal antibodies of particularly high potency regarding inhibition of IL-4Ro signaling pathway. Compared to anti-canine IL-4Ro monoclonal antibodies of the prior art, such potent antibodies could have the advantage of using lower doses for disease treatment and having a longer lasting effect, thereby allowing subjects to be treated less frequently. This will bring comfort to the subjects to be treated and lower the overall cost of the treatment.

[0051] The anti-canine IL-4Ro monoclonal antibodies of the present invention have a significantly higher potency than those disclosed in prior art. In particular, of all tested prior art antibodies, C146E2-H3L3 was found to be the more potent to inhibit the signaling from both canine IL-4 and IL-13. However, the antibodies according to the invention were found to be about 5 times more potent than C146E2-H3L3.

[0052] Anti-canine IL-4Ra antibody

[0053] 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:

[0054] - 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 (HASX4YWRGAGKIDA where X4is N or T), and

[0055] - 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). Throughout the present description, “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.

[0056] “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. Indeed, 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. These three CDR regions, and particularly the CDR3 region, are found in the part of the antibody that will be in contact with the antigen and are therefore very important for antigen recognition. Thus, 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. In a certain number of cases, 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. The CDR3 region is also followed by a framework region FR4.

[0057] 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, 5thEd., 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. Another method is referred to as “Abm”, which CDRs corresponds to a compromise between the Kabat and Chothia methods (Whitelegg & Rees, 2000 and 2004). Still another method was proposed by the IMGT, based on determining hypervariable regions. In this method, a unique numbering has been defined to compare variable regions regardless of the antigen receptor, the chain type or the species (Lefranc et al., 2003). 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). Throughout the present description, the CDR sequences are defined according to the Kabat nomenclature. In particular, 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.

[0058] Functional features

[0059] In the present invention, the antibody is directed against the canine interleukine- 4-receptor alpha subunit (IL-4Ro). The amino acid sequence of canine IL-4Ro polypeptide chain is shown in NCBI reference sequence XP_022275635.1 , as follows (SEQ ID NO: 7): MGRLCSGLTFPVSCLVLVWVASSGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVP ENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTE NHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNY YEPWEQHLPLGVSISCLVILAICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWK TCLTKLLPCLLEHGLGREEESPKTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEED KRSLCPSLEGSGGSFQEGREGIVARLTESLFLDLLGGENGGFCPQGLEESCLPPPSGSVGAQMPWAQFPRAGPRAA PEGPEQPRRPESALQASPTQSAGSSAFPEPPPVVTDNPAYRSFGSFLGQSSDPGDGDSDPELADRPGEADPGIPSAP QPPEPPAALQPEPESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSL LPGGATCPGTSGGEAGSGEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAG KEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHLCGHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRA PNVLPGGVLLEASLSPASLVP The amino acid sequence of canine IL-4Ro extra cellular domain mature polypeptide chain which was produced as a recombinant protein and used for immunization and screening is shown, this sequence has a six-histidine tag at the C- terminal end; which is as follows (SEQ ID NO: 8):

[0060] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQL DLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVY NVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHGSGHHHHHH

[0061] The “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. Subsequently, 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).

[0062] When bound by IL-4, 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. Alternatively, 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). Generally, type 1 IL-4R signaling is restricted to cells of hematopoietic origin, whereas type 2 signaling is more widely distributed (Murata et al., 1998). Both the interaction of IL-4 with its high-affinity receptor IL-4Ro and the subsequent recruitment of the IL-2Ry chain have been characterized (Zhang et al., 2002a, b), and ectodomain crystal structures are available for all three ligand-induced heterodimers (Hage et al., 1999; LaPorte et al., 2008).

[0063] 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. However, 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):

[0064] MKVLQEPTCVSDYMSISTCEWKMNGPTNCSTELRLLYQLVFLLSEAHTCIPENNGGAGCVCHLLMDDVVSADNYTL DLWAGQQLLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTYAVNIWSENDPADFRIYN VTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSEWSPSTKWHNSYREPFEQH

[0065] The terms “binds” or “binding” as used herein refer 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. The value of 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. Preferably, 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).

[0066] 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.

[0067] 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. Preferably 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. Preferably, 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. Preferably 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.

[0068] “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):

[0069] MERPARLCGLWALLLCAAGGRGGGVAAPTETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCTLRYFSHFDNKQ DKKIAPETHRSKEVPLNERICLQVGSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQCVWHNLSYMKCTWLPGR NTSPDTNYTLYYWHSSLGKILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQIVVKDNAGKIRPSFNIVPLTSHVKP DPPHIKRLFFQNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETNDIFYVEEAKCQNSEFEGNLEGTICFMVPGVLP DTLNTVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPTFYITMLLATPVIVAGAIIVLLLYLKRLKIIIFPPIPDPGKIF KEMFGDQNDDTLHWRKYDIYEKQTKEETDSVVLIENLKKASQ

[0070] Signaling pathways activated by IL-4 and IL-13 binding to their respective receptors are summarized in Figure 1.

[0071] 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.

[0072] 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. In particular, one or more STAT6 binding consensus sequences may be inserted just upstream of a minimal promoter. b) a sandwich immunoassay for quantitative detection of phospho-STAT6 (phosphorylated on Tyr641 ) in cellular lysates (referred to as an alphaLISA assay) using:

[0073] • two distinct anti-phospho-STAT6 antibodies, one of which is biotinylated and the other is labeled with a tag,

[0074] • Donor beads coated with streptavidin to capture the biotinylated anti- phospho-STAT6 antibody, wherein the donor beads contain a photosensitizer that converts ambient oxygen to singlet oxygen (an excited and reactive form of oxygen) upon illumination at a defined wavelength (for example, the photosensitizer may be phthalocyanine, which converts ambient oxygen to singlet oxygen upon illumination at 680 nm),

[0075] • 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).

[0076] Within its 4 psec half-life, singlet oxygen can diffuse approximately 200 nm in solution. Therefore, in the presence of phospho-STAT6, the two anti-phospho-STAT6 antibodies bring the Donor and Acceptor beads close together (less than 200 nm), and the singlet oxygen emitted by donor beads excites acceptor beads, which then emit light, thus generating signal. In the absence of phospho-STAT6, the donor beads and acceptor beads are not close enough for the singlet oxygen emitted by donor beads to excite acceptor beads. The amount of light emission is thus directly proportional to the amount of phosphoprotein present in the sample.

[0077] One can notably use the AlphaLISA SureFire Ultra p-STAT6 (Tyr641 ) Assay Kit available from Perkin Elmer. In this assay, 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.

[0078] No matter the assay used, it is preferably carried out with culture supernatants or with purified antibodies, and more preferably with purified antibodies. Structural features

[0079] 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.

[0080] 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:

[0081] - 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 (HASX4YWRGAGKIDA where X4is N or T), and

[0082] - 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).

[0083] Indeed, 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.

[0084] Preferred CDRs

[0085] Particularly preferred 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:

[0086] - 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

[0087] - 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).

[0088] Other preferred 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:

[0089] - 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).

[0090] Preferred heavy chain variable regions (VH)

[0091] The CDR-H sequences mentioned above are preferably integrated in the following VH framework regions:

[0092] VH FR1 : EVQLVESGGDLVKPGGTLRLSCVASGFTFS (SEQ ID NO: 13);

[0093] VH FR2: WVRQSPGKGLQWVA (SEQ ID NO: 14);

[0094] VH FR3: RATISRDDAKNTLYLQLNSLRAEDTAVYYCAK (SEQ ID NO: 15); and

[0095] VH FR4: WGQGTLVTVSS (SEQ ID NO: 16).

