Caninized il31rΑ antibodies with an extended half life

Abstract

The present invention generally relates to canine IgG antibodies or antigen binding proteins thereof, comprising a canine IgG modified fragment crystallizable region (Fc region) that has an enhanced half-life due to one or more specific amino acid substitutions in the Fc region. Specifically, the present invention relates to canine antibodies and antigen binding fragments thereof, that bind canine interleukin-31 receptor alpha (IL31RA), comprising a canine fragment crystallizable region (cFc) that comprises two, three, four or more specific amino acids at defined positions, such as 428, 434, 436, 438 and 440 in the cFc region. Pharmaceutical compositions that comprise these antibodies are also provided.

Description

[0001] CANINIZED IL31Ra ANTIBODIES WITH AN EXTENDED HALF LIFE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit to European Application No. 24218832.4, filed on December 10, 2024, the contents of which is hereby incorporated by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The present invention generally relates to caninized antibodies to canine IL-31 receptor alpha (clL-31 RA) that have an enhanced half-life due to the presence of one or more specific amino acid residues at defined positions in the canine fragment crystallizable region (Fc region) of the antibody. The present invention further provides the pharmaceutical compositions and nucleic acids sequence encoding the antibodies, and their use for the treatment of atopic dermatitis in dogs.

[0006] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0007] The contents of the electronic sequence listing (25949-EP.xml; Size: 50,978 bytes; and Date of Creation: December 6, 2024) are herein incorporated by reference in their entirety.

[0008] BACKGROUND

[0009] Atopic dermatitis (AD) is a common skin disease in companion animals, especially dogs, where its prevalence has been estimated to be approximately 10-15% of the canine population. The pathogenesis of atopic dermatitis in dogs was found to be very similar to that of humans (Nuttall et al., Veterinary Records 172(8):201-207 (2013)) including skin infiltration by a variety of immune cells and CD4+ Th2 polarized cytokine milieu including the preponderance of interleukins, particularly interleukin-31 (IL-31), interleukin-4 (IL-4) and interleukin- 13 (IL-13). Symptoms of canine AD are consequently also very similar to that of humans and includes relapsing, chronic pruritis and skin lesions.

[0010] To treat AD in dogs, canine immunoglobulin G (IgG) monoclonal antibodies (mABs) are being developed as effective therapeutics in veterinary medicine. These canine IgG mABs have been directed against some of the interleukins (IL) and / or the interaction with their respective receptors. For example, at canine IL-31 (W02013 / 011 ,407 A1 ; W02018 / 156,180 A1), of which there are commercial products available (e.g., Lokivetmab, commercialized under tradename Cytopoint®, proprietary product of Zoetis), or at canine IL-4 or its receptor canine IL-4 receptor alpha (clL-4 RA) (WO2021 / 123.089 A1 , WO2016 / 156,588 A1), or at canine IL-31 receptor alpha (clL-31 RA) (W02022 / 079, 139 A1). However, despite recent success in treating AD in dogs, none of the current therapies employed result in a rapid onset of antipruritic action concomitant with a significant effect on the skin inflammation with an improvement in skin barrier function.

[0011] One approach utilized in the art to improve antibody therapies has been to modulate the fragment crystallizable (Fc) regions of the IgGs. Specifically, the Fc region spanning the interface of CH2 and CH3 domains interacts with the FcRn on the surface of cells to regulate IgG homeostasis. The strong binding of the Fc region of the IgG to FcRn in endosomal compartment protects the antibody from degradation by proteolytic enzymes in endosomes and allows recycling of the receptor-bound antibody to the cell surface where the increased pH weakens the interaction and allow release of the antibody into circulation at physiological pH. Therefore, the interaction of the Fc with the neonatal Fc receptor (FcRn) prolongs the half-life of an IgG in a pH-dependent manner, through a recycling mechanism. Accordingly, the structural and functional properties of their Fc regions is of importance for the extension of half-life for canine IgG mABs. It is well known that extended half-lives of IgGs can have several therapeutic benefits, such as less frequent dosing and / or lower dose of the antibody drug, which in turn can reduce the number of veterinary visits, improves patient compliance, lowers the concentration-dependent cytotoxicity (CDC), and reduces the chances of adverse events occurring.

[0012] Although previous studies have shown certain mutations at certain positions alter the binding of human IgG to human FcRn [see e.g., US 7,083,784, US 7,658,921 , US 7,217,797, US 7,217,798, US 8,088,376, US 10,336,818 and WO2019 / 147973], human and dog IgG proteins have many differences in their amino acids sequences which give the residues in the human sequence different environments and / or different identities than in the dog or cat sequence. This variability makes it difficult to directly transfer characteristics of one IgG from one species to an IgG from another species. Consistently, a number of corresponding studies have more recently been conducted on companion animals [see, e.g., WO2018073185, W02020082048, WO20210116560, US20200362035, US20200216536, WO2020191289, WO2021231464, and US20210347854], W02023 / 021169 discloses antibodies and IgG Fc fusion proteins to IL-31 RA, IL-13Ra1 and IL-13Ra2 with enhanced half-lives. However, any particular mutation or combination of mutations may have drastically different effects on prolongation of half-life or other attributes of antibodies, including but not limited to changes in binding affinity to a particular target, thus making it difficult to predict the effect of such mutations on key antibody properties, including affecting the binding affinity to the target as well as the potential to induce anti-drug (antibody) antibodies.

[0013] Therefore, there is very little guidance in the art relating to increasing half-life of antibody therapeutics in canines. Any particular mutation or combination of mutations may have drastically different effects on prolongation of half-life or other attributes of antibodies, for example, particular mutations, even within the Fc region of an IgG may directly affect the binding affinity, target specificity and / or half-life of the antibody. Therefore, there remains a need to identify new specific mutations in canine IgGs that extend the half-life of therapeutic antibodies that bind CIL-31 RA and / or that improve the treatment of AD in dogs with therapeutic antibodies.

[0014] Accordingly, there exists a need for improved antibodies that bind clL-31 RA with specificity and / or for novel Fc constant regions of canine IgG monoclonal antibodies to improve the half-life of the antibodies that bind CIL-31 RA.

[0015] The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the instant application.

[0016] SUMMARY OF INVENTION

[0017] The technology herein relates to a caninized antibody or an antigen binding fragment thereof, that binds to canine interleukin-31 receptor alpha (clL-31 RA), that has an enhanced half-life as compared to the wild type CIL-31 RA antibody.

[0018] More specifically, the technology disclosed herein relates to canine or caninized interleukin-31 receptor alpha (clL-31 RA) antibodies, and functional fragments thereof and / or IgG Fc fusion proteins thereof, which comprises a variant of a canine fragment crystallizable region (cFc) region, where the variant cFc region comprises at least one amino acid modification as compared to the wild type (WT) cFc region, to result in an enhanced half-life in vivo as compared the half-life of a corresponding antibody comprising a WT cFc region (i.e., wherean unsubstituted cFc region is present). In particular, the technology disclosed herein relates, in part, to polypeptides, e.g., anticl L-31 RA antibodies or antigenic fragments thereof, that have an increased half-life in canines (i.e., in vivo) as compared to a corresponding anti-clL-31 RA antibody that comprises a wild type cFc region. For example, provided are binding molecules (e.g., antibodies or functional fragments thereof, and / or IgG Fc fusion proteins) that bind to canine CIL-31 RA with increased half-life relative to versions of these binding molecules attached to the non-modified Fc regions (i.e., a wild-type Fc region), or canine FcRn binding regions thereof, that does not comprise the half-life amino acid modifications. The variant cFc regions as disclosed herein, in addition to having an amino acid modification (relative to the wild type canine Fc region) that increase half-life, may also include other amino acid modifications that, e.g., increase effector function, decrease effector function, increase binding to CIL-31 RA and / or decrease heterogeneity of the polypeptide (e.g., by removing one or more post-translational modifications in the Fc region). The canine Fc region sequences can be from any canine antibody. In some instances, the canine Fc region sequences are from a canine IgG (e.g., IgGA, IgGB, IgGC, or IgGD).

[0019] Accordingly, the disclosure features a canine or caninized antibody and functional fragments thereof, and / or IgG Fc fusion proteins comprising (1) a binding region, e.g., a variable region, or a fragment thereof, that specifically binds to CIL-31 RA, wherein the binding domain is attached to (2) a variant cFc region, as disclosed herein, comprising a cFc region (CH2+CH3 region) or a canine FcRn binding region thereof. In some instances, the binding domain comprises (i) the six complementarity determining regions (CDRs) of a canine CIL-31 RA antibody or (ii) a soluble receptor-binding domain that binds a ligand, or a ligand-binding fragment thereof. In all aspects of the technology described herein, the Fc region is a cFc, where the canine Fc region sequences are from a canine IgG (e.g., IgGA, IgGB, IgGC, or IgGD).

[0020] Accordingly, one aspect of the present invention relates to a caninized anti-clL-31 RA antibody or functional fragment thereof, that comprises a light chain and a heavy chain, in which the heavy chain (HC) comprises a variant canine fragment crystallizable region (referred to herein as “variant cFc region”), where the variant cFc region comprises one or more amino acid modifications at the following amino acid residue positions: at amino acid residue position 252, at amino acid residue position 254, at amino acid residue position 256, at amino acid residue position 311 , at amino acid residue position 434, at amino acid residue position 436, at amino acid residue position 438, at amino acid residue position 440, or at any combination of these amino acid positions, numbered according to the Ell index as in Kabat. In some embodiments, the variant cFc comprises an amino acid modification at amino acid position 433, numbered according to Kabat. In some embodiments, the amino acid at position 433 is a histidine (H). In some embodiments, the technology herein relates to an antibody that comprises a light chain and a heavy chain, in which the heavy chain comprises a variant cFc region comprising two or more amino acid substitutions at two amino acid residue positions selected from: 252, 254, 256, 311 , 434, 436, 438, 440, as disclosed herein. In some embodiments, the technology herein relates to an antibody that comprises a light chain and a heavy chain, in which the heavy chain comprises a variant cFc region comprising three amino acid substitutions at three amino acid residue positions selected from: 252, 254, 256, 311 , 434, 436, 438, 440. In some embodiments, the variant cFc region of the heavy chain can comprise, one, two or three amino acid substitutions at e.g., residue positions 434, 438 and 440. In some embodiments, the variant cFc region of the heavy chain can comprise one, two or three amino acid substitutions at e.g., residue positions 252, 254 and 256. In some embodiments, the technology herein relates to an antibody that comprises a light chain and a heavy chain, in which the heavy chain comprises a variant cFc region comprising four amino acid substitutions at four amino acid residue positions selected from: 434, 436, 438 and 440, as disclosed herein. In some embodiments, an antibody that comprises a light chain and a heavy chain, in which the heavy chain comprises a variant cFc region comprising four amino acid substitutions at amino acid residue positions 434, 436, 438 and 440, can further comprise one an additional substitution at amino acid residue 428.

[0021] Another aspect of the technology disclosed herein relates to a IgG Fc fusion protein that comprises a variant cFc region comprising an amino acid substitution at an amino acid residue position numbered according to the Ell index as in Kabat at amino acid residue position 252, at amino acid residue position 254, at amino acid residue position 256, at amino acid residue position 311 , at amino acid residue position 434, at amino acid residue position 436, at amino acid residue position 438, at amino acid residue position 440, or at any combination of these amino acid positions. In some embodiments, the technology herein relates to a IgG Fc fusion protein that bind to CIL-31 RA that comprises a light chain and a heavy chain, in which the heavy chain comprises a variant cFc region comprising two or more amino acid substitutions at two amino acid residue positions selected from: 252, 254, 256, 311 , 434, 436, 438, 440, as disclosed herein. In some embodiments, the technology herein relates to a IgG Fc fusion protein that bind to CIL-31 RA which comprises a variant cFc region comprising three amino acid substitutions at three amino acid residue positions selected from: 252, 254, 256, 311 , 434, 436, 438, 440. In some embodiments, a Fc region of an IgG Fc fusion protein that binds to CIL-31 RA can comprise three amino acid substitutions at e.g., residue positions 434, 438 and 440. In some embodiments, a Fc region of a IgG Fc fusion protein that binds to CIL-31 RA can comprise three amino acid substitutions at e.g., residue positions 252, 254 and 256. In some embodiments, the technology herein relates to an IgG Fc fusion protein that binds to CIL-31 RA where the variant cFc region comprises four amino acid substitutions at four amino acid residue positions selected from: 434, 436, 438 and 440, as disclosed herein. In some embodiments, an antibody that comprises a light chain and a heavy chain, in which the heavy chain comprises a variant cFc region comprising four amino acid substitutions at amino acid residue positions 434, 436, 438 and 440, can further comprise one an additional substitution at amino acid residue 428. In certain embodiments of this type the Fc region is a canine Fc region (cFc), where the canine Fc region sequences are from a canine IgG (e.g., IgGA, IgGB, IgGC, or lgGD). Accordingly, in one aspect of the technology herein provides a caninized antibody or antigen binding fragment thereof, that binds canine interleukin-31 receptor alpha (clL-31 RA), wherein the antibody or antigen binding fragment thereof comprises a heavy chain that comprises a modified canine fragment crystallizable region (cFc) having one or more amino acid substitutions, wherein the one or more amino acid substitutions is at an amino acid residue position selected from the group consisting of 252, 254, 256, 311 , 428, 434, 436, 438, 440, or any combination thereof, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat. One aspect of the technology disclosed herein relates to caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL-31 RA), and where the antibody comprises CDRs having the amino acid sequences of SEQ ID NO: 13-18, and where the antibody has an improved or longer in vivo half-life as a result of comprising a variant cFc comprising specific amino acid modifications disclosed in International Application WO2 020 / 116560, which is incorporated herein in its entirety by reference.

[0022] Exemplary IL-31 RA antibodies comprising a cFc with a combination of at least four amino acid substitutions at amino acid residue positions 434, 438, 440 and 428 and / or 436.

[0023] One aspect of the technology described herein relates to a caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody comprising a canine fragment crystallizable region (cFc) that comprises at least four of: an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, an amino acid other than a serine residue at position 440, and an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436. wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0024] One aspect of all aspects of the technology described herein relates to a caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody comprising a cFc, the cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). In some embodiments, such an antibody is referred to herein in the Examples as A6 and comprises the amino acids: M428L / N434A / Y436T / Q438R / S440E.

[0025] One aspect of all aspects of the technology described herein relates to a caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody comprising a cFc, the cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). In some embodiments, such an antibody is referred to herein in the Examples as A7 and comprises the modifications M428L / N434A / Q438R / S440E.

[0026] One aspect of all aspects of the technology described herein relates to a caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody comprising a canine fragment crystallizable region (cFc), the cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). In some embodiments, such an antibody is referred herein in the Examples to as A5 and comprises the amino acids modifications N434A / Y436T / Q438R / S440E.

[0027] One aspect of all aspects of the technology described herein relates to a caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody comprising a canine fragment crystallizable region (cFc), the cFc comprising: a leucine amino acid residue at position 428 (M428L) and a serine amino acid residue at position 434 (N434S). In some embodiments, such an antibody is referred herein in the Examples to as A9 and comprises the amino acid modifications: M428UN434S.

[0028] One aspect of all aspects, a cFc present in a caninized antibody or antigen binding fragment thereof that binds CIL31 RA, further comprises the one or more residues selected from the group consisting of: a leucine amino acid residue at position 252, an alanine amino acid residue at position 254, a threonine amino acid residue at position 256, and a glutamine amino acid residue at position 311.