[0096] 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).

[0097] Particularly preferred anti-canine IL-4Ro antibodies according to the invention have a heavy chain comprising a variable region with of sequence: EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSS (SEQ ID NO:

[0098] 18).

[0099] Other preferred anti-canine IL-4Ro antibodies according to the invention have a heavy chain comprising a variable region with of sequence: EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASNYWRGAGKIDAWGQGTLVTVSS (SEQ ID NO:

[0100] 19).

[0101] Preferred light chain variable regions (VL)

[0102] The CDR-L sequences mentioned above are preferably integrated in the following VL framework regions:

[0103] VL FR1 : SSVLTQPPSVSVSLGQTATITC (SEQ ID NO: 20);

[0104] VL FR2: WX2QQKSPGQAPMTVIY (SEQ ID NO: 21 ), where X2is F (WFQQKSPGQAPMTVIY (SEQ ID NO: 22) or where X2is is Y (WYQQKSPGQAPMTVIY (SEQ ID NO: 23), and preferably X2is 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 Xi5is S (GIPDRFSGSGSGSTSTLTISGAQAEDEAEYFC (SEQ ID NO: 26)); and VL FR4: FGSGTQLTVL (SEQ ID NO: 27).

[0105] Preferably, 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.

[0106] Anti-canine IL-4Ro antibodies according to the invention preferably have a light chain comprises a variable region of sequence:

[0107] SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWX33QQKSPGQAPMTVIYDNDKRPSGIPDRFSGSX64SGS TX69TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL, where X33 is F or Y, X64is G or S et X69is S or H (SEQ ID NO: 28).

[0108] Particularly preferred anti-canine IL-4Ro antibodies according to the invention have a light chain comprises a variable region of sequence:

[0109] SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL (SEQ ID NO: 29).

[0110] Other preferred anti-canine IL-4Ro antibodies according to the invention have a light chain comprises a variable region of sequence:

[0111] SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWYQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSS SGSTHTLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL (SEQ ID NO: 30).

[0112] Preferred combinations of heavy and light chains variable regions (VH and VL)

[0113] Particularly preferred anti-canine IL-4Ro antibodies according to the invention comprise:

[0114] - 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) SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL, or

[0115] - 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) SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWYQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSSSGSTH TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL, or

[0116] - 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) SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL, or

[0117] - 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) SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWYQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSSSGSTH TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL.

[0118] Most preferred anti-canine IL-4Ro antibodies according to the invention comprise:

[0119] - a heavy chain comprising a variable region with a sequence (SEQ ID NO: 18) EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSS, and

[0120] - a light chain comprising a variable region with a sequence (SEQ ID NO: 29) SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVL.

[0121] Caninized antibodies

[0122] The anti-canine IL-4Ro antibodies of the present invention are preferably caninized antibodies. In particular, the above-described VH and VL amino acid sequences are caninized sequences.

[0123] 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.

[0124] 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. Similarly, 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. As used herein, "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). In addition, 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). As used herein the term "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. Based upon information on the structure of immunoglobulin variable regions, and with the guidance of an homology molecular model of the Fv of the parental monoclonal antibody, a few residues in the framework regions that are identified as having key roles in either maintaining the CDRs in the right conformation or in VH / VL packing, may or not be retained in caninized versions after comparing caninized versions retaining them with caninized versions substituting them with their canine germline counterparts. Under guidance of the homology molecular model, some CDR residues, as defined by KABAT, may also be substituted or not for their canine germline counterparts (so called germlining) in caninized versions when judged possible the CDR residues, in order to increase the degree of canineness (i.e. percentage sequence identity for both VH and VL between the caninized versions and the closest canine germline used as acceptor sequence for the CDR-grafting).

[0125] 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. Also, the structural models permit making more intelligent choices regarding back-mutations in light of the particular germlines involved. Note that the Kabat CDR definitions are not as strictly structural as those of other systems; thus, for some germlines the Kabat definitions are too broad. For both chains, heavy and light, we can usually be fairly confident that the assignment of residues from CDR1 and 2 as paratopic and non-paratopic, based upon the structural model, is correct. Similarly, 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.

[0126] Preferred constant regions

[0127] The constant regions of antibodies according to the invention are preferably canine constant regions.

[0128] 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.

[0129] Advantageously, the anti-canine IL-4Ro antibody according to the present invention is of canine isotype IgG, and more preferably of canine isotype IgGB. In 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”.

[0130] 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. For example, 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 , and 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. For example, the kappa light chain amino acid sequence has the accession number F1 NY2 in UniProtKB and the lambda light chain amino acid sequence can be found in IMGT at the following address; http: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genet able&species=dog&group=IGLC.

[0131] Thus, in one advantageous embodiment the heavy chain of the anti -canine IL-4Ro antibody according the invention comprises a wild type canine IgGB constant region (SEQ ID NO: 31 ): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG The anti-canine IL-4Ra antibody according to the invention can also be optimized for absence or reduction of certain effector functions, and especially in order to avoid or reduce ADCC (antibody-dependent cell cytotoxicity), ADCP (antibody-dependent phagocytosis); the cell-mediated reactions wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell and / or CDC (complement-dependent cytotoxicity); activation of the classical complement pathway by binding of protein C1q to bound antibody on a target cell and subsequently resulting in target cell lysis. Thus, 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. Generally, 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.

[0132] For example, it has been shown that several types of mutations in the 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).

[0133] In the context of the present invention, 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):

[0134] - hinge mutation K228P, to avoid Fab arm exchange and improve manufacturability of the molecule,

[0135] - CH2 mutation(s) M234A and / or L235A reducing binding to Fc gamma receptors, thus avoiding ADCP and ADCC,

[0136] - CH2 mutation D265A, which reduces binding to Fc gamma receptors, thus avoiding ADCP and ADCC,

[0137] - CH2 mutation P329G or P329A, which reduces binding of C1q complement protein;

[0138] - CH2 mutation 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.

[0139] In particular, preferred mutant canine IgGB heavy chain’s constant regions may contain:

[0140] - the three mutations M234A-L235A-P329G in order to 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: 32): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0141] - the three mutations M234A-L235A-P329A in order to reduce binding to Fc gamma receptors and avoid ADCP and ADCC (mutations M234A-L235A) and reduce binding of C1q complement protein (P329A), such as the sequence (SEQ ID NO: 33): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALASPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0142] - the four mutations 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 HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0143] - the four mutations K228P-M234A-L235A-P329A 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 (P329A), such as the sequence (SEQ ID NO: 35): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPPCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALASPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0144] - the two mutations 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 HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0145] - the two mutations 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 HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0146] - the two mutations 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 HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0147] - the two mutations 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 HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0148] - the two mutations K228P-N297G for improving manufacturability (K228P) and having a non-glycosylated (aglycosylated) Fc with reduced ADCP and ADCC activities (N297G), such as sequence (SEQ ID NO: 40): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPPCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFGGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0149] - the two mutations K228P-N297A for improving manufacturability (K228P) and having a non-glycosylated (aglycosylated) Fc with reduced ADCP and ADCC activities (N297A), such as sequence (SEQ ID NO: 41 ): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPPCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0150] - the two mutations D265A-N297G for reducing binding to both FcyR and C1q, such as sequence (SEQ ID NO: 42): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDP EDPEVQISWFVDGKQMQTAKTQPREEQFGGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0151] - the two mutations D265A-N297A for reducing binding to both FcyR and C1q, such as sequence (SEQ ID NO: 43): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDP EDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0152] - the single mutation N297G for reducing binding to both FcyR and C1q, such as sequence (SEQ ID NO: 44): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFGGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0153] - the single mutation N297A for reducing binding to both FcyR and C1 q, such as sequence (SEQ ID NO: 45): ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWP SETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDP EDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQA HQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQ RGDTFICAVMHEALHNHYTQESLSHSPG

[0154] Also, in one advantageous embodiment, 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.

[0155] In particular, 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) .

[0156] Alternatively, or in combination with mutations intended to limit the antibody’s effector (ADCC and / or CDC) functions, 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.

[0157] Preferred full-length antibodies

[0158] By fusing the preferred heavy and light chain constant regions to the C-terminus of preferred VH or VL domains, the preferred complete amino acid sequences of the antibodies according to the present invention are obtained, as described in Table 1 below:

[0159] Antigen-binding fragments or antigen-binding derivatives

[0160] The present invention also relates to an antigen-binding fragment or an antigenbinding derivative of the antibody according to the invention. By “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.

[0161] 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.

[0162] A "Fab'-SH fragment” refers to a Fab’ fragment containing a free sulfhydryl group.

[0163] A “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.

[0164] 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.

[0165] By “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.

[0166] By “scFv” or “single-chain variable fragment” or “single-chain 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 a peptide called a “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.

[0167] By “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.

[0168] 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).

[0169] 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).

[0170] By “scFab” or “single chain Fab”, it is referred to a Fab fragment derivative, in which the constant part of the light chain (CL) is further indirectly fused to the variable part of the heavy chain (VH) via a peptide linker to form a consecutive polypeptide chain.

[0171] 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).

[0172] Some of the 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”.

[0173] Antigen -binding derivatives of particular interest are:

[0174] - monovalent 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).

[0175] 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. In this case, 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.

[0176] - monovalent single chain Fab-Fc (scFab-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).

[0177] The monovalent scFab-Fc can be obtained using the Knobs-into-holes’ technology originally described by Ridgway et al. in 1996 as disclosed above.

[0178] 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).

[0179] Exemplary amino acid sequences of the heavy and light chains of a Fab-Fc fusion and a scFab-Fc fusion are presented in Table 2 below:

[0180] As for antigen-binding fragments as defined above, 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.

[0181] Bispecific antibodies

[0182] The above-mentioned antigen-binding fragments and antigen-binding derivatives may be used to produce bispecific antibodies, which also represent an aspect of the present invention. In another aspect, 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.

[0183] 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.

[0184] Nucleic acids encoding an antibody, antigen-binding fragment, antigen-binding derivative or bispecific antibody of the invention

[0185] 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.

[0186] All the different nucleic sequences, because of degeneration of the genetic code, encoding a particular amino acid sequence are within the scope of the invention.

[0187] In particular, the 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. Indeed, there are in general several three- nucleotide combinations encoding the same amino acid (except for methionine and tryptophan), called synonymous codons. However, 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).

[0188] This preference depends notably on the producing organism from which the cell is derived. Consequently, when a protein derived from one or more organisms is produced in a heterologous organism or a cell of such a heterologous organism, it may be useful to modify the nucleic sequence encoding the protein to use mainly the preferred codons of the heterologous organism. Data are available in the literature concerning the use of codons preferred by different species and a person skilled in the art knows how to optimize the expression of a given protein in a heterologous organism or a cell of a heterologous organism.

[0189] 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.

[0190] 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.

[0191] 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

[0192] The present invention also relates to a vector comprising a nucleic acid or combination of nucleic acids according to the invention. Such 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. In particular, for a vector designed for expression in eukaryotic cells, the vector advantageously comprises a Kozak consensus sequence, i.e. , a conserved sequence found at the translation start site of eukaryotic messenger RNA, around the AUG start codon (generally GCCGCCRCCATGG, SEQ ID NO: 57), the translation initiation codon being underlined). The vector can notably be a plasmid or viral vector. It is used to clone or express the nucleic acids according to the invention.

[0193] The one skilled in the art would routinely know and found vectors able to be used in the context of the invention, including the transcription unit to be used.

[0194] Host cells, transgenic non-human animals, transgenic plants

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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. 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).

[0199] 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.

[0200] Therapeutic uses and methods

[0201] 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.

[0202] 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.

[0203] In a first embodiment, 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.

[0204] In a second embodiment, 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] The present invention also concerns 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.

[0209] By “treatment” is meant an improvement, observed at the clinical or biochemical level, of the subject’s disease.

[0210] By “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.

[0211] Persons skilled in the art know, on the basis of their general knowledge, how to determine which clinical or biochemical manifestations are associated with a given disease and which are likely to be improved (treatment) or prevented, delayed or decreased in intensity (prevention). In the context of atopic dermatitis, 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.

[0212] The following examples aim at illustrating the present invention. EXAMPLES

[0213] Example 1 : Generation of MDCK nLuc Stat6 cell-based luciferase assay to measure IL4- or IL13-induced STAT6 transcription factor activation

[0214] For functional testing of antibody supernatants and recombinant anti-canine IL- 4Ralpha antibodies, 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.

[0215] Materials and Methods

[0216] MDCK cells were purchased from ATCC (parental MDCK cell line CCL-34™). 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.

[0217] In order to have a convenient and quantitative assay to measure canine IL4Ralpha activation, 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. 70% confluentMDCK cells (grown on 6-well plate) in serum free medium were transfected using 1 ug of linearized DNA construct mixed with 4ul of PEI transfection reagent. 48h post-transfection antibiotic selection was initiated by including 400ug / ml HygromycinB in growth medium. Antibiotic-containing medium was replaced after every 3 days and the selection lasted for 10 days. 70-100 small colonies were observed on the plate and cells from 36 distinct colonies were transferred into 96-well plate to expand the cells and later test inducibility of the nluc reporter. 16 clones demonstrated good recovery and growth after plating onto 96-well plate and they were transferred to 24-well plate for further cultivation. Each clone was plated in duplicate onto 48-well plate to perform inducibility test with canine IL-4. 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).

[0218] Out of the 16 clones tested, 2 clones (#13 and 25) showed good response of the nluc reporter. These two cell clones were then expanded and 12 vials of each clone was cryopreserved in liquid nitrogen for long-term storage. For clone #13 a concentration range of STAT6-nluc reporter assay responsiveness using serially-diluted canine IL-4 and canine IL-13 were performed (see Figure 3 and Figure 4, respectively).

[0219] From the results showed in Figure 3 and 4, a concentration of canine IL-4 and canine IL-13 around 5 and 1 .5 ng / ml, respectively, seems adequate for the screening and characterization of inhibitory potency of the anti-IL-4Ralpha antibodies in the MDCK cellular assay.

[0220] Conclusions

[0221] 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.

[0222] EXAMPLE 2: PRODUCTION AND PURIFICATION OF CANINE EXTRA CELLULAR DOMAIN IL-4 RECEPTOR ALPHA (c-ECD-IL-4Ra)

[0223] Recombinant extra cellular domain of canine IL-4 receptor alpha (cECD-IL-4Ro) was produced and purified 1 ) to be used for immunization and screening in order to develop antibodies against canine IL4Ro and 2) to be used for characterization of the binding of the antibodies to cECD-IL-4Ro.