[0029] In alternative embodiments, a cFc present in a caninized antibody or antigen binding fragment thereof that binds CIL31 RA, further comprises the one or more residues selected from the group consisting of: an amino acid other than a leucine residue at position 252, an amino acid other than an alanine residue at position 254, an amino acid other than a threonine residue at position 256, and an amino acid other than a glutamine (Q) residue at position 311 , where the numbering of amino acid residue positions is according to the Ell Index as in Kabat. For example, but not limited to, in some embodiments, the cFc comprises the one or more residues selected from the group consisting of: an amino acid selected from a phenylalanine (F), or tyrosine (Y) or tryptophan (W) or threonine (T) amino acid residues at position 252 (e.g., L252F, L252Y, L252W, L252T), a threonine (T) amino acid residue at position 254 (A254T), a glutamic acid (E) or aspartic acid (D) amino acid residue at position 256 (T256D, T256E), and a serine amino acid residue position 311 (Q311 S) , where the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0030] In alternative embodiments, a caninized antibody or antigen binding fragment thereof that binds CIL31 RA as disclosed herein, comprises a cFc that comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc comprises at least four of: an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, an amino acid other than a serine residue at position 440, and an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat. In alternative embodiments, a caninized antibody or antigen binding fragment thereof that binds CIL31 RA as disclosed herein, comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc is selected from the group consisting of: a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E);

[0031] (M428UN434A / Y436T / Q438R / S440E), a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E); M428L / N434A / Q438R / S440E), a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E); N434A / Y436T / Q438R / S440E), a cFc comprising: a leucine amino acid residue at position 428 (M428L), and a serine amino acid residue at position 434 (N434S). (M428UN434S), where the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0032] In all aspects of each embodiment, a caninized antibody or antigen binding fragment thereof that binds CIL31 RA as described herein, comprises a heavy chain variable domain (VH) and / or a light chain variable domain (VL), wherein the VH contains the following CDRs:

[0033] VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 13;

[0034] VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 14;

[0035] VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and the VL comprises a light chain variable domain that contains the following CDRs:

[0036] VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 16; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 17;

[0037] VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

[0038] In all aspects of each embodiment, a caninized antibody or antigen binding fragment thereof that binds CIL31 RA as described herein, comprises a heavy chain variable domain (VH) and / or a light chain variable domain (VL), where the VH contains the following CDRs: VH-CDR1 comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 13; VH-CDR2 comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 14; VH-CDR3 comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 15; and where the VL comprises a light chain variable domain that contains the following CDRs: VL-CDR1 comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 16; VL-CDR2 comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17; VL-CDR3 comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 18.

[0039] In some embodiments, a caninized antibody or antigen binding fragment thereof that binds CIL31 RA as disclosed herein, comprises any one or more of a) to d), where: a) cFc comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc comprises at least four of: an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, an amino acid other than a serine residue at position 440, and an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436, b) a cFc comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and where the cFc is selected from the groups (i), (ii), (iii) and (iv):

[0040] (I) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E);

[0041] (M428UN434A / Y436T / Q438R / S440E)

[0042] (ii) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E); (M428UN434A / Q438R / S440E)

[0043] (iii) a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E); (N434A / Y436T / Q438R / S440E)

[0044] (iv) a cFc comprising: a leucine amino acid residue at position 428 (M428L), and a serine amino acid residue at position 434 (N434S). (A9: M428L / N434S) where the numbering of amino acid residue positions is according to the Ell Index as in Kabat. c) a cFc comprising a hinge region that comprises an amino acid sequence that comprises at least 90%, 95%, 97%, 98%, 99% identity sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 or SEQ ID NO: 46, and d) a light chain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID Nos: 21 , 22 and 23.

[0045] In some embodiments, a caninized antibody or antigen binding fragment thereof that binds CIL31 RA as disclosed herein, comprises a heavy chain, where the heavy chain comprises an amino acid sequence comprising amino acid residues of SEQ ID NO: 28, 29, 30 or 31 , or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOs: 28, 29, 30 or 31.

[0046] One aspect of the technology as disclosed herein relates to a composition, e.g., a pharmaceutical composition, and methods of use, of a caninized antibody that binds canine interleukin-31 receptor alpha (clL31 RA), where the antibody selected from the group consisting of:

[0047] (i) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and wherein the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 30, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 30,

[0048] (ii) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and wherein the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 29, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 29,

[0049] (iii) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: an alanine amino acid residue at position 434 (N434A), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and wherein the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 28, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 28, and

[0050] (iv) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: a leucine amino acid residue at position 428 (M428L) and an alanine amino acid residue at position 434 (N434A), and wherein the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 31 , or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 31 , where the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0051] One aspect of the technology as disclosed herein relates to a nucleic acid molecule that encodes a caninized antibody or antigen binding fragment thereof as disclosed herein, and optionally, wherein the nucleic acid is present in an expression vector, and optionally, wherein the expression vector is present in a host cell. Other aspects relate to a nucleic acid molecule that encodes such a caninized IL-31 RA antibody or antigen binding fragment thereof, as well as expression vectors and / or host cells comprising the same.

[0052] Another aspect relates to a pharmaceutical composition comprising a caninized antibody or antigen binding fragment thereof as disclosed herein comprising a cFc region comprising at least four of: (i) an amino acid other than an asparagine residue at position 434, (ii) an amino acid other than a glutamine residue at position 438, (iii) an amino acid other than a serine residue at position 440, and (iv) an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436, and a pharmaceutically acceptable carrier.

[0053] Another aspect relates to a pharmaceutical composition comprising a caninized antibody or antigen binding fragment thereof as disclosed herein comprising a cFc region and a pharmaceutically acceptable carrier, where the Fc region is selected from any of: (i) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (referred to as

[0054] M428L / N434A / Y436T / Q438R / S440E), (ii) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (referred to as A7:

[0055] M428L / N434A / Q438R / S440E); (iii) a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (referred to as A5: N434A / Y436T / Q438R / S440E), and (iv) a cFc comprising: a leucine amino acid residue at position 428 (M428L), and a serine amino acid residue at position 434 (N434S) (referred to as A9: M428L / N434S)

[0056] Other embodiments relate to a pharmaceutical composition, a nucleic acid molecule, or expression vector encoding a caninized antibody or antigen binding fragment thereof as disclosed herein, or any combination thereof, and a pharmaceutically acceptable carrier. In some embodiments, the nucleic acid encodes a heavy chain comprising a cFc region, the Fc comprising at least four of: (i) an amino acid other than an asparagine residue at position 434, (ii) an amino acid other than a glutamine residue at position 438, (iii) an amino acid other than a serine residue at position 440, and (iv) an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436, and a pharmaceutically acceptable carrier. In some embodiments, the nucleic acid encodes a heavy chain, where the Fc region is selected from any of: (i) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (A6: M428L / N434A / Y436T / Q438R / S440E), (ii) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E); (A7: M428L / N434A / Q438R / S440E); (iii) a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (A5: N434A / Y436T / Q438R / S440E), and (iv) a cFc comprising: a leucine amino acid residue at position 428 (M428L), and a serine amino acid residue at position 434 (N434S). (A9: M428L / N434S). In some embodiments, the nucleic acid, or expression vector is present in a host cell as disclosed herein.

[0057] Other aspects relate to the use, in the treatment of atopic dermatitis in a canine, of a caninized antibody or antigen binding fragment thereof as disclosed herein comprising a cFc region, or a nucleic acid molecule encoding the same, or an expression vector comprising a nucleic acid encoding the same, or the pharmaceutical composition, the Fc comprising at least four of: (i) an amino acid other than an asparagine residue at position 434, (ii) an amino acid other than a glutamine residue at position 438, (iii) an amino acid other than a serine residue at position 440, and (iv) an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436.

[0058] Another aspect relates to the use in the treatment of atopic dermatitis in a canine, of a caninized antibody or antigen binding fragment thereof as disclosed herein comprising a cFc region, or a nucleic acid molecule encoding the same, or an expression vector comprising a nucleic acid encoding the same, or the pharmaceutical composition, where the cFc is selected from any of: (i) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (A6:

[0059] M428L / N434A / Y436T / Q438R / S440E), (ii) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E); (A7: M428L / N434A / Q438R / S440E); (iii) a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (A5: N434A / Y436T / Q438R / S440E), and (iv) a cFc comprising: a leucine amino acid residue at position 428 (M428L), and a serine amino acid residue at position 434 (N434S). (A9: M428L / N434S).

[0060] Another aspect of the technology described herein relates to a method of treating a canine that has atopic dermatitis comprising administering to a canine any one, or a combination of, caninized antibody or antigen binding fragment thereof as disclosed herein, or the pharmaceutical composition comprising the caninized antibody or antigen binding fragment thereof as disclosed herein comprising a cFc region, the cFc comprising at least four of: (i) an amino acid other than an asparagine residue at position 434, (ii) an amino acid other than a glutamine residue at position 438, (iii) an amino acid other than a serine residue at position 440, and (iv) an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436. In some embodiments, the cFc is selected from any of: (i) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (A6: M428L / N434A / Y436T / Q438R / S440E), (ii) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E); (A7: M428L / N434A / Q438R / S440E); (iii) a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E) (A5: N434A / Y436T / Q438R / S440E), and (iv) a cFc comprising: a leucine amino acid residue at position 428 (M428L), and a serine amino acid residue at position 434 (N434S). (A9: M428L / N434S).

[0061] In a further aspect there is provided for a nucleic acid molecule encoding the caninized antibody, or antigen binding fragment thereof, according to the invention.

[0062] In a further aspect there is provided for an expression vector comprising the nucleic acid molecule encoding the caninized antibody, or antigen binding fragment thereof, according to the invention. In a further aspect there is provided for a host cell that comprises the expression vector. In a further aspect there is provided for a method of producing the caninized antibody, or antigen binding fragment thereof, according to the invention, comprising culturing the host cell or a population of host cells. In a further aspect there is provided for a pharmaceutical composition comprising the caninized antibody, or antigen binding fragment thereof, according to the invention, the nucleic acid molecule, the expression vector, the host cell or any combination thereof, and a pharmaceutical carrier. In a further aspect there is provided for the caninized antibody, or antigen binding fragment thereof, according to the invention, the nucleic acid molecule, the expression vector, the host cell or any combination thereof for us in the treatment of atopic dermatitis in a canine. In a further aspect there is provided for a method of treating a canine that has atopic dermatitis comprising administering anyone, or a combination of, the caninized antibody, or antigen binding fragment thereof, according to the invention, the nucleic acid molecule, the expression vector, the host cell or the pharmaceutical composition.

[0063] In certain exemplary embodiments, the isolated caninized antibody has an altered serum half-life compared to a corresponding caninized antibody with the same CDR sequences, comprising a wild-type Fc domain or unsubstituted cFc domain. In certain exemplary embodiments, the isolated caninized antibody has an increased serum half-life compared to a corresponding caninized antibody having the same CDR sequences comprising a wild-type Fc domain or unsubstituted cFc.

[0064] In certain exemplary embodiments, the isolated caninized antibody has an altered serum half-life compared to a corresponding caninized antibody having the same CDR sequences and comprising a Fc domain of SEQ ID NO: 3. In certain exemplary embodiments, the isolated caninized antibody has an increased serum half-life compared to a corresponding caninized antibody comprising a Fc domain comprising the double mutation D31A / N63A (referred to herein as IgG-Bm Fc), according to Kabat numbering. In certain exemplary embodiments, the isolated caninized antibody has an increased serum half-life as compared to a corresponding caninized antibody comprising a Fc domain comprising the amino acid sequence of SEQ ID NO: 4 (“referred to herein as “IgG-Bm Fc”), according to Kabat numbering.

[0065] The present invention further provides individual nucleic acids comprising individual nucleotide sequences that encode the individual antibody heavy chains, antibody light chains, or IgG fusion proteins of the invention. Accordingly, the present invention provides a nucleic acid comprising a nucleotide sequence that encodes an antibody heavy chain, an antibody light chain, or an IgG fusion protein of the invention. In particular embodiments, the present invention provides nucleic acids comprising a nucleotide sequence that encodes the heavy chain of the CIL-31 RA caninized antibody as disclosed herein, where nucleic acid encodes a heavy chain with an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, or encodes a heavy chain with an amino acid sequence that comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40. In related embodiments, the present invention provides nucleic acids comprising a nucleotide sequence that encodes any one of the heavy chains of a cl L-31 RA caninized antibody. The present invention also provides nucleic acids comprising a nucleotide sequence that encodes any one of the light chains of the CIL-31 RA caninized antibody. In addition, the present invention provides nucleic acids comprising multiple nucleotide sequences that each encode an antibody heavy and a light chain of the present invention. The present invention further provides nucleic acids comprising multiple nucleotide sequences that each encode an IgG fusion protein of the invention. The present invention further provides vectors that comprise these nucleic acids. In particular embodiments, the vector is an expression vector. The present invention further provides host cells that comprise any of the vectors of the present invention.

[0066] The present invention also provides pharmaceutical compositions comprising antibodies and IgG Fc fusion proteins with enhanced half-lives of the invention and a pharmaceutically acceptable carrier. This enhanced half-life is due to, at least in part, the antibodies and IgG Fc fusion proteins comprising a fragment crystallizable region (Fc) that comprises one or more amino acid substitutions in their Fc regions. In more specific embodiments, the pharmaceutical comprises a cl L-31 RA caninized antibody of the invention and a pharmaceutically acceptable carrier.

[0067] The present invention further provides methods of treating a canine that has atopic dermatitis comprising administering to the canine any one or more of the pharmaceutical compositions of the present invention.

[0068] The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

[0069] DEFINITIONS

[0070] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

[0071] As used herein the singular forms of words such as "a", "an" and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0072] In the specification and claims, the numbering of the amino acid residues in an immunoglobulin heavy chain is that of the Eu index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The “EU index as in Kabat” refers to the residue numbering of the IgG antibody.

[0073] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. An antibody can be a monomer, dimer, or larger multimer. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (mABs) (including full length mABs), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and camelized single domain antibodies. Unless otherwise specifically indicated, an antibody of the present invention is an IgG antibody.

[0074] As used herein, an antibody of the present invention that "blocks" or is "blocking" or is "blocking the binding" of e.g., a canine receptor to its binding partner (e.g., its ligand), is an antibody that blocks (partially or fully) the binding of the ligand and its receptor and vice versa, as determined in standard binding assays (e.g., BIACore®, ELISA, or flow cytometry). Typically, an antibody or antigen binding fragment of the invention retains at least 10% of its canine antigen binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen binding fragment according to the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine antigen binding affinity as the parental antibody.

[0075] As used herein, the term “chimeric antibody" includes monovalent, divalent or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated through disulfide bridges with a chimeric light chain. A divalent chimeric antibody is a tetramer formed by two heavy chain-light chain dimers associated through at least one disulfide bridge. A chimeric heavy chain of an antibody for use in canines comprises an antigen-binding region derived from the heavy chain of a non-canine antibody, which is linked to at least a portion of a canine heavy chain constant region, such as CH1 or CH2. A chimeric light chain of an antibody for use in canine comprises an antigen binding region derived from the light chain of a non-canine antibody, linked to at least a portion of a canine light chain constant region (CL). Antibodies, fragments or derivatives having chimeric heavy chains and light chains of the same or different variable region binding specificity, can also be prepared by appropriate association of the individual polypeptide chains, according to known method steps. With this approach, hosts expressing chimeric heavy chains are separately cultured from hosts expressing chimeric light chains, and the immunoglobulin chains are separately recovered and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin or fragment or both the heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, e.g., U.S. Pat. Nos. 6,284,471 ; 5,807,715; 4,816,567; and 4,816,397).

[0076] As used herein, the term "canine" includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.