[0224] Materials and Methods

[0225] 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.

[0226] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQL DLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVY NVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHGSGHHHHHH (SEQ ID NO: 8).

[0227] On day four after transfection additional feed was added and temperature was shifted from 37° C to 30° C for production. Overall protein production time was 12 days. At the end of production, the supernatants were clarified by centrifugation and phenylmethylsulfonyl fluoride (PMSF) was added. The supernatants were frozen and kept at -20 ° C until purification.

[0228] Recombinant cECD-IL-4Ra was then purified by IMAC using HisTrap™ Excel columns (GE Healthcare) followed by preparative gel filtration with Superdex 200 Increase 10 / 300 (GE Healthcare).

[0229] Results

[0230] Purified material was analysed by SDS-PAGE under non-reducing conditions. In Figure 5, a broad band spreading below and above the 35 Kd molecular weight marker is clearly visible on the gel indicative of a highly glycosylated protein. The theoretical molecular weight for cECD-IL-4Ra is 24,9 Kd and the protein has seven potential N- glycosylation sites which is in accordance with the broad band seen on the gel.

[0231] Conclusions

[0232] Altogether, 35 mg of purified cECD-IL4Ra was obtained, aliquoted and stored at - 80° C.

[0233] EXAMPLE 3: ISOLATION OF RECOMBINANT MONOCLONAL ANTIBODIES BY HYBRIFREE TECHNOLOGY

[0234] Materials and Methods

[0235] Monoclonal recombinant blocking antibodies against canine IL4Ro were developed from immunized chickens and rabbits using HybriFree Technology (Kivi et al. 2016).

[0236] A total of 4 chickens and 4 rabbits were immunized with recombinant canine ECD- IL4Ra (clL4Ra) produced in CHO and purified on HiTrap column followed by gel filtration. Immune response of chickens or rabbits after 3rdimmunization with clL4Ra was evaluated by ELISA titer, after using different immunization strategies to increase diversity of the developed antibodies.

[0237] 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).

[0238] 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.

[0239] All test animals had a clear immune response against canine IL4Ra, with especially a high antibody titer for rabbits.

[0240] Results

[0241] Immuno modules (Thermo Scientific™) 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. Amplified 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.

[0242] In total 296 panning reactions with different panning conditions using spleen cells from immunized animals were performed and 113 ELISA positive hlgG1 antibody pools were identified. From the three immunized rabbits, 38 clones with unique sequences were isolated and from the three immunized chickens 26 clones with unique sequences were isolated.

[0243] Selected unique antigen-binding antibody clone supernatants were analyzed on MDCK cell-based assay.

[0244] Conclusions

[0245] Altogether, 8 rabbit-derived antibodies and 2 chicken-derived antibodies were identified that, in a concentration-dependent manner, suppressed IL4-induced STAT6- nluc reporter activity. All 10 lead antibodies suppressed STAT6-nluc reporter activity.

[0246] Example 4: Preparation and characterization of chimeric antibodies

[0247] 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.

[0248] Materials and Methods

[0249] 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).

[0250] In order to produce chimeric antibodies, 15x106CHOEBNAL 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. At the end of production phase 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.

[0251] 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.

[0252] For performing cell-based STAT6-nluc reporter activation assays, 5ng / ml 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. For most experiments clgGB concentration range from 5000-2.3 ng / ml was used. For 31 H9 and 27E1 antibodies, 2500-1.1 ng / ml range was also used for IC50 calculation experiments. For the 4H3, reference antibody (W02016 / 156588, Intervet), the concentration range was much higher, between 108-0.05 pg / ml. Serially-diluted antibodies were applied in triplicates for IC50 concentration calculation from cell-based- assay experiments. IC50 value was calculated at least from 3 independent experiments. GraphPad Prism software was used for calculation of IC50 concentrations.

[0253] Binding of the purified antibodies to cECD-IL-4Ra was analyzed using the Octet system (BLI technology, Fortebio). The following protocol was used:

[0254] 1 ) 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.

[0255] 2) For binding measurement, sensors were loaded with biotinylated canine-ECD-IL-4R alpha (5 pg / ml in kinetic buffer) for 30 sec.

[0256] 3) Afterwards sensors were transferred to kinetic buffer for 120 sec. for baseline establishment.

[0257] 4) Next, 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.

[0258] 5) Then sensors were transferred to kinetic buffer for 300 sec. for off-rate measurement. Kinetics data were fit using a 1 :1 binding local model of data analysis software provided by Forte Bio.

[0259] Results

[0260] The binding measurements and inhibition of canine IL-4-induced STAT6-nluc reporter activation of chimeric antibodies were performed. In relative affinity measurement using Octet, 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). In the MDCK-nluc STAT6 reporter assay, 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.

[0261] 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.

[0262] 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.

[0263] The titration curves and the average calculated IC50 concentrations for 27E1 antibody are annexed as Figure 6 for IL-4 induction and as Figure 7 for IL-13 induction, respectively.

[0264] The relevant parameters measured for 27E1 show the potency of this antibody.

[0265] Several chimeric variants for 27E1 within the CDRs as defined by the Kabat nomenclature were also tested and show similar advantageous compared with the wildtype (original chicken VH and VL sequences) chimeric version (two separate experiments for IL-4 inhibition measure) (data not shown). The choice of the positions of the amino acid residues to be mutated, deleted or inserted was the results of a thorough inspection of the homology molecular model of antibody 27E1 that was built in order to guide the caninization design.

[0266] Conclusions

[0267] 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. In particular, the chicken antibody 27E1 were found to be the most potent lead candidate.

[0268] EXAMPLE 5: preparation and characterization of Fab fragments of 27E1

[0269] Materials and Methods

[0270] 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). Briefly, 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 HisTrap™ Excel columns (GE Healthcare) followed by preparative gel filtration with Superdex 200 Increase 10 / 300 (GE Healthcare).

[0271] For performing MDCK cell-based STAT6-nluc reporter activation assays, 6 ng / ml of canine IL-4 was used for induction on serum-starved (7-8hrs) 60-80% confluent MDCK reporter cells, grown in 96-well plates in serum free culture medium. Media volume was 100pl per well. Small aliquots of purified anti-canine IL-4Ralpha Fab molecules 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. 20 hours later media was discarded and MDCK reporter cells were lysed in 40pl of lysis buffer. The concentration range of Fab molecules were from 6pg to 2.7ng / ml. IC50 value was calculated from two separate experiments. GraphPad Prism software was used for calculation of Fab IC50 concentrations.

[0272] Results

[0273] Graphs showing the titration curves for Fab fragment of antibody 27E1 are shown in Figure 8. The average calculated IC50 concentrations was 308 pM.

[0274] Conclusions

[0275] Compared to the whole IgG format, and although being on average 2 times less potent, Fab fragment of 27E1 still have a subnanomolar IC50 which makes them very powerful antagonist molecules.

[0276] Example 6: preparation and characterization of caninized versions of 27E1

[0277] 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. In order to guide the caninization of the chicken (Gallus gallus domesticus) antibody 27E1 , an homology molecular model of the parental 27E1 antibody has been constructed. Several examples of the ability of the structural model to impact the caninisation design by CDR-grafting can be revealed by inspection of the model of the chicken 27E1 antibody. This is because inspection of selected regions of the model, in light of the sequences of the canine germline candidates for engrafting, can aid in the avoidance of potential design errors that might arise from a design based purely upon sequence considerations.

[0278] One of the challenges of a successful caninization design is the selection of residues from the canine framework to be ‘back-mutated’, i.e. to be substituted with the parental chicken amino acid, in order to prevent loss of affinity or antibody stability due to structural incompatibility of the framework with the engrafted chicken CDR sequences. Typically, these residues 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.

[0279] Materials and Methods

[0280] Without being restricted to any specific approach, 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 suitable expression plasmid, and transfect the plasmids containing desired caninized H and L chains into CHO cells, viii) Purify expressed caninized antibody from CHO supernatant, ix) Test purified caninized antibody for binding to canine IL-4 receptor alpha chain by Octet and potency in the MDCK cellular assay using both canine IL-4 and IL-13 for induction. The application of the above outlined steps resulted in a set of caninized VH and VL sequences.

[0281] Results

[0282] Generally, the construction of the homology model was uneventful except for the selection of suitable templates for VL CDR1 and VH CDR1 . In the case of 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. It is plausible that the expression of a functional VLA chain with such unique features in avians may be a hallmark of an evolutionary pressure designed to facilitate a structural complementation between the IgY short VL CDR1 and a long VH CDR3 (27E1 has a 14 amino acid long VH CDR3) (Conroy et al., 2014). In the case of 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.

[0283] An example of a vernier location is the heavy chain residue D(H73) (Kabat numbering). In the candidate germline for the caninization design, IGHV3-38, position 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 ). Thus, 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.

[0284] An example of a canonical residue is the light chain residue G(L64) (Kabat numbering). In one of the candidate germlines for the caninization design, IGKV3-29, (L64) is occupied by serine (S) as opposed to the 27E1 glycine (G). 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). Thus, G(L64) appears to be crucial to the adoption of a specific three-dimensional conformation by VL CDR2. The serine substitution would preserve all of these conformationally stabilizing mainchain contacts, in addition to acquiring a sidechain contact to the Vernier residue I (L48) , albeit with slight strain. Thus, in the IGKV3-29 germline it is probably not necessary that position (L64) be a candidate for back-mutation to the parental 27E1 residue, glycine.

[0285] Parental antibody 27E1 having VH and VL amino acid sequences of SEQ ID NOs: 58 (AVTLDESGGGLQTPGGALSLVCKASGFTFSSYGMGWMRQAPGKGLEWVAVINPAVSGSRQGYAPAVKGRATISR DDGQSTLRLQLNNLRTEDTGTYYCAKHASNYWNGAGRIDAWGHGTEVIVSS) and 59

[0286] (ALTQPSSVSANPGETVKITCSGGSGNDYGWFQQKSPGSAPVTVIYDNDKRPSNIPSRFSGSGSGSTSTLTITGVQA EDEAVYFCGGYDRHTYDAFGAGTTLTVL), respectively, 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). The sequences of the canine germlines are as follows: IGHV3-38*01 : EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYDMSWVRQSPGKGLQWVAVIWNDGSSTYYADAVKGRFTISRDNA KNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 60) IGHJ4*01 : NFDYWGQGTLVTVSS (SEQ ID NO: 61 ) IGLJ4*01 : YVFGSGTQLTVL (SEQ ID NO: 62)

[0287] In 27E1 caninized VH version M (338-VHM), as compared to the parental 27E1 chicken VH,2 residues in CDR3 were mutated; asparagine (N) at Kabat position H100A was mutated to arginine (R) (destruction of the potential NG motif at Kabat positions H100A / H100B) and arginine (R) at Kabat position H100E was mutated to lysine (K) .

[0288] In 27E1 caninized version N (338-VHN), as compared to 338-VHM, 1 extra residue in CDR3 was mutated; asparagine (N) at Kabat position H98 was substituted with threonine (T) in order to destroy the potential deamidation site NY (N at Kabat L98 and Y at Kabat L99; NY to TY) .

[0289] In 27E1 caninized VL version A (329-VLA), based on canine IGLV3-29*01 germline , eight (8) original chicken residues in the framework (FR) sequences were conserved; in framework 2 (FR2) phenylalanine (F) at Kabat position L36 and serine (S) at Kabat L39A; in framework 3 (FR3), glycine (G) at Kabat position L64; glycine (G) at Kabat position L66; glycine (G) at Kabat position L68; serine (S) at Kabat position L69; serine (S) at Kabat position L71 and phenylalanine (F) at Kabat position L87. The sequence of the canine germline IGLV3-29*01 is: SSVLTQPPSVSVSLGQTATITCSGESLSRYYAQWYQQKPGQAPMTVIYGDRERPSGIPDRFSSSSSENTHTLTISGAQ AEDEAEYYCEIWDASADD (SEQ ID NO: 63) . In 27E1 IGLV3-29*01 caninized VL version N (329-VLN), as compared to version A, three (3) residues were germlined, in framework 2 (FR2) phenylalanine (F) at Kabat position L36 was substituted with its corresponding canine germline IGLV3-29*01 residue tyrosine (Y; F L36 Y substitution); and in framework 3 (FR3) glycine (G) at Kabat position L66 and serine (S) at Kabat position L71 were substituted with their corresponding canine germline IGLV3-29*01 residues; serine (S; G L66 S substitution) and histidine (H; S L71 H substitution), respectively.

[0290] Many 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.

[0291] Among the different combinations produced, not all retained the relevant improved inhibition activity. The IC50 values obtained for the most relevant caninized versions for both canine IL-4 and IL-13 are shown in Table 4 below. In all individual assays, the chimeric version of antibody 27E1 has been used as the reference molecule.

[0292] Conclusions

[0293] Starting with 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.

[0294] Example 7: in vivo experiments

[0295] An acute canine atopic dermatitis (cAD) house dust mite (HDM) -sensitized dogs model was used. In order to establish a proof of concept of the therapeutic interest of an anti-canine IL-4 receptor alpha antibody in the treatment of AD, 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).

[0296] Materials and Methods

[0297] • Test item, reference item and allergenic element

[0298] 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) in the form of 20mg tablets administered orally and the allergenic element was Dermatophagoides farinae (Allergic element) as a powder, applied to the clipped sternal / abdominal / inguinal region of all dogs.

[0299] • Test system

[0300] 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:

[0301] 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

[0302] 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

[0303] 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

[0304] Group 4: untreated control

[0305] 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.

[0306] • In life-phase

[0307] 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,..).

[0308] All the animals were weighed during the clinical examination performed on day - 6 for each group in relation to treatment administration, ie on Day 0 for group 1 , on Day 4 for group 2 and Day 6 for group 3. 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.

[0309] In order to induce an allergic skin reaction in these sensitised dogs, they were all exposed (groups 1 to 4) to 50 mg of Allergic element, applied once a day on three consecutive days (D7 to D9) to the sternal / abdominal / inguinal region that had been clipped 5 days before the first application of the allergen. Immediately before the first application of the allergen, the clipped area of each dogs was tape-stripped.

[0310] A dermatological examination took place once a day from study days D7 to D14. The scoring used throughout the study was a modified version of the CADESI scoring system. It took into account erythema, papules and excoriation of the skin each on a scale of 0 to 3 (0= no erythema / papules or excoriation, 1= mild erythema / papules or excoriation, 2= moderate erythema / papules or excoriation and 3 severe erythema / papules or excoriation), assed on the 11 most frequently affected body areas (sternum, abdomen, perigenital area, and left and right axilla, thorax, inguinal area, and inner thigh). The schedule of events is described in Table 5 below and in Figure 9.