[0077] As used herein, the term "caninized antibody" refers to forms of IgG antibodies that contain amino acid sequences originating from both canine and non-canine (e.g., mouse, rat, or human) IgG antibodies. In general, the caninized antibody will comprise substantially all of at least one or more typically, two variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-canine IgG (e.g., comprising 6 CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) are those of a canine IgG sequence. A modified canine frame comprises one or more amino acids changes as exemplified herein that further optimize the effectiveness of the caninized antibody, e.g., to increase its binding to its canine antigen and / or its ability to block the binding of that canine antigen to the canine antigen 's natural binding partner. As exemplified below, a caninized antibody comprises both the three heavy chain CDRs and the three light chain CDRS from a mouse anti-canine antigen antibody together with a modified canine frame. As detailed below, the cFc of the caninized antibody can also comprise amino acid substitutions that lead to a greater half-life of the caninized antibody.

[0078] Caninized antibodies that bind canine interleukin-31 receptor alpha (cIL 31 RA or clL-31 Ra) include, but are not limited to antibodies for use in the present invention that comprise canine IgGA, IgGB, IgGC, or IgGD heavy chains, and modified forms of the canine IgGA, IgGB, IgGC, or IgGD heavy chains, including the modified Fes that are disclosed herein, particularly those caninized antibodies that also show an increased binding affinity for its neonatal Fc receptor (FcRn) at pH 5.5 - pH 6.5.

[0079] As used herein, the term "caninized antibody to canine interleukin-31 receptor alpha" is used interchangeably with a "caninized CIL-31RA antibody" and a "clL-31RA caninized antibody" and is a caninized antibody of a mammalian antibody raised in a non-canine mammal (e.g., a mouse or a rat) against CIL-31 RA

[0080] As used herein the term "canine frame" refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the hypervariable region residues defined herein as CDR residues. With regard to a caninized antibody the amino acid sequences of the native canine CDRs are largely replaced with the corresponding foreign CDRs (e.g., those from a mouse, rat, or human) in both chains. Optionally the heavy and / or light chains of the canine antibody may contain some foreign non-CDR residues, e.g., so as to preserve the conformation of the foreign CDRs within the canine antibody, and / or to modify the Fc region function.

[0081] The "fragment crystallizable region" abbreviated as "Fc” and used interchangeably with "Fc region" refers to a C-terminal region of an immunoglobulin heavy chain and corresponds, generally, to the CH2-CH3 portion of an antibody that interacts with cell surface receptors called Fc receptors. In some embodiments, variants comprise only portions of the Fc region and can include or not include the carboxy-terminus. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In some embodiments, variants having one or more of the constant domains are contemplated. In other embodiments, variants without such constant domains (or with only portions of such constant domains) are contemplated.

[0082] As used herein a "canine fragment crystallizable region" is interchangeably abbreviated as "cFc region" or just "cFc" and corresponds to a canine fragment crystallizable region from a canine antibody. The canine fragment crystallizable region (cFc) of each of the four canine IgGs were first described by Tang etal. Vet. Immunol. Immunopathol. 80: 259-270 (2001); see also, Bergeron et al., Vet. Immunol. Immunopathol. 157: 31-41 (2014).

[0083] As used herein, the term a “functional Fc region" possesses an “effector function” of a native sequence Fc region. At least one effector function of a polypeptide comprising a variant Fc region of the present invention may be enhanced or diminished with respect to a polypeptide comprising a native Fc region or the parent Fc region of the variant. Examples of effector functions include, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-depended cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be operably linked to a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assay, ADCC assays, CDC assays, target cell depletion from whole or fractionated blood samples, etc.). As used herein, the term "framework' or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0084] As used herein, the term a “variant Fc region” or “variant cFc” or “variant cFc region" are used interchangeably herein with “modified Fc region” and “modified cFc region” and refers to an amino acid sequence that differs from that of “wild-type Fc” or an unsubstituted cFc (or fragment thereof) by virtue of at least one “amino acid modification” as defined herein. In preferred embodiments, the variant Fc region has at least one amino acid substitution as compared to a Fc region of a parent Fc region. A parent Fc region can be a wild-type Fc region, or an unsubstituted cFc. Preferably a variant Fc region has 1 , 2, 3, 4 or 5 amino acid modifications s in a unsubstituted cFc or in the cFc region of the parent polypeptide. In an alternative embodiment, a variant Fc region may be generated according to the methods herein disclosed and this variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or a nonantibody polypeptide, e.g., binding domain of a receptor or ligand, e.g., for a Fc-fusion protein, as that term is defined herein.

[0085] As used herein, the term a "wild-type Fc" is used interchangeably with a "wild-type Fc region" or “native sequence Fc region” refers to an amino acid sequence of a Fc region (e.g., cFc region) commonly found in nature that does not comprise any amino acid modifications disclosed herein that enhance the half-life of an antibody that comprises such a wild-type Fc region. As noted herein, a Fc region comprising one or more amino acid modifications that result in diminishing the antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the antibody, such as the canine IgG-Bm region, is not found in nature and therefore, is not a wildtype Fc region. However, an antibody comprising such an Fc region (e.g., a canine IgG-Bm Fc region as disclosed herein) should have the same half-life as the corresponding antibody that comprises the corresponding wild-type Fc region found in nature (i.e. , the canine IgG-B Fc region) in the absence of any amino acid modifications made to enhance the half-life of an antibody comprising the canine IgG-Bm region.

[0086] As used herein, a “unsubstituted cFc” as used herein refer to a cFc that comprises one or more amino acid modifications to the wild-type Fc, where the modifications affect only the antibody’s ADCC and CDC function, and the cFc does not comprise any amino acid modifications in the Fc region that affect the half-life of an antibody. Accordingly, exemplary “unsubstituted cFc” encompasses, for example, the sequences of IgG-Am Fc region, IgG-Bm Fc region, IgG-Cm Fc region and IgG-Dm Fc region, as defined herein, and correspond to SEQ ID NOs: 2, 4, 6 and 8. As used herein, cFc an unsubstituted cFc comprises the amino acid sequence of SEQ ID NO: 4, which is the amino acid sequence of canine IgGB Fc but comprising two (2) amino acid residue modifications, D31A and N63A, in the amino acid sequence of SEQ ID NO: 3 of IgG B (see below). Accordingly, both the aspartic acid residue (D) at position 31 of SEQ ID NO: 3 and the asparagine residue (N) at position 63 of SEQ ID NO: 3, are substituted by an alanine residue (A) in the amino acid sequence of IgG Bm, i.e. , SEQ ID NO: 4. These two amino acid residue modifications serve to significantly diminish the ADCC and CDC of the naturally occurring canine IgG B (see, WQ2015 / 091 , 910 A2 the contents of which are hereby incorporated by reference in their entirety). Analogous modifications can be made in the canine IgG A, IgG C, and IgG D Fc regions.

[0087] As used herein, the term " hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e., CDRL1 (or LCDR1), CDRL2 (or LCDR2), and CDRL3(or LCDR3) in the light chain variable domain and CDRH1 (or HCDR1), CDRH2 (or HCDR2), and CDRH3 (or HCDR3) in the heavy chain variable domain (Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) defines the CDR regions of an antibody by sequence; Chothia and Lesk, 1 Moi. Biol. 196: 901-917 (1987) defines the CDR regions of an antibody by structure).

[0088] As used herein, the term “modification’’ and "substitution" are used interchangeably herein, and when used in reference to a modification of an amino acid residue, refers to the replacement of a particular amino acid in a protein (i.e., an amino acid sequence) with a different amino acid. Stated differently, it is a change of one amino acid with another amino acid residue in an amino acid sequence of an antibody for example, and is equivalent to "replacing an amino acid residue" with another amino acid residue. In particular, the term denotes thatthat a particular amino acid residue at a specific position in the amino acid sequence has been replaced by (or substituted for) by a different amino acid residue e.g., by recombinant DNA technology. Such substitutions can be particularly designed i.e., purposefully replacing an asparagine (N) with a histidine (H) at a specific position in the amino acid sequence of an Fc region e.g., at position 434, as numbered according to the Ell index as in Kabat. The amino acid substitutions can be made, for example, to alter the half-life of a given antibody, and / or as noted herein.

[0089] As used herein, an "IgG antibody" is an immunoglobulin G antibody that comprises two heavy chains and two light chains. IgG antibodies of the present invention include caninized antibodies, fully canine antibodies and optional chimeric antibodies. As used herein an “Fc fusion protein" is used interchangeably with the term “IgG Fc fusion protein” and is an artificial protein that joins the Fc region of an IgG antibody, which can further include a hinge region, e.g., the canine IgG B hinge region-CH2-CH3, with another biologically active protein domain to generate a molecule with unique structure and therapeutic utility.

[0090] As used herein a “cFc fusion protein" is used interchangeably with the term “canine IgG Fc fusion protein” and is an artificial protein that joins the cFc of a canine IgG antibody, which can include a hinge region, e.g., the IgG B hinge region-CH2-CH3, with another biologically active protein domain to generate a molecule with unique structure and therapeutic utility. For example, a canine IL 31 RA cFc fusion protein (cIL 31 RA cFc fusion protein) comprises the extracellular domain (ECD) of canine IL-31 RA linked to the N terminus of a canine IgG Fc (cFc). The ECD of the IL- 31 RA may be linked to the N terminus of the cFc by a canine hinge region. The cFc fusion proteins of the present invention, although exemplified by the use of the IgGB hinge region and the IgGB cFc, are in no way so limited, but rather they include the corresponding fusion proteins with the eFes of IgGA, IgGC, and IgGD and optionally the hinge regions of IgGA, IgGC, and IgGD. Accordingly, the canine Fc fusion protein cIL 31 RA clgGB-Fc is one species of the cIL 31 RA cFc genus, which also includes cIL 31 RA clgGA-Fc, cIL 31 RA a2 clgGC-Fc, cIL 31 RA clgGD-Fc and modified fusion proteins thereof.

[0091] The term “Sequence identity’ refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. As used herein one amino acid sequence is 100% " identical" to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% "identical" to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared.

[0092] As used herein, the term “derivative" in the context of polypeptides refers to a polypeptide that comprises and amino acid sequence which has been altered by introduction of an amino acid residue modification. The term “derivative” as used herein also refers to a polypeptide which has been modified by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative polypeptide may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative polypeptide possesses a similar or identical function as the polypeptide from which it was derived. It is understood that a polypeptide comprising a modified cFc region of the present invention may be a derivative as defined herein, preferably the derivatization occurs within the cFc region. As used herein, the term "Conservatively modified variants" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side-chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), such that the changes frequently can be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity (Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table 1 directly below.

[0093] TABLE 1: Exemplary Conservative Amino Acid Substitutions

[0094] The term "Function-conservative variants," as used herein, refers to the IgG Fc antibodies in which one or more amino acid residues have been changed without altering a desired property, such an antigen affinity and / or specificity. Such variants include, but are not limited to, replacement of an amino acid with one having similar properties, such as the conservative amino acid substitutions of Table 1 above.

[0095] The terms “Fc receptor1’ or “FcR” are used to describe a receptor that binds to an Fc region (e.g., the Fc region of an antibody). The preferred FcR is a native sequence FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc gamma Rl, Fc gamma RII, Fc gamma RIH subclasses, including allelic variants and alternatively spliced forms of these receptors. Another preferred FcR includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

[0096] The phrase “antibody-dependent cell-mediated cytotoxicity’ and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) that express FcRs (e.g., Natural Killer (“NK”) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cells. The primary cells for mediating ADCC, NK cells, express Fc gamma MIT only, whereas monocytes express Fc gamma Rl, Fc gamma Ml and Fc gamma MIT

[0097] As used herein, the phrase “effector cells’’ refers to leukocytes (preferably canine) which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc gamma MIT and perform ADCC effector function. Examples of leukocytes which mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils. The effector cells may be isolated from a native source (e.g., from blood or PBMCs). A variant polypeptide with “altered’ FcRn binding affinity is one which has either enhanced (i.e., increased, greater or higher) or diminished (i.e., reduced, decreased or lesser) FcRn binding affinity compared to the variant's parent polypeptide or to a polypeptide comprising a native Fc region when measured at pH 6.0. A variant polypeptide which displays increased binding or increased binding affinity to an FcRn binds FcRn with greater affinity than the parent polypeptide. A variant polypeptide which displays decreased binding or decreased binding affinity to an FcRn, binds FcRn with lower affinity than its parent polypeptide. Such variants which display decreased binding to an FcRn may possess little or no appreciable binding to an FcRn, e.g., 0-20% binding to the FcRn compared to a parent polypeptide. A variant polypeptide which binds an FcRn with “enhanced affinity” as compared to its parent polypeptide, is one which binds FcRn with higher binding affinity than the parent polypeptide, when the amounts of variant polypeptide and parent polypeptide in a binding assay are essentially the same, and all other conditions are identical. For example, a variant polypeptide with enhanced FcRn binding affinity may display from about 1.10 fold to about 100 fold (more typically from about 1.2 fold to about 50 fold) increase in FcRn binding affinity compared to the parent polypeptide, where FcRn binding affinity is determined, for example, in an ELISA assay or other method available to one of ordinary skill in the art.

[0098] The term “expression vector1’ as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.

[0099] A nucleic acid is "operably linked' when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0100] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0101] As used herein, the term “host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, CHO cells, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in situ, or in vivo.

[0102] As used herein, "inhibit" or "treat" or "treatment" includes a postponement of development of the symptoms associated with a disorder or condition and / or a reduction in the severity of the symptoms of such disorder or condition. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject (e.g., a canine) with a disorder, condition and / or symptom, or with the potential to develop such a disorder, disease or symptom.

[0103] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, e.g., canine or feline, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A therapeutically effective dose further refers to that amount of the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess severity of the condition. DESCRIPTION OF EMBODIMENTS

[0104] Binding of an antibody and / or a fusion protein comprising a Fc region (herein referred to a “IgG Fc fusion protein”) to FcRn is highly dependent on pH. Specifically, the binding occurs with high affinity at moderately acidic pH in the endosomal compartments, but with a significant lower binding affinity at physiological pH at the cell surface. The strong binding of IgG antibodies to FcRn in the endosomal compartment both protects the antibody from the degradative action of proteolytic enzymes in endosomes and allows the recycling of the receptor-bound antibody at the cell surface, whereas the higher pH at the cell surface (i.e., physiological pH) weakens that binding and thereby allows the release of the antibody into the circulation. Substituting one or more amino acids in the Fc region of an antibody, or a fusion protein comprising the Fc can serve to increase the binding affinity of antibodies and / or IgG Fc fusion proteins to FcRn at moderately acidic pH, and thereby increase the half-life of the antibody and / or IgG Fc fusion protein in vivo.

[0105] While some substitutions increase the half-life of the antibodies, such mutations or variants may also adversely decrease the binding affinity of the antibody or the IgG Fc fusion protein to the target protein. Moreover, while one a single specific amino acid substitution may increase the halflife of the antibody or Fc-containing fusion protein, one or more additional substitutions may counter this effect. Therefore, the challenge is identify specific amino acid substitutions, or combinations thereof, in the IgG Fc region that both enhance the half-life of the protein and do not adversely affect (i.e., do not significantly decrease) the binding affinity of the antibody or IgG Fc fusion protein to the target epitope.

[0106] Details of embodiments and of further aspects of the invention will be described below.