[0311] Results

[0312] Results of dermatologic scores on average for the four groups are summarized in Table 6 below and depicted in Figure 10.

[0313] Conclusions

[0314] In vivo experiments show that anti-IL4Ralpha 27E1 antibody as described herein is at least as much effective for treating atopic dermatitis in dogs as glucocorticoids (Dermipred®).

[0315] Example 8: Potency comparison between prior art anti-canine IL-4R antibodies

[0316] Generation of MDCK cell-reporter gene assay (MDCK 5x STAT6 Luc2P)

[0317] In order to have a convenient and quantitative assay to measure and properly compare the activation of the canine interleukin IL4 / 13 pathway, a MDCK -cell reporter gene assay (MDCK 5x STAT6 Luc2P) was generated to measure IL4-induced STAT6 transcription factor activation.

[0318] 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.

[0319] To generate a monoclonal cell line from the polyclonal pool of stable cells limited dilution cloning was used.

[0320] 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.

[0321] Then a screening in bioluminescence for each clone will be determine clones that have a good response to stimulation in terms of luminescence activity.

[0322] Thawing and culture medium:

[0323] DMEM 500mL (Gibco; 11960044)

[0324] Penicillin 100 units / mL- Streptomycin 100pg / mL (Gibco; 15140122)

[0325] Glutamax 1X (Gibco; 35050061 )

[0326] Non essential amino acids 1X (Gibco; 11140035)

[0327] Fetal Bovine Serum, qualified, heat inactivated 10% (Gibco; 10500064) Hygromycin B Gold 175 pg / mL (Invivogen; ant-hg-1 )

[0328] Bioassay medium:

[0329] DMEM 500mL (Gibco; 11960044)

[0330] Penicillin 100 units / mL- Streptomycin 100pg / mL (Gibco; 15140122)

[0331] Glutamax 1X (Gibco; 35050061 )

[0332] Non essential amino acids 1X (Gibco; 11140035)

[0333] Hygromycin B Gold 175 pg / mL (Invivogen; ant-hg-1 )

[0334] Ranking of the prior art antibodies potency

[0335] The inhibitory activity (potency) 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:

[0336] 152H11 and 146E2 candidates described in applications W02021 / 123089 and

[0337] W02021 / 123091 ;

[0338] M3, M8 and M9 candidates described in application W02021 / 188631 ;

[0339] Clone B and Clone I candidates described in application W02021 / 188631 ; 4H3 candidates described in application W02016 / 156588;

[0340] Dupi-H2L2 described in application W02017 / 102920.

[0341] 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. Then added 25pl of IL4 at 500ng / mL or 25pl of IL13 at 1 g / mL was added into each well that need IL4 or IL13 stimulation, respectively. After that plate was incubated at 37° C, 5% CO2 for 22h for IL4 and 24h for IL13. Then the plate was put at room temperature 10 minutes before the end of incubation. After that 50pl of Promega Bright-Glo™ Luciferase Assay reagent was added into each well of the plate. Relative luciferase units (RLU) were finally recorded by the Spark plate reader.

[0342] The four-parameter model was used to fit the dose response curve that correlates RLU and value of antibodies concentration.

[0343] Results

[0344] The IC50s obtained for all candidates are depicted in Figure 11 for IL4 stimulation and in Figure 12 for IL13 stimulation. The results demonstrated that 152H11 and 146E2 candidates described in applications W02021 / 123089 and W02021 / 123091 are the more potent prior art antibodies to inhibit the signaling from both canine IL-4 and IL-13, and in particular that 146E2 is found to be the best one.

[0345] Example 9: Comparison with the most potent prior art anti-canine IL-4R antibody

[0346] Based on results described in Example 8, 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-34™ 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 .

[0347] Materials and Methods

[0348] 1 . 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.

[0349] 3. 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.

[0350] 4. 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.

[0351] 5. In parallel, 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.

[0352] 6. Medium was removed from the plate and 100 pL per well of freshly prepared 1x Lysis Buffer from the AlphaLISA p-STAT-6 Assay Kit were added. The plate was agitated on a plate shaker at 350 rpm for 10 minutes at room temperature.

[0353] 7. 10pL of the lysates were transferred to a 384-well Proxiplate for assay.

[0354] 8. 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.

[0355] 9. 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.

[0356] 10. The plate was read using the AlphaScreen settings on the BMG Clariostar.

[0357] Results

[0358] The IC50 obtained for all candidates are depicted in Figure 13 and shown in Table 7 below. Conclusions

[0359] The results show that 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.

[0360] 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)

[0361] The efficacy of a monoclonal antibody targeting the canine IL-4R in comparison with a reference molecule in a model of canine reconstructed atopic epidermis (RCE-AD). In this RCE model, atopic dermatitis is induced through a cocktail of pro-inflammatory cytokines (IL-4, IL-13 and TNF-o).

[0362] Different concentrations of the monoclonal antibody CAN-27E1 -338-VHN / 329-VLA were tested (1 , 3, 10, 100 pm / mL).

[0363] The reference molecule used is oclacitinib and was tested at 10 pM concentration.

[0364] 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).

[0365] Histological morphological analysis

[0366] Analysis of the morphology by histological staining of negative control shows a normal appearance of the epithelium (stratum basale, stratum spinosum, stratum granulosom and stratum corneum) with no signs of inflammation or pathological modification (acanthosis and spongiosis) and the presence of numerous keratohyaline granules (Figure 14). Histological morphological analysis of positive control shows an altered appearance of the epithelium characterized by a moderate to severe hyperplastic, acanthosic and spongiotic epidermis with rare keratohyaline granules (Figure 15).

[0367] Histological morphological analysis of RCE-AD treated with the monoclonal antibody targeting the IL-4R (Figure 16) or with the reference molecule (Figure 17) shows a mild alteration of the epithelium characterized by a mild spongiosic epidermis with numerous keratohyaline granules. Evaluation of inflammation

[0368] The inflammation was evaluated by ELISA quantification of IL-8 in the medium.

[0369] Results are shown in Figure 18.

[0370] 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.

[0371] Conclusion

[0372] The above results performed on a model of canine reconstructed atopic epidermis (RCE-AD) evidently highlights an inhibitory effect of the mAb targeting the IL-4R according to the invention on the pro-inflammatory effect of the cytokine cocktail. The histological morphological analysis of the RCE-AD mAb IL-4R treated samples shows a decrease of the hyperplasia, spongiosis and the acanthosis compared to the positive control. Moreover, an increase of the keratohyaline granules is observed.

[0373] Taken together, these results suggest that 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).

[0374] Example 11 : Preparation of antigen-binding derivatives

[0375] The following antigen-binding derivatives of the anti-canine IL-4R antibodies according to the invention were generated.

[0376] Monovalent Fab-27E1 -Fc-canine IgGB (27E1-mono-1)

[0377] Monovalent Fab Fc format (i.e. IgG-like format but with only one arm) of caninized 27E1 anti-canine IL4Ralpha antibody (version VHN / VLA) fused with 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.

[0378] 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.

[0379] The corresponding amino acid sequences are the following:

[0380] 27E1 -338-VHN-canlgGB-MALAPG-Y349C-T366S-L368A-Y407V

[0381] EVQLVESGGDLVKPGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVK GRATISRDDAKNTLYLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSSASTTAPSVFPL APSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETF TCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVD LDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIE RTISKARGQAHQPSVCVLPPSREELSKNTVSLSCAIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDED GSYFLVSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 49)

[0382] Hinge-canlgGB-MALAPG-S354C-T366W

[0383] RVPRPPDCPKCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTA KTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIERTISKARGQAHQPSVYVLPPCRE ELSKNTVSLWCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFI CAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 50)

[0384] 27E1 -329-VLA-LAMBDA

[0385] SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDF YPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAP AECS (SEQ ID NO: 51 )

[0386] Monovalent scFab-27E1 -Fc-canine IgGB (27E1 -mono-2)

[0387] Monovalent single chain Fab (scFab) Fc format (i.e. IgG-like format but with only one arm) of caninized 27E1 anti-canine IL4Ralpha antibody (version VHN / VLA) fused with canine IgGB Fc containing the mutations M234A, L235A and P329G to reduce Fc related effector functions. 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.

[0388] 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).

[0389] 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.

[0390] The corresponding amino acid sequences are the following: scFab27E1 -VHN-canlgGB-MALAPG-Y349C-T366S-L368A-Y407V

[0391] SSVLTQPPSVSVSLGQTATITCSGGSGNDYGWFQQKSPGQAPMTVIYDNDKRPSGIPDRFSGSGSGSTS TLTISGAQAEDEAEYFCGGYDRHTYDAFGSGTQLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDF YPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHECSTVEKKVAP AECSGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGEVQLVESGGDLVK PGGTLRLSCVASGFTFSSYGMGWVRQSPGKGLQWVAVINPAVSGSRQGYAPAVKGRATISRDDAKNTL YLQLNSLRAEDTAVYYCAKHASTYWRGAGKIDAWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVAL ACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVD KPVPKRENGRVPRPPDCPKCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFV DGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIERTISKARGQAHQPS VCVLPPSREELSKNTVSLSCAIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLVSKLSVDKSR WQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 52)

[0392] Hinge-canlgGB-MALAPG-S354C-T366W RVPRPPDCPKCPAPEAAGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTA KTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALGSPIERTISKARGQAHQPSVYVLPPCRE ELSKNTVSLWCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFI CAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 50) Example 12: INHIBITORY POTENCY OF ANTIGEN-BINDING DERIVATIVES FOR IL4 STIMULATION

[0393] 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.

[0394] Results

[0395] The 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 .

[0396] Example 13: INHIBITORY POTENCY OF ANTIGEN-BINDING DERIVATIVES FOR IL13 STIMULATION

[0397] 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.

[0398] Results

[0399] The 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 .

[0400] BIBLIOGRAPHIC REFERENCES

[0401] Boulay, J.-L., O’Shea, J. J. and Paul, W. E. 2003. Molecular phylogeny within type I cytokines and their cognate receptors. Immunity 19, 159-163.

[0402] Carter, P. Bispecific human IgG by design. J. Immunol. Methods 2001 , 248, 7-15.

[0403] Chothia C, Lesk a M. 1987 Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. 196:901-17.

[0404] 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.

[0405] Conroy, P. J. et al. 2014 Reconciling the Structural Attributes of Avian Antibodies. J Biol Chem 289, 15384-15392.

[0406] Edelman, G.M., Cunningham BA, Gall WE, Gottlieb PD, Rutischauser U, Waxdal MJ. 1969. The covalent structure of an entire gammaG immunoglobulin molecule. Proc. Natl. Acad. USA, 63: 78-85eu

[0407] Fan G, Wang Z, Hao M, Li J. 2015. Bispecific antibodies and their applications. J Hematol Oncol 8:130.

[0408] Fisher R, et al. Vaccine 21 (2003) 820-825.

[0409] Gedon NKY and Mueller RS. 2018, Atopic dermatitis in cats and dogs: a difficult disease for animals and owners. Clin Transl Allergy.;8:41 .

[0410] Giudicelli V., Chaume D. and Lefranc M.-P. 2005 IMGT / GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes Nucl. Acids Res., 33(S1 ):D256-D261.

[0411] Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A. & Ruddle, F. H. 1980, Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A 77, 7380-7384.

[0412] Griffin CE and DeBoer DJ. 2001 , The ACVD task force on canine atopic dermatitis (XIV): clinical manifestations of canine atopic dermatitis. Vet Immunol Immunopathol.; 81 (3-4) :255-69.

[0413] Hage, T., Sebald,W. and Reinemer, P. 1999. Crystal structure of the interleukin- 4 / receptor alpha chain complex reveals a mosaic binding interface. Cell 97, 271 -281.

[0414] Hamilton JD, Ungar B, Guttman-Yassky E., 2015. Drug evaluation review: Dupilumab in atopic dermatitis. Immunotherapy 7:1043-58.

[0415] Kelly-Welch, A. E., E. M. Hanson, M. R. Boothby, and A. D. Keegan. 2003. Interleukin-4 and interleukin-13 signaling connections maps. Science 300:1527-1528. Kivi, G. et al. 2016 HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species. BMC Biotechnol 16, 2.

[0416] LaPorte, S. L., Juo, Z. S., Vaclavikova, J., Coif, L. A., Qi, X., Heller, N. M., Keegan, A. D. and Garcia, K. C. 2008. Molecular and structural basis of cytokine receptor pleiotropy in the interleukin-4 / 13 system. Cell 132, 259-272.

[0417] Larrick JW, Danielsson L, Brenner CA, Abrahamson M, Fry KE, Borrebaeck CA.Larrick JW, et al. 1989 Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction. Biochem Biophys Res Commun. ; 160(3) : 1250-6.

[0418] 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.

[0419] Leonard, W. J. and O’Shea, J. J. 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16, 293-322.

[0420] Liu AY, Robinson RR, Hellstrom KE, Murray ED Jr, Chang CP, Hellstrom I. Liu AY, et al. 1987. Chimeric mouse-human lgG1 antibody that can mediate lysis of cancer cells.. Proc Natl Acad Sci U S A. ;84(10) :3439-43.

[0421] Mineshige T, Kamiie J, Sugahara G, Shirota K.Mineshige T, et al. 2018. A study on periostin involvement in the pathophysiology of canine atopic skin. J Vet Med Sci.;80(1 ):103-111 .

[0422] Marsella R and Girolomoni, G. 2009 Canine models of atopic dermatitis: a useful tool with untapped potential. J Invest Dermatol.; 129(10):2351 -7.

[0423] Marsella R and De Benedetto A. 2017. Atopic Dermatitis in Animals and People: An Update and Comparative Review. Vet Sci . ;4(3) :37.

[0424] Marsella R. 2012 An update on the treatment of canine atopic dermatitis. Vet Med (Auckl).;3:85-91.

[0425] Murata, T., Taguchi, J. and Puri, R. K. 1998. Interleukin-13 receptor alpha’ but not alpha chain: a functional component of interleukin-4 receptors. Blood 91 , 3884-3891 .

[0426] Nelms, K., Keegan, A. D., Zamorano, J., Ryan, J. J. and Paul, W. E. 1999. The IL- 4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17, 701 - 738.

[0427] Nuttall TJ, Marsella R, Rosenbaum MR, Gonzales AJ, Fadok VA. et al. 2019. Update on pathogenesis, diagnosis, and treatment of atopic dermatitis in dogs. J Am Vet Med Assoc.;254(11 ):1291 -1300. Olivry T, Mayhew D, Paps JS, Linder KE, Peredo C, Rajpal D, Hofland H, Cote-Sierra J et al. 2016. Early Activation of Th2 / Th22 Inflammatory and Pruritogenic Pathways in Acute Canine Atopic Dermatitis Skin Lesions. J Invest Dermatol.; 136(10): 1961 -1969.