[0107] The present inventors surprisingly found that by substituting or changing certain amino acid residues at certain amino acid positions for certain, other, amino acid residues in the canine fragment crystallizable region (cFc) of a caninized antibody, or antigen binding fragment thereof, can result in the antibody having an enhanced half-life. It was surprisingly found that specific amino acid residues at specific position, and combinations of such amino acids in the Fc region of the antibody, or antigen binding fragment thereof, resulted in the antibody having an enhanced half-life (e.g., longer half-life) as compared to a wild-type cFc and / or unsubstituted cFc, such as a wild-type Fc that only contains substitutions affecting the antibody’s ADCC and CDC. Moreover, it was found that some particular amino acids at designated positions, and / or particular combinations of specific amino acids at designated positions, increased (extend) the half-life of the antibody, whilst other amino acid substitutions or combinations of substitutions decreased (shorten) the half-life of the antibody. Consequently, it was surprising that only some amino acid substitutions, or combinations of substitutions, were found to drastically extend the half-life of antibodies, whereas other substitutions or combination of amino acid substitutions had the opposite effect and reduced the half-life of the antibody. This differentiating effect on the prolonging of half-life of the antibody makes it difficult to predict the effect of such mutations on key antibody properties, as well as the effect of the mutation on other properties, including the potential to induce anti-drug (antibody) antibodies.

[0108] In one aspect, there is provided for a caninized antibody or antigen binding fragment thereof, that binds CIL-31 RA, wherein the antibody comprises a modified cFc comprising one or more amino acid substitutions relative to a wild-type cFc, wherein the modified cFc comprises one or more amino acid substitutions at an amino acid residue position selected from the group consisting of 252, 254, 256, 311 , 428, 434, 436, 438, 440, or any combination thereof. The numbering of amino acid residue positions is according to the Ell Index as in Kabat (Sequences of Proteins of Immunological Interest, 5th ed., Kabat et al., National Institutes of Health, Bethesda, Md. (1991) of the Ell Index).

[0109] Accordingly, the present invention provides an antibody that comprises a light chain comprising a set of three light chain complementary determining regions (LCDRs) and a heavy chain comprising a set of three heavy chain complementary determining regions (HCDRs), in which the heavy chain comprises a Fc region comprising an amino acid modification relative to a wild-type canine Fc (cFc), wherein the modified cFc comprises an amino acid modification at amino acid residue position 252, at amino acid residue position 254, at amino acid residue position 256, at amino acid residue position 311 , at amino acid residue position 428, at amino acid residue position 434, at amino acid residue position 436, at amino acid residue position 438, at amino acid residue position 440, or at any combination of these amino acid positions, numbered according to the Ell index as in Kabat. In certain embodiments the Fc region is a canine Fc (cFc). In certain embodiments the Fc region is a canine Fc (cFc) of IgGA, IgG B, IgG C or IgG D. In some embodiments, the Fc region is an unsubstituted variant cFc, including but not limited to IgG-Am, IgG-Bm, IgG-Cm or IgG-Dm cFc. In some embodiments, the Fc region is IgG-Bm unsubstituted variant cFc.

[0110] When referring to a caninized antibody or antigen binding fragment thereof binding CIL-31 RA it is understood that the antibody binds to canine IL-31 RA and blocks the binding of canine IL-31 to canine IL-31 RA. It is contemplated that the antibody thereof can bind canine IL-31 RA with specificity, and when bound to canine IL-31 RA, the antibody binds to an epitope on canine IL- 31 RA that are specific for the antibodies. In particular embodiments the caninized antibody or antigen binding fragment thereof according to the invention comprises one or more amino acid modifications, wherein one or more of the following amino acids is substituted: L252, A254, T256, Q311 , M428, N434H, Y436, Q438 and S440, numbered according to the Ell index as in Kabat.

[0111] In particular embodiments, the caninized antibody or antigen binding fragment thereof according to the invention comprises a modified cFc comprising one amino acid modification relative to a wild-type cFc or an unsubstituted variant cFc.

[0112] In certain embodiments, the modified cFc comprises a phenylalanine (F) at amino acid position 252. In other embodiments, the modified cFc comprises a tyrosine (Y) at amino acid position 252. In other embodiments, the modified cFc comprises a tryptophan (W) at amino acid position 252. In other embodiments, the modified cFc comprises a threonine (T) at amino acid position 252. In other embodiments, the modified cFc comprises a threonine (T) at amino acid position 254. In other embodiments, the modified cFc comprises an aspartic acid (D) at amino acid position 256. In other embodiments, the modified cFc comprises a glutamic acid (E) at amino acid position 256. In other embodiments, the modified cFc comprises a serine (S) at amino acid position 311. In other embodiments, the modified cFc comprises a leucine (L) at amino acid position 428. In other embodiments, the modified cFc comprises an alanine (A) at amino acid position 434. In other embodiments, the modified cFc comprises a histidine (H) at amino acid position 434. In other embodiments, the modified cFc comprises a serine (S) at amino acid position 434. In other embodiments, the modified cFc comprises a threonine (T) at amino acid position 436. In other embodiments, the modified cFc comprises an arginine (R) at amino acid position 438. In other embodiments, the modified cFc comprises a glutamic acid (E) at amino acid position 440, numbered according to the Ell index as in Kabat.

[0113] In a particular embodiment, the caninized antibody or antigen binding fragment thereof according to the invention comprises one or more amino acid modifications in the cFc region selected from:

[0114] L252F, L252Y, L252W, L252T, A254T, T256D, T256E, Q311S, M428L, N434A, N434H, N434S, Y436T, Q438R and S440E, or any combination thereof, numbered according to the Ell index as in Kabat.

[0115] In one aspect of the technology described herein, a modified cFc comprises a methionine at amino acid residue position 252. In one aspect of the technology described herein, the modified cFc comprises a serine at amino acid residue position 252. In other embodiments, the modified cFc comprises a serine residue at amino acid residue position 254. In other embodiments, the modified cFc comprises an alanine residue at amino acid residue position 256. In other embodiments, the modified cFc comprises a glutamine residue at amino acid residue position 256. In other embodiments, the modified cFc comprises a tyrosine residue at amino acid residue position 311. In other embodiments, the modified cFc comprises a isoleucine residue at amino acid residue position 428. In other embodiments, the modified cFc comprises a valine residue at amino acid residue position 428, numbered according to the Ell index as in Kabat.

[0116] In other embodiments, the modified cFc comprises a glycine residue at amino acid residue position 434. In other embodiments, the modified cFc comprises a serine residue at amino acid residue position 434. In other embodiments, the modified cFc comprises an asparagine residue at amino acid residue position 434. In other embodiments, the modified cFc comprises a glutamic acid residue at amino acid residue position 434. In other embodiments, the modified cFc comprises a serine residue at amino acid residue position 436. In other embodiments, the modified cFc comprises a lysine residue at amino acid residue position 438. In other embodiments, the modified cFc comprises a histidine residue at amino acid residue position 438. In other embodiments, the modified cFc comprises an aspartic acid residue at amino acid residue position 440. In other embodiments, the modified cFc comprises a glutamine residue at amino acid residue position 440, numbered according to the Ell index as in Kabat.

[0117] Exemplary IL-31 RA antibodies with extended half life comprising a cFc region, with a combination of at least four amino acid modifications at amino acid residue positions 434, 438, 440 and 428 and / or 436:

[0118] One aspect of the disclosure relates to a caninized antibody or antigen binding fragment thereof that binds cl L31 RA, the antibody comprising a cFc that comprises at least four of: an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, an amino acid other than a serine residue at position 440, and an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat. In some embodiments, the cFc comprises an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, an amino acid other than a serine residue at position 440, and an amino acid other than a methionine residue at position 428. In some embodiments, the cFc comprises an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, an amino acid other than a serine residue at position 440, and an amino acid other than a tyrosine residue at position 436. In some embodiments, the cFc comprises an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, an amino acid other than a serine residue at position 440, an amino acid other than a methionine residue at position 428 and an amino acid other than a tyrosine residue at position 436.

[0119] Another aspect of the disclosure relates to a caninized antibody or antigen binding fragment thereof that binds CIL31 RA, the antibody comprising a cFc which is selected from the group consisting of:

[0120] (i) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E);

[0121] (ii) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E).

[0122] (iii) a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E).

[0123] (iv) a cFc comprising: a leucine amino acid residue at position 428 (M428L), a serine amino acid residue at position 434 (N434S) wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0124] Other aspect of the technology include, but are not limited, the following embodiments.

[0125] In some embodiments, a caninized antibody that binds CIL31 RA as disclosed herein comprises a cFc, the cFc comprising at least two amino acid modifications, selected from the group consisting of: an amino acid other than a methionine residue at position 428, an amino acid other than an asparagine residue at position 434, an amino acid other than a tyrosine residue at position 436, an amino acid other than a glutamine residue at position 438, and an amino acid other than a serine residue at position 440, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0126] In some embodiments, a caninized antibody that binds CIL31 RA as disclosed herein comprises a cFc, where the cFc comprises at least three amino acid modifications, selected from the group consisting of: an amino acid other than a methionine (M) residue at position 428, an amino acid other than an asparagine (N) residue at position 434, an amino acid other than a tyrosine (Y) residue at position 436, an amino acid other than a glutamine (Q) residue at position 438, and an amino acid other than a serine (S) residue at position 440, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0127] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a cFc where the amino acid at position 428 in the cFc is selected from any of: a Leucine (L) residue, an isoleucine (I) or valine (V), where the preferred embodiment is a Leucine (L) residue, and where the numbering of amino acid residue positions is according to the EU Index as in Kabat.

[0128] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a cFc, where the amino acid residue at position 434 in the cFc is selected from any of: alanine (A), serine (S), histidine (H), phenylalanine (F), tyrosine (Y), or a conservative amino acid thereof (e.g., selected from any of: Glycine (G), serine (S), Asparagine (N), Glutamine (Q), Tyrosine (Y), Methionine (M) or Thr), wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0129] In some embodiments as disclosed herein, a caninized antibody that binds CIL31 RA as disclosed herein can comprise a cFc, where the amino acid residue at position 434 in the cFc is not a histadine, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0130] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a cFc, where the amino acid residue at position 435 in the cFc is a threonine (T), or a conservative amino acid thereof (e.g., a lysine (K) or histidine (H)) (Y435T).

[0131] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a cFc, where the amino acid residue at position 438 in the cFc is an arginine (R) residue, or a serine (S) (Q438R).

[0132] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a cFc, where the amino acid residue at position 440 in the cFc is a glutamic acid (E) residue, or a conservative amino acid thereof (e.g., asparagine (N) or a glutamine (Q)) (S440E).

[0133] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises the one or more residues selected from the group consisting of: a leucine amino acid residue at position 252, an alanine amino acid residue at position 254, a threonine amino acid residue at position 256, a glutamine amino acid residue at position 311 , a methionine amino acid residue at position 428, a histidine amino acid residue at position 433, an asparagine at position 434, a tyrosine at position 436, a glutamine at position 438, and a serine at position 440, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0134] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises three or more of the amino acid residues at the specific positions, selected from the group consisting of: (i) a leucine (L) amino acid residue at position 428 (M428L), (ii) an alanine amino acid (A) residue or histidine (H) amino acid residue at position 434 (N434A or N434H), (iii) a threonine amino acid (T) residue at position 436 (Y436T), (iv) an arginine amino acid (R) residue at position 438 (Q438R) and (v) a glutamic acid (E) amino acid residue at position 440 (S440E).

[0135] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R) and a glutamic acid amino acid residue at position 440 (S440E)

[0136] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R) and a glutamic acid amino acid residue at position 440 (S440E).

[0137] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R) and a glutamic acid amino acid residue at position 440 (S440E).

[0138] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc further comprises the one or more residues selected from the group consisting of: an amino acid other than a leucine residue at position 252, an amino acid other than an alanine residue at position 254, an amino acid other than a threonine residue at position 256, an amino acid other than a glutamine (Q) residue at position 311 and an amino acid other than a methionine residue at position 428, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0139] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises the one or more residues selected from the group consisting of: a leucine at position 252, an alanine at position 254, a threonine at position 256, a glutamine (Q) at position 311 , wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0140] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises the one or more residues selected from the group consisting of: an amino acid other than a leucine residue at position 252, an amino acid other than an alanine residue at position 254, an amino acid other than a threonine residue at position 256 and an amino acid other than a glutamine (Q) residue at position 311 wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0141] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises the one or more residues selected from the group consisting of: an amino acid selected from a phenylalanine (F), or tyrosine (Y) or tryptophan (W) or threonine (T) amino acid residues at position 252, (L252F, L252Y, L252W, L252T), a threonine (T) amino acid residue at position 254 (A254T), a glutamic acid (E) or aspartic acid (D) amino acid residue at position 256 (T256D, T256E), and a serine amino acid residue position 311 (Q311 S), wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0142] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the variant cFc comprises at least two amino acid modifications, selected from the group consisting of: an amino acid other than a methionine residue at position 428, an amino acid other than an asparagine residue at position 434, an amino acid other than a tyrosine residue at position 436, an amino acid other than a glutamine residue at position 438, and an amino acid other than a serine residue at position 440, or a conservative amino acid thereof.

[0143] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the variant cFc is selected from the group consisting of: a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R) and a glutamic acid amino acid residue at position 440 (S440E). a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E)

[0144] In all embodiments of all aspects as disclosed herein, a caninized antibody that binds CIL31 RA as disclosed herein can comprise a variant cFc, the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), where the heavy chain variable domain that contains the following CDRs:

[0145] VH-CDR1 comprising at least 90% sequence identity with SEQ ID NO: 13;

[0146] VH-CDR2 comprising at least 90% sequence identity with SEQ ID NO: 14;

[0147] VH-CDR3 comprising at least 90% sequence identity with SEQ ID NO: 15; and / or where the light chain variable domain that contains the following CDRs:

[0148] VL-CDR1 comprising at least 90% sequence identity with SEQ ID NO: 16; VL-CDR2 comprising at least 90% sequence identity with SEQ ID NO: 17;

[0149] VL-CDR3 comprising at least 90% sequence identity with SEQ ID NO: 18.

[0150] In all embodiments of all aspects as disclosed herein, a caninized antibody that binds CIL31 RA as disclosed herein can comprise a variant cFc, the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), where the heavy chain variable domain that contains the following CDRs:

[0151] VH-CDR1 comprising amino acids residues of SEQ ID NO: 13;

[0152] VH-CDR2 comprising amino acids residues of SEQ ID NO: 14;

[0153] VH-CDR3 comprising amino acids residues of SEQ ID NO: 15; and / or where the a light chain variable domain that contains the following CDRs:

[0154] VL-CDR1 comprising amino acids residues of SEQ ID NO: 16;

[0155] VL-CDR2 comprising amino acids residues of SEQ ID NO: 17;

[0156] VL-CDR3 comprising amino acids residues of SEQ ID NO: 18.

[0157] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein can comprise a variant cFc, where the variant cFc can comprise a hinge region that comprises an amino acid sequence that comprises at least 90%, 95%, 97%, 98%, 99% identity sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 or SEQ ID NO: 46.

[0158] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein has an increased half-life compared to the half-life of the corresponding caninized antibody or antigen binding fragment thereof that comprises a wild-type cFc, or comprises a cFc that is not a variant cFc as disclosed herein.

[0159] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein comprises an IgG constant domain comprising the cFc, wherein the IgG is selected from IgGA, IgGB, IgGC or IgGD.

[0160] In some embodiments of all aspects as disclosed herein, a caninized antibody that binds cl L31 RA as disclosed herein comprises a heavy chain and / or a light chain. For example, in some embodiments, the heavy chain comprises an amino acid sequence comprising amino acid residues of SEQ ID NO: 28, 29, 30 or 31 , or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOs: 28, 29, 30 or 31.

[0161] In some embodiments as disclosed herein, a caninized antibody that binds CIL31 RA as disclosed herein comprises a heavy chain, where the heavy chain comprises an amino acid sequence comprising amino acid residues of SEQ ID NO: 29, or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOs: 29 and wherein the cFc comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). (M428UN434A / Y436T / Q438R / S440E), In some embodiments, such a caninized antibody further comprises a light chain, the light chain comprises the amino acid sequence selected from the group consisting of: SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID Nos: 21 , 22 and 23.