[0428] Renert-Yuval Y, Guttman-Yassky et al. 2019. What's New in Atopic Dermatitis. Dermatol Clin.; 37(2) :205-213.

[0429] Ridgway, J. B. B.; Presta, L. G.; Carter, P. ’Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996, 9, 617-621 .

[0430] Riechmann L, Clark M, Waldmann H, Winter G. 1988. Reshaping human antibodies for therapy. Nature. ;332(6162) :323-7.

[0431] Ryan TM, Ciavatta DJ, Townes TM. 1997. Knockout-transgenic mouse model of sickle cell disease. Science; 278: 873-876

[0432] Santoro D. 2018. Evaluation of the secretion of antimicrobial peptides and antimicrobial effect of skin wash in atopic and healthy dogs: a preliminary study. Vet Dermatol. ;29(5):402-e132.

[0433] Santoro D. 2019. Therapies in Canine Atopic Dermatitis: An Update. Vet Clin North Am Small Anim Pract.;49(1 ):9-26.

[0434] Schillberg S., Twyman RM, Fischer R. 2005.; Opportunities for recombinant antigen and antibody expression in transgenic plants-technology assessment. Vaccine. ;23(15):1764-9.

[0435] Stoger E, Sack M, Fischer R, Christou P.Stoger E, et al. 2002 Plantibodies: applications, advantages and bottlenecks. Curr Opin Biotechnol. ;13(2):161 -6.

[0436] Tamamoto-Mochizuki C, Paps JS, Olivry T. 2019. Proactive maintenance therapy of canine atopic dermatitis with the anti-IL-31 lokivetmab. Can a monoclonal antibody blocking a single cytokine prevent allergy flares? Vet Dermatol, doi: 10.1111 / vde.12715. Online ahead of print.

[0437] Tang L. 2001 . Molecular cloning of canine IL-13 receptor alpha chain (alphal and alpha2) cDNAs and detection of corresponding mRNAs in canine tissues. Vet Immunol Immunopathol. ;79(3-4) :181 -95.

[0438] Whitelegg NR, Rees AR. et al. 2000. WAM: an improved algorithm for modelling antibodies on the WEB. Protein Eng. ; 13(12):819-24.

[0439] Whitelegg N, Rees AR, et al. 2004 Antibody variable regions: toward a unified modeling method. Methods Mol Biol. ;248:51 -91 .

[0440] Wilhem S, Kovalik M, Favrot C. 2011 Breed -associated phenotypes in canine atopic dermatitis. Vet Dermatol. ;22(2): 143-149.

[0441] Weidemann, T., Ho' finger, S., Muller, K. and Auer, M. 2007. Beyond dimerization: a membrane-dependent activation model for interleukin-4 receptor-mediated signalling. J. Mol. Biol. 366, 1365-1373. Whitty, A. and Riera, T. V. 2008. New ways to target old receptors. Curr. Opin. Chem. Biol. 12, 427-433

[0442] Winter G. and Harris WJ. (1993), Trends Pharmacol., 14:139-43,

[0443] Zhang, J.-L., Buehner, M. and Sebald,W. 2002a. Functional epitope of common gamma chain for interleukin-4 binding. Eur. J. Biochem. 269, 1490-1499.

[0444] Zhang, J.-L., Simeonowa, I., Wang, Y. and Sebald, W. 2002b. The high-affinity interaction of human IL-4 and the receptor alpha chain is constituted by two independent binding clusters. J. Mol. Biol. 315, 399-407.

[0445] Patent literature cited

[0446] WO2017 / 102920

[0447] WO2016 / 156588

[0448] W02021 / 123089

[0449] W02021 / 123091

[0450] W02021 / 188631

[0451] W02003 / 101485

[0452] WO00 / 26357

[0453] W090 / 04036

[0454] WO95 / 17085

[0455] WO01 / 26455

[0456] W02004 / 050847

[0457] W02005 / 033281

[0458] W02007 / 048077

[0459] W02007 / 106078

Claims

CLAIMS1. An anti-canine IL-4Ra antibody, in which heavy and light chains respectively comprises CDR-H and CDR-L with the following amino acid sequences:- a heavy chain comprising three CDR-H (heavy chain CDR) with the following amino acid sequences CDR1 -H-27E1 : SEQ ID NO: 1 , CDR2-H-27E1 : SEQ ID NO: 2, and CDR3-H- 27E1 : SEQ ID NO: 3, and- a light chain comprising three CDR-L (light chain CDR) with the following amino acid sequences CDR1 -L-27E1 : SEQ ID NO: 4, CDR2-L-27E1 : SEQ ID NO: 5, and CDR3-L-27E1 : SEQ ID NO: 6.

2. The anti-canine IL-4Ro antibody according to claim 1 , wherein the heavy chain comprises a variable region of sequence SEQ ID NO: 17.

3. The anti-canine IL-4Ro antibody according to claims 1 or 2, wherein the heavy chain comprises a variable region of sequence SEQ ID NO: 18.

4. The anti-canine IL-4Ro antibody according to any one of claims 1 to 3, wherein the light chain comprises a variable region of sequence SEQ ID NO: 28.

5. The anti-canine IL-4Ro antibody according to any one of claims 1 to 4, wherein the light chain comprises a variable region of sequence SEQ ID NO: 29.

6. The anti-canine IL-4Ro antibody according to any one of claims 1 to 5, comprising- a heavy chain comprising a variable region with a sequence SEQ ID NO: 19 and a light chain comprising a variable region with a sequence SEQ ID NO: 29, or- a heavy chain comprising a variable region with a sequence SEQ ID NO: 19 and a light chain comprising a variable region with a sequence SEQ ID NO: 30, or- a heavy chain comprising a variable region with a sequence SEQ ID NO: 18, and a light chain comprising a variable region with a sequence SEQ ID NO: 29, or- a heavy chain comprising a variable region with a sequence SEQ ID NO: 18, and a light chain comprising a variable region with a sequence SEQ ID NO: 30.

7. The anti-canine IL-4Ro antibody according to claim 6, comprising:- a heavy chain comprising a variable region with a sequence SEQ ID NO: 18, and- a light chain comprising a variable region with a sequence SEQ ID NO: 29.

8. An antigen-binding fragment or an antigen-binding derivative of the antibody according to any one of claims 1 to 7.

9. A bispecific antibody comprising an antigen-binding fragment or an antigenbinding derivative according to claim 8 directed to one other target relevant for treating atopic dermatitis.

10. Nucleic acid or combination of two nucleic acids encoding the antibody according to any one of claims 1 to 7 or the antigen-binding fragment or the antigenbinding derivative thereof according to claim 8 or the bispecific antibody according to claim 9.

11. Vector comprising the nucleic acid(s) according to claim 10.

12. Host cell comprising the nucleic acid(s) according to claim 10 or the vector according to claim 11 .

13. The anti-canine IL-4o antibody according to any one of claims 1 to 7 or the antigen-binding fragment or derivative thereof according to claim 8, or the bispecific antibody according to claim 9, for use as a medicinal product.

14. The anti-canine IL-4o antibody according to any one of claims 1 to 7 or the antigen-binding fragment or derivative thereof according to claim 8, or the bispecific antibody according to claim 9, for use in the treatment and / or prevention of itch and / or inflammatory skin due to atopic dermatitis and / or allergies in dogs, preferably in the treatment of canine atopic dermatitis.

Images