[0162] One aspect of the technology disclosed herein relates to a caninized antibody that binds cl L31 RA, the antibody comprising a heavy chain, the heavy chain comprising a variant cFc that comprises: an amino acid other than a methionine residue at position 428, an amino acid other than an asparagine residue at position 434, an amino acid other than a tyrosine residue at position 436, an amino acid other than a glutamine residue at position 438, and an amino acid other than a serine residue at position 440, and where the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 29, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 29, and wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0163] One aspect of the technology disclosed herein relates to a caninized antibody that binds cl L31 RA, the antibody comprising a heavy chain and a variant cFc that comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and where the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 29, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 29, and wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat. One aspect of the technology disclosed herein relates to a caninized antibody that binds CIL31 RA, the antibody comprising a heavy chain and a variant cFc, where the variant cFc is selected from: a cFc that comprises: an amino acid other than a methionine residue at position 428, an amino acid other than an asparagine residue at position 434, an amino acid other than a glutamine residue at position 438, and an amino acid other than a serine residue at position 440, or a cFc that comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and where the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 30, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 30, and wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0164] One aspect of the technology disclosed herein relates to a caninized antibody that binds CIL31 RA, the antibody comprising a heavy chain and a variant cFc, where the variant cFc is selected from: a cFc that comprises: an amino acid other than an asparagine residue at position 434, an amino acid other than a tyrosine residue at position 436, an amino acid other than a glutamine residue at position 438, and an amino acid other than a serine residue at position 440, or a cFc that comprises: an alanine amino acid residue at position 434 (N434A), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and where the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 28, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 28, and wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0165] One aspect of the technology disclosed herein relates to a caninized antibody that binds CIL31 RA, the antibody comprising a heavy chain and a variant cFc, where the variant cFc is part of the heavy chain and is selected from: (i) a cFc that comprises: an amino acid other than a methionine residue at position 428, an amino acid other than an asparagine residue at position 434, or (ii) a cFc that comprises: a leucine amino acid residue at position 428 (M428L) and an alanine amino acid residue at position 434 (N434A), and where the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 31 , or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 31 , and wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0166] Another aspect of the technology disclosed herein relates to a nucleic acid molecule that encodes any of the caninized antibody or antigen binding fragment thereof disclosed herein. In some embodiments, the nucleic acid encodes a heavy chain comprising the amino acid selected from any of: SEQ ID NO: 28, 29, 30 or 31. In some embodiments, the nucleic acid molecule encodes a heavy chain comprising the amino acid that has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOs: 28, 29, 30 or 31.

[0167] In some embodiments, the nucleic acid encodes a light chain, where the light chain has an amino acid selected from the amino acid sequences of: SEQ ID NO: 21 , 22 and 23, or a light chain with an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID Nos: 21 , 22 and 23.

[0168] Another aspect of the technology disclosed herein relates to an expression vector comprising the nucleic acid molecule disclosed herein. In some embodiments, the expression vector comprises a nucleic acid encoding a heavy chain comprising the amino acid selected from any of: SEQ ID NO: 28, 29, 30 or 31 or a heavy chain comprising the amino acid that has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOs: 28, 29, 30 or 31 . In some embodiments, the expression vector comprises a nucleic acid that encodes a light chain having amino acid sequence selected from the amino acid sequences of: SEQ ID NO: 21 , 22 and 23, or a light chain with an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID Nos: 21 , 22 and 23. In some embodiments, the expression vector comprises (i) a nucleic acid encoding a encoding a heavy chain comprising the amino acid selected from any of: SEQ ID NO: 28, 29, 30 or 31 or a heavy chain comprising the amino acid that has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOs: 28, 29, 30 or 31 , and (ii) a nucleic acid that encodes a light chain having amino acid sequence selected from the amino acid sequences of: SEQ ID NO: 21 , 22 and 23, or a light chain with an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID Nos: 21 , 22 and 23.

[0169] Another aspect of the technology disclosed herein relates to a host cell comprising such an expression vector, for use in the manufacturer of an antibody as disclosed herein.

[0170] Another aspect relates to a pharmaceutical composition comprising the caninized antibody as disclosed herein, and a pharmaceutically acceptable carrier. Another aspect relates to a caninized antibody that binds CIL31 RA as disclosed herein or a pharmaceutical composition comprising the same, for use in the treatment of atopic dermatitis in a canine.

[0171] Another aspects relates to method of treating a canine that has atopic dermatitis comprising administering to the canine at least one, or a combination of any of the caninized antibodies as disclosed herein that binds CIL31 RA, or a pharmaceutical composition comprising the same.

[0172] / / . Other Exemplary half-life mutations and combinations thereof:

[0173] In particular embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising two or more amino acid modifications relative to a wild-type cFc or an unsubstituted cFc. In certain embodiments, the modified cFc comprises a phenylalanine (F) at amino acid position 252 and an aspartic acid (D) at amino acid position 256. In certain embodiments, the modified cFc comprises a tyrosine (Y) at amino acid position 252 and an aspartic acid (D) at amino acid position 256. In certain embodiments, the modified cFc comprises a tyrosine (Y) at amino acid position 252 and an glutamic acid (E) at amino acid position 256. In certain embodiments, the modified cFc comprises a serine at amino acid position 311 and a histidine at amino acid position 434. In certain embodiments, the modified cFc comprises a leucine at amino acid position 428 and a serine at amino acid position 434, numbered according to the Ell index as in Kabat.

[0174] In particular embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least three amino acid modifications relative to a wild-type cFc or an unsubstituted variant cFc. In certain embodiments, the modified cFc comprises at least three amino acid modifications are selected from any one of: an alanine at amino acid position 434, an arginine at amino acid position 438 and a glutamic acid at amino acid position 440, numbered according to the Ell index as in Kabat.

[0175] In particular embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising a combination of modifications at the amino acid residue positions selected from the group consisting of, numbered according to the Ell index as in Kabat:

[0176] N434A, Y436T, Q438R and S440E;

[0177] M428L, N434A, Y436T, Q438R and S440E; M428L, N434A, Q438R and S440E.

[0178] L252F and T256D;

[0179] L252F, A254T and T256E; or

[0180] Q311S and N434H.

[0181] In other embodiments, a modified cFc comprises an alanine (A) residue at amino acid residue position 434. In other embodiments, a modified cFc comprises a histidine (H) residue at amino acid residue position 434. In other embodiments, a modified cFc comprises a threonine (T) residue at amino acid residue position 436. In other embodiments, a modified cFc comprises an arginine (R) residue at amino acid residue position 438. In other embodiments, a modified cFc comprises a glutamic acid (E) residue at amino acid residue position 440, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat. In one aspect of the technology described herein, an antibody or antibody binding fragment thereof comprises a leucine residue (L) at amino acid residue position 428.

[0182] In one aspect of the technology described herein, a modified cFc further comprises a Phenylalanine (F) residue at amino acid residue position 252. In other embodiments, a modified cFc comprises a tyrosine (Y) residue at amino acid residue position 252. In other embodiments, a modified cFc comprises a tryptophan (W) residue at amino acid residue position 252. In other embodiments, a modified cFc comprises a threonine (T) residue at amino acid residue position 252, wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

[0183] In other embodiments, a modified cFc can comprise a threonine (T) residue at amino acid residue position 254. In other embodiments, a modified cFc comprises a glutamic acid (E) residue at amino acid residue position 256. In other embodiments, a modified cFc comprises an aspartic acid (D) residue at amino acid residue position 256. In other embodiments, a modified cFc comprises a serine (S) residue at amino acid residue position 311.

[0184] In other aspects, an isolated antibody as disclosed herein comprises a modified Fc domain, where the modified cFc selected from:

[0185] (i) a modified cFc comprising two amino acid modifications selected from the group consisting of: L252 / T256, Q311 / N434 or M428 / N434, and where a Leucine (L) is not at amino acid position 252, a threonine (T) is not at amino acid position 256, a glutamine (Q) is not at amino acid position 311 , a methionine (M) is not at amino acid position 428, and an asparagine (N) is not at position 434, or

[0186] (ii) a modified cFc comprising three amino acid modifications L252 / A254 / T256, where a Leucine (L) is not at amino acid position 252, an alanine (A) is not at amino acid position 254, and a threonine (T) is not at amino acid position 256, or

[0187] (iii) a modified Fc domain comprising four amino acid modification selected from the group consisting of: N434 / Y436 / Q438 / S440 or M428 / N434 / Q438 / S440, where a methionine (M) is not at amino acid position 428, an asparagine (N) is not at position 434, a tyrosine (Y) is not at position 436, a glutamine (Q) is not at amino acid position 438 and a serine (S) is not at position 440, or

[0188] (iv) a modified Fc domain comprising five amino acid modification of: M428 / N434 / Y436 / Q438 / S440, where a methionine (M) is not at amino acid position 428, an asparagine (N) is not at position 434, a tyrosine (Y) is not at position 436, a glutamine (Q) is not at amino acid position 438 and a serine (S) is not at position 440, wherein the amino acid modifications are numbered according to the Ell index as in Kabat.

[0189] In other aspects, an antibody as disclosed herein comprises a modified cFc domain, the modified cFc comprising two amino acid modifications selected from the group consisting of: L252 / T256, Q311 / N434, M428 / N434, where: the Leucine (L) at amino acid position 252 is substituted with a phenylalanine (F) or a conservative amino acid thereof, e.g., Tyrosine (Y) or methionine (M), or alternatively, a Leucine (L) at amino acid position 252 is substituted with a tyrosine (Y) or a tryptophan (W) or a conservative amino acid of tyrosine or tryptophan (W), or alternatively, a Leucine (L) at amino acid position 252 is substituted with a threonine (T) or a conservative amino acid of threonine e.g., serine (S); the threonine (T) at amino acid position 256 is substituted with a glutamic acid (E) or a conservative amino acid thereof, e.g., A or Q, or alternatively, a threonine (T) at amino acid position 256 is substituted with a aspartic acid (D) or a conservative amino acid of aspartic acid; the glutamine (Q) at amino acid position 311 is substituted with a serine (S) or a conservative amino acid thereof e.g., threonine (T), the methionine (M) at amino acid position 428 is substituted with leucine (L) or a conservative amino acid thereof e.g., isoleucine (I) or valine (V), and the asparagine (N) at position 434 is substituted with an alanine (A) or a conservative amino acid thereof e.g., Glycine (G) or Serine (S), or alternatively, an asparagine (N) at position 434 is substituted with an phenylalanine (F) or a conservative amino acid thereof e.g., tyrosine (Y) or methionine (M); or alternatively, an asparagine (N) at position 434 is substituted with an tyrosine (Y) or a conservative amino acid thereof, or alternatively, an asparagine (N) at position 434 is substituted with an histidine (H) or a conservative amino acid thereof, e.g., asparagine (D) or glutamine (Q), or alternatively, an asparagine (N) at position 434 is substituted with a serine (S) or a conservative amino acid thereof, e.g., threonine (T).

[0190] In other aspects, an antibody as disclosed herein comprises a modified cFc domain, the modified cFc comprising three amino acid modifications at positions L252 / A254 / T256, where a Leucine (L) at amino acid position 252 is substituted with a phenylalanine (F) or a conservative amino acid thereof, e.g., Tyrosine (Y) or methionine (M), or alternatively, a Leucine (L) at amino acid position 252 is substituted with a tyrosine (Y) or a tryptophan (W) or a conservative amino acid of tyrosine or tryptophan (W), or alternatively, a Leucine (L) at amino acid position 252 is substituted with a threonine (T) or a conservative amino acid of threonine e.g., a serine (S); an alanine (A) at amino acid position 254 is substituted with a threonine (T) or a conservative amino acid of threonine e.g., serine (S); and a threonine (T) at amino acid position 256 is substituted with a glutamic acid (E) or a conservative amino acid thereof, e.g., A or Q, or alternatively, a threonine (T) at amino acid position 256 is substituted with an aspartic acid (D) or a conservative amino acid of aspartic acid.

[0191] In other aspects, an antibody as disclosed herein comprises a modified cFc domain, the modified cFc comprising four amino acid modifications at positions selected from the group consisting of: N434 / Y436 / Q438 / S440 or M428 / N434 / Q438 / S440, where: a methionine (M) at amino acid position 428 substituted with leucine (L) or a conservative amino acid thereof e.g., isoleucine (I) or valine (V), and an asparagine (N) at position 434 substituted with an alanine (A) or a conservative amino acid thereof e.g., Glycine (G) or Serine (S), or alternatively, an asparagine (N) at position 434 is substituted with an phenylalanine (F) or a conservative amino acid thereof e.g., tyrosine (Y) or methionine (M); or alternatively, an asparagine (N) at position 434 is substituted with an tyrosine (Y) or a conservative amino acid thereof, or alternatively, an asparagine (N) at position 434 is substituted with an histidine (H) or a conservative amino acid thereof, e.g., asparagine (D) or glutamine (Q), or alternatively, an asparagine (N) at position 434 is substituted with a serine (S) or a conservative amino acid thereof, e.g., threonine (T), a tyrosine (Y) at position 436 substituted with a threonine (T) or a conservative amino acid thereof, e.g., a serine (S), a glutamine (Q) at amino acid position 438 substituted with an arginine (R) or a conservative amino acid thereof e.g., a lysine (K) or histidine (H), a serine (S) at position 440 substituted with a glutamic acid (E) or a conservative amino acid thereof, e.g., aspartic acid (R) or glutamine (Q).

[0192] In other aspects, an antibody as disclosed herein comprises a modified cFc domain, the modified cFc comprising five amino acid modifications at positions M428 / N434 / Y436 / Q438 / S440, where: a methionine (M) at amino acid position 428 substituted with leucine (L) or a conservative amino acid thereof e.g., isoleucine (I) or valine (V), and an asparagine (N) at position 434 substituted with an alanine (A) or a conservative amino acid thereof e.g., Glycine (G) or Serine (S), or alternatively, an asparagine (N) at position 434 is substituted with an phenylalanine (F) or a conservative amino acid thereof e.g., tyrosine (Y) or methionine (M); or alternatively, an asparagine (N) at position 434 is substituted with an tyrosine (Y) or a conservative amino acid thereof, or alternatively, an asparagine (N) at position 434 is substituted with an histidine (H) or a conservative amino acid thereof, e.g., asparagine (D) or glutamine (Q), or alternatively, an asparagine (N) at position 434 is substituted with a serine (S) or a conservative amino acid thereof, e.g., threonine (T), a tyrosine (Y) at position 436 substituted with a threonine (T) or a conservative amino acid thereof, e.g., a serine (S), a glutamine (Q) at amino acid position 438 substituted with a arginine (R) or a conservative amino acid thereof e.g., a lysine (K) or histidine (H), a serine (S) at position 440 substituted with a glutamic acid (E) or a conservative amino acid thereof, e.g., aspartic acid (R) or glutamine (Q).

[0193] In other aspects, an antibody or antibody fragment thereof as disclosed herein comprises a modified cFc domain, the modified cFc comprising a two amino acid modifications located at the positions selected from the group consisting of: L252 / T256, Q311 / N434, M428 / N434, where: a Leucine (L) at amino acid position 252 substituted with a phenylalanine (F) or a conservative amino acid thereof, e.g., Tyrosine (Y) or methionine (M), a threonine (T) at amino acid position 256 substituted with a aspartic acid (D) or a conservative amino acid of aspartic acid, a glutamine (Q) at amino acid position 311 is substituted with a serine (S) or a conservative amino acid thereof e.g., threonine (T), a methionine (M) at amino acid position 428 is substituted with leucine (L) or a conservative amino acid thereof e.g., isoleucine (I) or valine (V), and an asparagine (N) at position 434 is substituted with a serine (S) or a conservative amino acid thereof, e.g., threonine (T), or alternatively, a N at position 434 a histidine (H) or a conservative amino acid thereof, e.g., asparagine (D) or glutamine (Q).

[0194] In other aspects, an antibody or antibody fragment thereof as disclosed herein comprises a modified cFc domain, the modified cFc comprising three amino acid modifications at positions L252 / A254 / T256, where: a Leucine (L) at amino acid position 252 is substituted with a phenylalanine (F) or a conservative amino acid thereof, e.g., Tyrosine (Y) or methionine (M), an alanine (A) at amino acid position 254 is substituted with a threonine (T) or a conservative amino acid of threonine e.g., serine (S); and a threonine (T) at amino acid position 256 is substituted with a glutamic acid (E) or a conservative amino acid thereof, e.g., A or Q,

[0195] In other aspects, an antibody or antibody fragment thereof as disclosed herein comprises a modified cFc domain, the modified cFc comprising four amino acid modifications at positions selected from the group consisting of: N434 / Y436 / Q438 / S440 or M428 / N434 / Q438 / S440, where a methionine (M) at amino acid position 428 is substituted with leucine (L) or a conservative amino acid thereof e.g., isoleucine (I) or valine (V), and an asparagine (N) at position 434 is substituted with an alanine (A) or a conservative amino acid thereof e.g., Glycine (G) or Serine (S), a tyrosine (Y) at position 436 is substituted with an threonine (T) or a conservative amino acid thereof, e.g., a serine (S), a glutamine (Q) at amino acid position 438 is substituted with a arginine (R) or a conservative amino acid thereof e.g., a lysine (K) or histidine (H), a serine (S) at position 440 is substituted with an glutamic acid (E) or a conservative amino acid thereof, e.g., aspartic acid (R) or glutamine (Q).

[0196] In other aspects, an antibody or antibody fragment thereof as disclosed herein comprises a modified cFc domain, the modified cFc comprising five amino acid modifications at positions selected from the group consisting of: M428 / N434 / Y436 / Q438 / S440, where: a methionine (M) at amino acid position 428 is substituted with leucine (L) or a conservative amino acid thereof e.g., isoleucine (I) or valine (V), and an asparagine (N) at position 434 is substituted with an alanine (A) or a conservative amino acid thereof e.g., Glycine (G) or Serine (S), a tyrosine (Y) at position 436 substituted with an threonine (T) or a conservative amino acid thereof, e.g., a serine (S), a glutamine (Q) at amino acid position 438 is substituted with a arginine (R) or a conservative amino acid thereof e.g., a lysine (K) or histidine (H), and a serine (S) at position 440 substituted with an glutamic acid (E) or a conservative amino acid thereof, e.g., aspartic acid (R) or glutamine (Q).

[0197] Exemplary modified cFc regions comprising a single amino acid modification to extend half-life:

[0198] In particular embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising a single amino acid modification at residue position L252, where the modification is selected from the group consisting of, numbered according to the Ell index as in Kabat: L252F, or L252Y L252W, L252T. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that has a single amino acid modification at residue position L252 does not comprise another mutation in the Fc region that extends the half-life of the Fc. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises a single amino acid modification at residue position L252, e.g., a modification selected from the group consisting of: L252F, L252Y, L252W, L252T can comprise an amino acid modification at residue position D31 and / or N63. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises a single amino acid modification at residue position L252, e.g., a modification selected from the group consisting of: L252F, L252Y, L252W, L252T can comprise an amino acid modification selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that has a single amino acid modification at residue position L252, can comprise at least one or more additional amino acid modifications at amino acid residue positions A254, Q311 , M428, N434, Y436, Q438 and S440, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that has a single amino acid modification at residue position L252, can comprise at an additional amino acid modification at amino acid residue position T256, where the amino acid modification is T256E, numbered according to the Ell index as in Kabat.

[0199] Exemplary modified cFc regions comprising a combination of two amino acid modifications to extend half-life:

[0200] In particular embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least two amino acid modifications at residue positions L252 and T256. In some embodiments, the least two amino acid substitutions at residue positions L252 and T256 are amino acid substitutions selected from the group consisting of: L252F / T256D (FD), L252Y / T256D (YD), L252T / T256D, L252W / T256D, L252F / T256E (FE), L252Y / T256E, L252T / T256E and L252W / T256E, numbered according to the EU index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises a two amino acid substitutions at residue positions L252 and T256, e.g., a substitution selected from the group consisting of: L252F / T256D (FD), L252Y / T256D (YD), L252T / T256D, L252W / T256D, L252F / T256E (FE), L252Y / T256E, L252T / T256E and L252W / T256E can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises two amino acid substitutions at residue positions L252 and T256, can comprise at least one or more additional amino acid substitutions at amino acid residue positions A254, Q311 , M428, N434, Y436, Q438 and S440, numbered according to the EU index as in Kabat. In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least two amino acid substitutions at residue positions Q311 and N434. In some embodiments, the least two amino acid substitutions at residue positions Q311 and N434 are amino acid substitutions selected from the group consisting of: Q311S / N434H or Q311S / N434F or Q311S / N434Y or Q311S / N434A and Q311S / N434S, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises two amino acid substitutions at residue positions Q311 and N434, e.g., a substitution selected from the group consisting of: Q311S / N434H or Q311S / N434F or Q311S / N434Y or Q311S / N434A and Q311S / N434S can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises two amino acid substitutions at residue positions Q311 and N434, can comprise at least one or more additional amino acid substitutions at amino acid residue positions L252, A254, T256, M428, N434, Y436, Q438 and S440, numbered according to the Ell index as in Kabat.

[0201] Exemplary combination of three amino acid substitutions in Fc:

[0202] In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least three amino acid substitutions at residue positions L252, A254 and T256. In some embodiments, the least three amino acid substitutions at residue positions L252, A254 and T256 are amino acid substitutions selected from the group consisting of: L252F / A254T / T256E, L252Y / A254T / T256E, L252W / A254T / T256E,

[0203] L252T / A254T / T256E, L252F / A254T / T256D, L252Y / A254T / T256D, L252W / A254T / T256D and L252T / A254T / T256D, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises three amino acid substitutions at residue positions L252, A254 and T256, e.g., a substitution selected from the group consisting of: L252F / A254T / T256E, L252Y / A254T / T256E, L252W / A254T / T256E, L252T / A254T / T256E, L252F / A254T / T256D, L252Y / A254T / T256D, L252W / A254T / T256D and L252T / A254T / T256D can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises three amino acid substitutions at residue positions L252, A254 and T256, can comprise at least one or more additional amino acid substitutions at amino acid residue positions Q311 , M428, N434, Y436, Q438 and S440, numbered according to the Ell index as in Kabat. In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at three amino acid substitutions at residue positions selected from any three of: M428, N434, Y436, Q438 and S440E. In some embodiments, the least three amino acid substitutions at residue positions selected from: M428, N434, Y436, Q438 and S440E are selected from the group of: M428L / N434A / Y436T, M428L / N434A / Q438R, M428L / N434A / S440E, M428L / Y436T / Q438R, M428L / Y436T / S440E, M428L / Q438R / S440E, N434A / Y436T / Q438R, N434A / Y436T / S440E, N434A / Q438R / S440E and Y436T / Q438R / S440E, numbered according to the Ell index as in Kabat.

[0204] In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at four amino acid substitutions at residue positions selected from any four of: M428, N434, Y436, Q438 and S440E. In some embodiments, the least four amino acid substitutions at residue positions selected from: M428, N434, Y436, Q438 and S440E are selected from the group of: M428L / N434A / Y436T / Q438R, M428L / N434A / Y436T / S440E, M428L / N434A / Q438R / S440E, M428L / Y436T / Q438R / S440E,

[0205] N434A / Y436T / Q438R / S440E, numbered according to the Ell index as in Kabat. In some embodiments, the amino acid substitution at amino acid position N434, e.g., the substitution N434A can be replaced with any of the substitutions selected from: N434F, N434Y, N434S, N434A or N434H, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises any three or four amino acid substitutions at residue M428, N434, Y436, Q438 and S440E, can further comprise at least one or more additional amino acid substitutions at amino acid residue positions L252, A254, T256 and Q311 , numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises any three or four amino acid substitutions selected from residue positions M428, N434, Y436, Q438 and S440E can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A.

[0206] Exemplary combination of four amino acid substitutions in Fc:

[0207] In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least four amino acid substitutions at residue positions N434, Y436, Q438 and S440. In some embodiments, the least four amino acid substitutions at residue positions N434, Y436, Q438 and S440 are amino acid substitutions selected from the group consisting of: N434A / Y436T / Q438R / S440E; or N434F / Y436T / Q438R / S440E or N434Y / Y436T / Q438R / S440E or N434S / Y436T / Q438R / S440E or N434H / Y436T / Q438R / S440E, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions N434, Y436, Q438 and S440, e.g., a substitution selected from the group consisting of: N434A / Y436T / Q438R / S440E; or N434F / Y436T / Q438R / S440E or N434Y / Y436T / Q438R / S440E or N434S / Y436T / Q438R / S440E or N434H / Y436T / Q438R / S440E can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions N434, Y436, Q438 and S440, can comprise at least one or more additional amino acid substitutions at amino acid residue positions L252, A254, T256, Q311 and M428, numbered according to the Ell index as in Kabat.

[0208] In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least four amino acid substitutions at residue positions N434, Y436, Q438R and S440E. In some embodiments, the least four amino acid substitutions at residue positions N434, Y436, Q438 and S440 are amino acid substitutions selected from the group consisting of: N434A / Y436T / Q438R / S440E,

[0209] N434F / Y436T / Q438R / S440E, N434Y / Y436T / Q438R / S440E, N434S / Y436T / Q438R / S440E and N434H / Y436T / Q438R / S440E, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions N434, Y436, Q438R and S440E, e.g., a substitution selected from the group consisting of: N434A / Y436T / Q438R / S440E, N434F / Y436T / Q438R / S440E, N434Y / Y436T / Q438R / S440E, N434S / Y436T / Q438R / S440E and N434H / Y436T / Q438R / S440E can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions N434, Y436, Q438R and S440E, can comprise at least one or more additional amino acid substitutions at amino acid residue positions L252, A254, T256, Q311 and M428, numbered according to the Ell index as in Kabat.

[0210] In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least four amino acid substitutions at residue positions M428, N434, Q438R and S440E. In some embodiments, the least four amino acid substitutions at residue positions M428, N434, Q438R and S440E are amino acid substitutions selected from the group consisting of: M428LZ N434A / Q438R / S440E, M428L / N434F / Q438R / S440E, M428L / N434Y / Q438R / S440E, M428L / N434S / Q438R / S440E and M428L / N434H / Q438R / S440E, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions M428, N434, Q438R and S440E, e.g., a substitution selected from the group consisting of: M428L / N434A / Q438R / S440E, M428L / N434F / Q438R / S440E, M428L / N434Y / Q438R / S440E, M428L / N434S / Q438R / S440E and M428L / N434H / Q438R / S440E can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions M428, N434, Q438R and S440E., can comprise at least one or more additional amino acid substitutions at amino acid residue positions L252, A254, T256, Q311 and Y436, numbered according to the Ell index as in Kabat.

[0211] Exemplary combination of five amino acid substitutions in Fc:

[0212] In some embodiments, the caninized antibody or antigen binding fragment according to the invention comprises a modified cFc comprising at least five amino acid substitutions at residue positions M428, N434, Y436, Q438 and S440E. In some embodiments, the least five amino acid substitutions at residue positions M428, N434, Y436, Q438 and S440E are amino acid substitutions selected from the group consisting of: M428L / N434A / Y436T / Q438R / S440E, M428L / N434F / Q438R / S440E, M428L / N434Y / Q438R / S440E, M428L / N434S / Q438R / S440E and M428L / N434H / Q438R / S440E, numbered according to the Ell index as in Kabat. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions M428, N434, Y436, Q438 and S440E, e.g., a substitution selected from the group consisting of: M428L / N434A / Y436T / Q438R / S440E, M428L / N434F / Q438R / S440E, M428L / N434Y / Q438R / S440E, M428L / N434S / Q438R / S440E and M428L / N434H / Q438R / S440E can comprise an amino acid substitution selected from D31A or N63A, or D31A and N63A. In some embodiments, a caninized antibody or antigen binding fragment comprising a modified cFc that comprises four amino acid substitutions at residue positions M428, N434, Y436, Q438 and S440E, can comprise at least one or more additional amino acid substitutions at amino acid residue positions L252, A254, T256 and Q311 , numbered according to the Ell index as in Kabat.

[0213] In other aspects, an antibody as disclosed herein comprising a modified Fc domain comprises two amino acid substitutions, numbered according to the Ell index as in Kabat, selected from the groups of two amino acid substitutions listed in Table 2: Table 2: n other aspects, an antibody as disclosed herein comprising a modified Fc domain comprises three amino acid substitutions, numbered according to the Ell index as in Kabat, selected from the groups of three amino acid substitutions listed in Table 3: Table 3:

[0214] In other aspects, an antibody as disclosed herein comprising a modified Fc domain comprises four amino acid substitutions, numbered according to the Ell index as in Kabat, selected from the groups of combination of four amino acid residue substitutions as listed in Table 4: Table 4:

[0215] In other aspects, an antibody as disclosed herein comprising a modified Fc domain comprises three amino acid substitutions selected from: L252Y or L252F or L252W or L252T, A254T, T256E or T256D, Q311S and N434A or N434H, numbered according to the Ell index as in Kabat, selected from the groups of combination of three amino acid residue substitutions as listed in

[0216] Table 5:

[0217] Table 5:

[0218] In other aspects, an antibody as disclosed herein comprising a modified Fc domain comprises four amino acid substitutions selected from: L252Y or L252F or L252W or L252T, A254T, T256E or T256D, Q311S and N434A or N434H, numbered according to the Ell index as in Kabat, selected from the groups of combination of four amino acid residue substitutions as listed in Table 6: Table 6:

[0219] The wild-type cFc is selected from an IgG A (or “IgGA” or “IgG-A”) cFc, IgG B (or “IgGB” or “IgG- B”) cFc, IgG 0 (or “IgGC” or “IgG-C”) cFc or IgG D (or “IgGD” or “IgG-D”) cFc. In an embodiment, the cFc of the caninized antibody or antigen binding fragment thereof according to the invention may further comprise a mutation relative to a wild-type cFc, but which comprises one or more amino acid substitutions made to diminish the antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the antibody, that is not found in nature and therefore, is not a wild-type Fc region. However, it is contemplated herein that an antibody comprising such a Fc region that comprises one or more amino acid substitutions made to diminish the antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the antibody should have the same half-life as the corresponding antibody that comprises the corresponding wild-type Fc region found in nature in the absence of any amino acid substitutions made to enhance the half-life of an antibody comprising the modified cFc region. The modified cFc is selected from an IgG Am cFc, IgG Bm cFc, IgG Cm cFc or IgG Dm cFc.

[0220] The modified cFc comprises the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. In these sequences two (2) amino acid residue substitutions are introduced relative to the amino acid sequences of respectively, SEQ ID NO: 1 , 3, 5 and 7. These amino acid residue substitutions are D31 A and N63A, numbered according to Kabat. For example, in the amino acid sequence of SEQ ID NO: 3 of IgG-B (see below). Accordingly, both the aspartic acid residue (D) at position 31 of SEQ ID NO: 3 and the asparagine residue (N) at position 63 of SEQ ID NO: 3, are substituted by an alanine residue (A) in the amino acid sequence of IgG-Bm, i.e. , SEQ ID NO: 4. These two amino acid residue substitutions serve to significantly diminish the antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the naturally occurring canine IgG-B (see, U.S. 10,106,607 B2, the contents of which are hereby incorporated by reference in their entirety). Analogous substitutions can be made in the IgG-A, IgG-C, and IgG-D Fe regions, which are denoted as IgG-Am, IgG-Cm, and IgG-Dm.

[0221] In one embodiment, the caninized antibody or antigen binding fragment thereof comprises that the cFc comprises an amino acid sequence selected from the group consisting of: SEQ ID Nos: SEQ ID NOS: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOS: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc comprises one or more amino acid substitutions relative to a wild-type cFc at any one or more amino acid positions selected from the group of: 252, 254, 256, 311 , 428, 434, 436, 438 and 440.

[0222] In one embodiment, the caninized antibody or antigen binding fragment thereof comprises that the antibody or fragment thereof comprises a heavy chain variable domain that contains the following CDRs:

[0223] VH-CDR1 comprising at least 90% sequence identity with SEQ ID NO: 13;

[0224] VH-CDR2 comprising at least 90% sequence identity with SEQ ID NO: 14;

[0225] VH-CDR3 comprising at least 90% sequence identity with SEQ ID NO: 15; and / or a light chain variable domain that contains the following CDRs:

[0226] VL-CDR1 comprising at least 90% sequence identity with SEQ ID NO: 16; VL-CDR2 comprising at least 90% sequence identity with SEQ ID NO: 17;

[0227] VL-CDR3 comprising at least 90% sequence identity with SEQ ID NO: 18.

[0228] In one embodiment, the caninized antibody or antigen binding fragment thereof comprises that the antibody or fragment thereof comprises a heavy chain variable domain that contains the following CDRs:

[0229] VH-CDR1 comprises the amino acid residues of SEQ ID NO: 13;

[0230] VH-CDR2 comprises the amino acid residues of SEQ ID NO: 14;

[0231] VH-CDR3 comprises the amino acid residues of SEQ ID NO: 15; and / or a light chain variable domain that contains the following CDRs:

[0232] VL-CDR1 comprises the amino acid residues of SEQ ID NO: 16;

[0233] VL-CDR2 comprises the amino acid residues of SEQ ID NO: 17;

[0234] VL-CDR3 comprises the amino acid residues of SEQ ID NO: 18.

[0235] In one embodiment, the caninized antibody or antigen binding fragment thereof according to the invention, wherein the cFc comprises a hinge region that comprises an amino acid sequence that comprises at least 90%, 95%, 97%, 98%, 99% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 or SEQ ID NO: 46.

[0236] In one embodiment, the caninized antibody or antigen binding fragment thereof according to the invention that has an increased half-life compared to the half-life of the corresponding caninized antibody or antigen binding fragment thereof that comprises a wild-type cFc.

[0237] In one embodiment, the caninized interleukin-31 receptor alpha (clL-31 RA) antibodies and IgG Fc fusion proteins thereof with enhanced half-lives according to the invention comprise an IgG constant domain comprising the cFc, wherein the IgG is selected from IgGA, IgGAm, IgGB, IgGBm, IgGC, IgGCm, IgGD or IgGDm.

[0238] In one embodiment, the caninized interleukin-31 receptor alpha (clL-31 RA) antibodies and IgG Fc fusion proteins thereof with enhanced half-lives according to the invention comprises a heavy chain and / or a light chain. Preferably, the caninized interleukin-31 receptor alpha (clL-31 RA) antibodies and IgG Fc fusion proteins thereof with enhanced half-lives according to the invention comprise a heavy chain and a light chain. Accordingly, in some embodiments, the caninized interleukin-31 receptor alpha (clL-31 RA) antibodies and IgG Fc fusion proteins thereof with enhanced half-lives according to the invention comprise a heavy chain that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35,

[0239] 36, 37, 38, 39 and 40, or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40. In some embodiments, the caninized interleukin-31 receptor alpha (clL-31 RA) antibodies and IgG Fc fusion proteins thereof with enhanced half-lives according to the invention comprise a heavy chain that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 24, 25, 26 and 27 that comprise the two or more amino acid substitutions as follows, as disclosed herein, e.g., at least two or three substitutions selected from any of: L252F, L252Y, L252W, L252T, A254T, T256D, T256E, Q311 S, M428L, N434A, N434H, N434S, Y436T, Q438R and S440E, or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 3, 4, 24, 25, 26 and 27 that comprise the two or more amino acid substitutions as follows, as disclosed herein, e.g., at least two or three substitutions selected from any of: L252F, L252Y, L252W, L252T, A254T, T256D, T256E, Q311 S, M428L, N434A, N434H, N434S, Y436T, Q438R and S440E.

[0240] In one embodiment, the caninized interleukin-31 receptor alpha (clL-31 RA) antibodies and IgG Fc fusion proteins thereof with enhanced half-lives according to the invention comprises a light chain that comprises the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or a light chain comprising an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0241] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain with an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36,

[0242] 37, 38, 39 and 40, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0243] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence comprising SEQ ID NO: 28, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 28 and also comprising the following modifications N434A / Y436T / Q438R / S440E (ATRE) , and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0244] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 29, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 29 and also comprising the modifications of M428L / N434A / Y436T / Q438R / S440E (LATRE), and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0245] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 30, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 30 and also comprising the modifications of M428L / N434A / Q438R / S440E) (LARE), and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0246] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 31 , or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 31 and also comprising the modifications of M428L / N434S (LS), and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0247] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 32, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 32 and also comprising the N434H (H) modification, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0248] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 33, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 33 and also comprising the Q311S (S) modification, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0249] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 34, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 34 and also comprising the residue modifications of Q311S and N434H (SH), and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0250] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprises an amino acid sequence of SEQ ID NO: 35, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 35 and also comprising the residue modifications of L252F / A254T / T256E) (FTE), and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0251] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence comprising SEQ ID NO: 36, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 36 and also comprising the residue modifications of (L252F / T256D) (FD), and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0252] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence comprising SEQ ID NO: 37, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 37 and comprising a residue modification of L252F, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0253] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 38, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 38 and also comprising the residue modification of L252Y, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0254] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 39, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 39 and having the residue modification of L252T, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0255] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 40, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 40 and having the residue modification of L252W, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0256] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain with an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, and a light chain comprising the amino acid sequence of SEQ ID NO: 21 or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

[0257] 21.

[0258] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain with an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, and a light chain comprising the amino acid sequence of SEQ ID NO: 22 or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

[0259] 22.

[0260] In some embodiments, a caninized interleukin-31 receptor alpha (clL-31 RA) antibody with enhanced half-lives according to the invention comprises a heavy chain with an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, and a light chain comprising the amino acid sequence of SEQ ID NO: 23 or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 23.

[0261] III. Nucleic acids

[0262] Also included in the present invention are the nucleic acids that encode the IgG Fc fusion proteins, the antibodies, and the antigen binding fragments of the antibodies provided of the present invention, comprising amino acid sequences that are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of the caninized antibodies provided herein when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give an exact match between the respective sequences over the entire length of the respective reference sequences, i.e. , 95% identical means that 95% of the amino acids in the two sequences are identical. The present invention further provides nucleic acids that encode the fusion proteins and / or the immunoglobulin polypeptides which comprise nucleic acid sequences that are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference nucleic acid sequences when the comparison is performed with a BLAST algorithm, wherein the parameters of the algorithm are selected to give the exact match, between the respective nucleotide sequences over the entire length of the respective reference sequences, i.e., at least 98% identical means that at least 98% of the nucleotides in the two nucleic acid sequences are identical, are also included in the present invention.

[0263] As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program using the default parameters.

[0264] The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, T.L., et al., Meth. Enzymol. 266:131-141(1996); Altschul, S.F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J.C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J.M. et al., Comput. Appl. Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, (1978); Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3." (1978), M.O. Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., J. Mol. Biol. 219:555-565 (1991); States, D.J., et al., Methods 3:66-70(1991); Henikoff, S., et al., Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992); Altschul, S.F., et al., J. Mol. Evol. 36:290-300 (1993); ALIGNMENT STATISTICS: Karlin, S., et al., Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990); Karlin, S., et al., Proc. Natl. Acad. Sci. USA 90:5873- 5877 (1993); Dembo, A., et al., Ann. Prob. 22:2022-2039 (1994); and Altschul, S.F. "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).

[0265] In one aspect there is provided for a nucleic acid molecule that encodes the caninized antibody or antigen binding fragment thereof of any one of the previous claims.

[0266] In one aspect there is provided for an expression vector comprising the nucleic acid encoding a heavy chain with an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40. In one aspect there is provided for an expression vector comprising the nucleic acid encoding a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23. In one aspect herein, there is provided for an expression vector comprising a nucleic acid encoding a heavy chain with an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, or encodes a heavy chain with an amino acid sequence that comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40, and where the expression vector further comprises a nucleic acid encoding a light chain comprising the amino acid sequence of SEQ ID NO: 21 , 22 and 23, or a nucleic acid encoding a light chain with an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOS: 21 , 22 or 23.

[0267] The caninized antibody or antigen binding fragment thereof according to the invention can be produced by methods of producing antibodies, known in the field, such as but not limited to culturing host cells for the expression of the antibody or fragment thereof. Cell lines suitable as host cells are well known in the art. A host cell can comprise the expression vector as disclosed herein. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.

[0268] Accordingly, in one aspect provides a cell, e.g., host cell, comprising an expression vector. The IgG Fc fusion proteins, the antibodies, and the antigen binding fragments of the antibodies of the present invention can be produced recombinantly by methods that are known in the field. Mammalian cell lines available as hosts for expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and / or antigen-binding fragment thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.

[0269] Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0216 846, 0256055, and 0323997 and European Patent Application No. 89303964.4.

[0270] IV. Pharmaceutical Compositions and Administration

[0271] To prepare pharmaceutical or sterile compositions comprising the IgG Fc fusion proteins, the antibodies, and the antigen binding fragments of the antibodies of the present invention, can be admixed with a pharmaceutically acceptable carrier or excipient. [See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0272] Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions [see, e.g., Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY], In one embodiment, pharmaceutical compositions comprising the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention are diluted to an appropriate concentration in a sodium acetate solution pH 5-6, and NaCI or sucrose is added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

[0273] Toxicity and therapeutic efficacy of the IgG Fc fusion proteins, the antibodies, and the antigen binding fragments of the antibodies of the compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD50 / ED50). In particular aspects, the IgG Fc fusion proteins, the antibodies, or the antigen binding fragments of the antibodies of the present invention exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in the subjects, e.g., canines or felines. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

[0274] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial. In particular embodiments, pharmaceutical compositions comprising the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention can be administered by an invasive route such as by injection. In further embodiments of the invention, pharmaceutical compositions comprising the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention are administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.

[0275] Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector. The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos.: 6,620,135; 6,096,002; 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824 or 4,596,556.

[0276] The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules form administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0277] Alternatively, one may administer compositions comprising the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention in a local rather than systemic manner, often in a depot or sustained release formulation.

[0278] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies, the level of symptoms, the immunogenicity of the therapeutic IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies to effect improvement in the target disease / condition state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibodies, and / or fusion proteins and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available [see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341 :1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)].

[0279] Determination of the appropriate dose is made by the veterinarian, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of the symptoms.

[0280] The compositions comprising the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention, either alone or with the antibodies used in the present invention may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 pg / kg body weight, more generally at least 0.2 pg / kg, 0.5 pg / kg, 1 pg / kg, 10 pg / kg, 100 pg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg or more [see, e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother. 52:133-144 (2003)]. Doses may also be provided to achieve a pre-determined target concentration of the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention in the subject’s serum, such as 0.1 , 0.3, 1 , 3, 10, 30, 100, 300 pg / ml or more. In other embodiments, the IgG Fc fusion proteins, the antibodies, and / or the antigen binding fragments of the antibodies of the present invention are administered subcutaneously or intravenously, on a weekly, biweekly, "every 4 weeks," monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject.

[0281] One aspect of the technology disclosed herein relates to a pharmaceutical composition comprising the caninized antibody or antigen binding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier. Another aspect of the technology relates to a pharmaceutical composition comprising a nucleic acid as disclosed herein encoding a heavy chain of a caninized antibody or antigen binding fragment according to the invention comprises a modified cFc with the extended half life as compared to a wild type cFc as disclosed herein. Another aspect of the technology relates to a pharmaceutical composition comprising an expression vector comprising a nucleic acid as disclosed herein. Another aspect of the technology relates to a pharmaceutical composition comprising a host cell comprising an expression vector as disclosed herein.

[0282] One aspect of the technology disclosed herein relates to the use of a caninized antibody or antigen binding fragment thereof as disclosed herein, or a pharmaceutical composition comprising the same, or a nucleic acid encoding a heavy chain and / or light chain of such an caninized antibody or antigen binding fragment thereof, or an expression vector comprising such a nucleic acid, or a host cell as disclosed herein, or any combination thereof, for use in the treatment of atopic dermatitis in a canine.

[0283] One aspect of the technology disclosed herein relates to a method of treating a canine with atopic dermatitis comprising administering to a canine, a composition comprising a caninized antibody or antigen binding fragment thereof as disclosed herein, or a pharmaceutical composition comprising the same, or a nucleic acid encoding a heavy chain and / or light chain of such an caninized antibody or antigen binding fragment thereof, or an expression vector comprising such a nucleic acid, or a host cell as disclosed herein, or any combination thereof, wherein the administration is in an effective amount to reduce at least one symptom of atopic dermatitis in the canine.

[0284] Throughout the detailed description and examples of the invention the following abbreviations will be used:

[0285] ADCC Antibody-dependent cellular cytotoxicity

[0286] CDC Complement-dependent cyotoxicity

[0287] CDR Complementarity determining region in the immunoglobulin variable regions, defined using the Kabat numbering system cFc Canine fragment crystallizable region

[0288] CHO Chinese hamster ovary

[0289] EC50 concentration resulting in 50% efficacy or binding

[0290] ECD Extracellular domain ELISA Enzyme-linked immunosorbent assay

[0291] Ell index The widely used numbering according to: Sequences of Proteins of Immunological Interest, 5th ed., Kabat et al., National Institutes of Health, Bethesda, Md. (1991), also referred to as the Ell index, the Ell index of Kabat, or the Ell numbering scheme. cFc Canine fragment crystallizable region

[0292] FR Antibody framework region: the immunoglobulin variable regions excluding the CDR regions.

[0293] IC50 concentration resulting in 50% inhibition

[0294] IgG Immunoglobulin G

[0295] Kabat An immunoglobulin alignment and numbering system pioneered by Elvin

[0296] A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] mAb Monoclonal antibody (also Mab or MAb)

[0297] PCR Polymerase chain reaction

[0298] PK Pharmacokinetics

[0299] V region The segment of IgG chains which is variable in sequence between different antibodies.

[0300] VH Immunoglobulin heavy chain variable region

[0301] VL Immunoglobulin light chain variable region

[0302] Vk Immunoglobulin kappa light chain variable region

[0303] VI Immunoglobulin lambda light chain variable region cCK canine kappa light chain cCL canine lambda light chain

[0304] It is contemplated that any product, method, use, or composition described herein can be implemented with respect to any other product, method, use, or composition described herein. Embodiments disclosed in the context of products, methods, uses or compositions of the invention may be employed with respect to any other product, method, use, or composition described herein. Thus, an embodiment pertaining to one product, method, use or composition may be applied to other products, methods, uses or compositions of the invention as well.

[0305] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications, such as specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. However merely for conciseness, not every possible combination is outlined herein in full.

[0306] All references cited herein, including journal articles or abstract, published, or corresponding patent applications, patents, or any other references, are incorporated by reference herein in its entirety, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

[0307] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0308] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting to the present invention. Further aspects and embodiments will be apparent to those skilled in the art. The invention will now be further described by the following, non-limiting, examples.

[0309] EXAMPLES

[0310] EXAMPLE 1

[0311] CONSTRUCTION, EXPRESSION, AND PURIFICATION OF MODIFIED CANINE IgG Fc ANTIBODIES

[0312] BACKGROUND Atopic dermatitis (AD) is a relapsing pruritic and chronic inflammatory skin disease in humans and companion animals that is characterized by immune system dysregulation and epidermal barrier abnormalities. The binding of interleukin-31 (IL-31) to its receptor, the IL-31 receptor a (IL-31 RA), initiates the pruritic effect of atopic dermatitis in both humans and dogs. Blocking the binding of IL-31 to IL-31 RA with an antibody that binds IL-31 RA serves to block the signaling through IL- SI RA and ameliorates the pruritic effect of atopic dermatitis. An example of an advantage of extending the half-life of IL-31 RA antibodies is that it allows the corresponding potential drugs to be used at relatively lower doses and / or with reduced dosing frequency.

[0313] METHODS

[0314] Amino acid residue modifications were genetically engineered in the canine Fc regions of the heavy chain of IgG antibodies. The nucleic acid encoding the artificial signaling peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 45) and the amino acid sequence of the heavy chain of the antibody comprised the hinge region of canine IgG-B Fc and the canine IgG-Bm Fc region. Nucleic acids encoding the antibodies (canine heavy chain as described above or the corresponding canine light chain) were cloned into suitable vectors, e.g., pcDNA3.4 vectors. Each vector was transfected into suitable host cells, e.g., ExpiCHO cells, and the harvested supernatant was purified on suitable affinity columns, e.g., recombinant Protein A affinity columns. Subsequently, proteins were eluted in a suitable eluent, e.g., 0.1 M Glycine-HCI, pH 2.7, and then adjusted to pH 6.0 using a suitable buffer, e.g., 1 M Tris Buffer, pH 8.0.

[0315] EXAMPLE 2

[0316] AMINO ACID SEQUENCES OF EXEMPLARY ANTIBODY HALF-LIFE ENHANCING MODIFICATIONS

[0317] In order to increase the half-life of caninized anti-canine IL-31 RA antibodies, the canine IgG-Bm Fc regions of the canine heavy chains of the present invention were modified relative to the corresponding canine IgG-Bm Fc of SEQ ID NO: 27. Such a modified cFc region comprises one or more amino acid substitutions relative to the cFc region of IgG-Bm, at one or more amino acid residue positions selected from: 252, 254, 256, 311 , 428, 434, 436, 438 and 440 (numbered according to Sequences of Proteins of Immunological Interest, 5th ed., Kabat et al., National Institutes of Health, Bethesda, Md. (1991); the EU Index). Particular examples include one or more of the following modifications: L252F, L252Y, L252W, L252T, A254T, T256E, T256D, Q311S, M428L, N434A, N434S, N434Y, N434H, Y436T, Q438R, S440E, numbered according to the Ell Index as in Kabat.

[0318] Amino acid sequences of the heavy chains of exemplary CIL-31RA antibodies

[0319] Below are shown exemplary sequences (SEQ ID NO: 28 to SEQ ID NO: 40) of modified canine heavy chains of an anti-canine IL-31 RA antibody according to the invention, the amino acid residues comprising a mutation are shown in bold.

[0320] C218D9VH4 clgGBm A5 (N434A / Y436T / Q438R / S440E) (ATRE) [SEQ ID NO: 28]

[0321] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0322] VFIFPPKPKDTLLIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0323] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVM HEALHAHTTREELSHSPGK

[0324] C218D9VH4 clgGBm A6 (M428L / N434A / Y436T / Q438R / S440E (LATRE) [SEQ ID NO: 29]

[0325] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0326] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0327] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0328] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0329] VFIFPPKPKDTLLIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0330] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0331] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVL

[0332] HEALHAHTTREELSHSPGK

[0333] C218D9VH4 clgGBm A7 (M428L / N434A / Q438R / S440E) (LARE) [SEQ ID NO: 30] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0334] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0335] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0336] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0337] VFIFPPKPKDTLLIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0338] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0339] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVL

[0340] HEALHAHYTREELSHSPGK

[0341] C218D9VH4 clgGBm A9 (M428L / N434S) (LS) [SEQ ID NO: 31]

[0342] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0343] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0344] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0345] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0346] VFIFPPKPKDTLLIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0347] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0348] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVL

[0349] HEALHSHYTQESLSHSPGK

[0350] C218D9VH4 clgGBm N434H (H) [SEQ ID NO: 32]

[0351] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0352] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0353] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0354] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0355] VFIFPPKPKDTLLIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0356] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0357] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVM

[0358] HEALHHHYTQESLSHSPGK

[0359] C218D9VH4 clgGBm A13 (Q311S) (S) [SEQ ID NO: 33] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0360] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0361] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0362] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0363] VFIFPPKPKDTLLIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0364] SVLPIGHSDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0365] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVM HEALHNHYTQESLSHSPGK

[0366] C218D9VH4 clgGBm A16 (Q311S / N434H) (SH) [SEQ ID NO: 34]

[0367] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0368] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0369] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0370] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0371] VFIFPPKPKDTLLIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0372] SVLPIGHSDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0373] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVM HEALHHHYTQESLSHSPGK

[0374] C218D9VH4 clgGBm A14 (L252F / A254T / T256E) (FTE) [SEQ ID NO: 35]

[0375] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0376] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0377] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0378] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0379] VFIFPPKPKDTLFITREPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRW

[0380] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0381] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVM

[0382] HEALHNHYTQESLSHSPGK

[0383] C218D9VH4 clgGBm A15 (L252F / T256D) (FD) [SEQ ID NO: 36] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0384] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0385] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0386] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0387] VFIFPPKPKDTLFIARDPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVV

[0388] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0389] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVM HEALHNHYTQESLSHSPGK

[0390] C218D9VH4 clgGBm A17 (L252F) (F) [SEQ ID NO: 37]

[0391] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0392] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0393] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0394] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0395] VFIFPPKPKDTLFIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRW

[0396] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0397] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVL

[0398] HEALHNHYTQESLSHSPGK

[0399] C218D9VH4 clgGBm A18 (L252Y) (Y) [SEQ ID NO: 38]

[0400] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0401] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0402] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0403] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0404] VFIFPPKPKDTLYIARTPEVTCVWALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRW

[0405] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0406] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVL

[0407] HEALHNHYTQESLSHSPGK

[0408] C218D9VH4 clgGBm A20 (L252T) (T) [SEQ ID NO: 39] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0409] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0410] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0411] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0412] VFIFPPKPKDTLTIARTPEVTCWVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRW

[0413] SVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTC

[0414] LIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVL HEALHNHYTQESLSHSPGK

[0415] C218D9VH4 clgGBm A19 (L252W) (W) [SEQ ID NO: 40]

[0416] EVTLQESGPGLVKPSQTLSLTCSFSGFSLSTFGRGVGWIRQRPGRGLEWMGHIWWDDDKYY

[0417] NPALKSRLSITKDTAKNQVFLQLSSMTTEDTAVYYCARIAGGLRRAPYAMDSWGQGTLVTVSS

[0418] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLY

[0419] SLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPS

[0420] VFIFPPKPKDTLWIARTPEVTCVWALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRV

[0421] VSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLT

[0422] CLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAV LHEALHNHYTQESLSHSPGK

[0423] EXAMPLE 3

[0424] PHARMACOKINETIC STUDIES IN DOGS

[0425] Examples 3-6 shows the results from three studies conducted in dogs to determine the pharmacokinetics of the exemplary anti-canine IL-31 RA antibodies as disclosed herein, comprising specific modifications in the cFc regions as compared to a control caninized anticanine IL-31 RA antibody referred to as “c218D9VH4-clgGBm-VL3cCK”, which comprises heavy chain (HC) c218D9VH4-clgGBm having the amino acid sequence of SEQ ID NO: 27, and the light chain c218D9VL3cCK having the amino acid sequence of SEQ ID NO: 23.

[0426] In Example 3 are the results of the half-life of 8 different antibody variants (referred to as A5 to A12), which comprise two or more amino acid substitutions at specific positions in the cFc region, are compared to a control anti-canine IL-31 RA antibody with that lacks the same amino acid substitutions in the Fc region (designated as c218D9VH4-clgGBm). That is, the C218D9VH4- clgGBm control antibody comprises a unsubstituted variant Fc.

[0427] Each antibody was administered to 6 dogs by the subcutaneous (SC) route at 3 mg / kg of each antibody. Animals were bled at various time points throughout the study and their sera collected and used to measure the serum concentration of each antibody was tested against canine IL- 31 RA by ELISA. The serum concentration of each antibody was determined by ELISA as follows:

[0428] Procedure for the ELISA:

[0429] Coat 96-well plates with IL-31 RA (1 pg / mL in PBS buffer), 25 pL / well.

[0430] Incubate the plates at 4°C overnight.

[0431] Wash the plates 3 times with PBST (PBS +0.05% Tween 20)

[0432] Block the plates with blocking buffer [PBS with 5% fetal bovine serum (FBS)], 25pl / well for 30 minutes at room temperature.

[0433] Transfer 25 pl / well hybridoma supernatant to the 96-well plates, incubate 60 minutes at room temperature.

[0434] Wash the plates 3 times by PBST.

[0435] Add 25pl / well anti-mouse HRP, 1 :4000 dilution in blocking buffer, to the plates and incubate 60 minutes at room temperature.

[0436] Wash the plates 5 times by PBST.

[0437] Add TMB based reagent to the plates for colorimetric reaction for 2-3 minutes.

[0438] Stop the reactions with 0.16M sulfuric acid.

[0439] Read the plates by plate reader.

[0440] As the concentration of the antibody in the serum at the timepoints was determined by binding to canine IL-31 RA in an ELISA, this also confirmed that the antibody variants comprising amino acid substitutions in the cFc regions maintained their binding affinity to bind to canine IL-31 RA. The binding affinity of the control unsubstituted canine anti IL-31 R antibody with the CDRs of c218D9 (antibody c218D9VH4-clgGBm) is disclosed in International Application WO2023 / 111148 and WO2023 / 021169, which are incorporated herein in their entirety. The effect of amino acid substitutions in the cFc region on the half-life of each antibody variant is shown in Table 7 below. These antibodies contain specific amino acid substitutions in the Fc of 218D9 antibody (c218D9VH4-clgGBm) which is specific to canine IL-31 receptor alpha chain. The results demonstrate a clear increase in half-life as a result of specific combinations of amino acid substitutions in the cFc region of the antibodies.

[0441] Table 7: The average (n=6) half life of the antibody variants A5 to A12.

[0442] Amino acid substitutions numbered according to the Ell index as in Kabat. **ND= Not determined. These antibody variants were eliminated too quickly and therefore half-life for these antibodies could not be calculated. Antibody variant c218D9VH4-clgGBm-A9 comprising two amino acid substitutions at positions M428L / N434S had a longer half-life by about 1 .4 days as compared to the unsubstituted control antibody. Surprisingly, increasing the number of amino acid substitutions in the cFc region to four substitutions increased the half life to 9.36 days and 11.64 days, respectively for antibody variants c218D9VH4-clgGBm-A5 and c218D9VH4-clgGBm-A7, respectively. This is unexpected given that increasing the number of substitutions can frequently affect, often decreasing, the affinity of the antibody for the target, e.g., IL-31 RA. Here, increasing the number of substitutions from modifications at two positions (M428 and N434) to four amino acid positions (N434 / Y436 / Q438 / S440 (A5) or M428 / N434 / Q438 / S440 (A7)) increases the antibody half life. Moreover, antibody variant c218D9VH4-clgGBm-A6 had the longest half-life of 13.75 days in Example 3, which was particularly surprising given this variant has 5 different amino acid substitutions at amino acid positions 428, 434, 436, 438 and 440, whereas antibody C218D9VH4- clgGBm-A12, which also has 5 amino acid substitutions resulted in a decrease in half-life as compared to the half-life of the unsubstituted variant control antibody c218D9VH4-clgGBm.

[0443] EXAMPLE 4

[0444] In Example 4, the pharmacokinetics of half life in vivo of IL-31 RA antibody variants listed in Table 8 were evaluated. These antibodies contain specific amino acid substitutions in the Fc of 218D9 antibody (c218D9VH4-clgGBm) which is specific to canine IL-31 receptor alpha chain. The antibody variants were administered to dogs. Each antibody variant was administered to 5 dogs by SC route at 3 mg / kg of each antibody. Animals were bled at various time points throughout the study and their sera collected and used to measure the serum concentration of each antibody by ELISA, using the same procedure in Example 3. The effect of amino acid substitutions on antibody half-life is shown in Table 8 below. The results demonstrate the indicated amino acid substitutions led to varying degrees of increase in antibody half-life when compared to the antibody without amino acid substitutions.

[0445] Table 8:

[0446] Amino acid substitutions numbered according to the Ell index as in Kabat.

[0447] Antibody variant c218D9VH4-clgGBm-A13 comprising a serine at amino acid position 311 had a half-life of 10.4 days as compared to the unsubstituted control antibody. Surprisingly, adding an additional modification of N434H, which has been previously been reported to extend half-life in vivo, actually decreased the half-life to 9.8 days as compared to the antibody comprising the single modification of Q311S, as shown by the results of the c218D9VH4-clgGBm-A16 antibody. Antibody variant c218D9VH4-clgGBm-A15 had a similar half life to c218D9VH4-clgGBm-A16 of 9.3 days, whereas antibody variant c218D9VH4-clgGBm-A14, which has three modifications at amino acid residue positions 252, 254 and 256 in the cFc region has a superior half life of 12.4 days. This demonstrates that combinations of amino acid substitutions at specific residue positions can impact the half life of the IL-31 RA antibody in dogs in vivo, and that prior reports of improved half-life extension of specific amino acid modifications in different antibody molecules and different antibodies binding to different target molecules cannot be relied upon to predict the effect of the same amino acid modifications in a different antibody.

[0448] EXAMPLE 5

[0449] In Example 5, the pharmacokinetics of half life in vivo of IL-31 RA antibody variants listed in Table 9 were evaluated, where each variant had an amino acid other than lysine (L) at position 252. In particular, these antibodies contain specific amino acid substitutions at position 252 in the Fc of 218D9 antibody (c218D9VH4-clgGBm) which is specific to canine IL-31 receptor alpha chain. Each antibody variant was administered to 5 dogs by SC route at 3 mg / kg. Animals were bled at various time points throughout the study and their sera collected and used to measure the serum concentration of each antibody by ELISA. The effect of amino acid substitutions on antibody halflife is shown in Table 9 below.

[0450] Table 9:

[0451] The results demonstrate that different amino acid residues at position 252 led to varying degrees of increase in antibody half-life or in one case a decrease in antibody half-life, when compared to the antibody without amino acid substitutions (unmodified or unsubstituted c218D9VH4-clgGBm has an average half-life ranging from 7.4 - 8.31 days).

[0452] Antibody variant c218D9VH4-clgGBm-A17 comprising a phenylalanine (F) at amino acid position 252 had a half-life of 14.1 days as compared to the unsubstituted control antibody. Conversely, antibody variant c218D9VH4-clgGBm-A19 comprising a tryptophan (W) at amino acid position 252 had a lower half-life (5.7 days), as compared to the unsubstituted control antibody, demonstrating that certain specific amino acid residues at position 252 can contribute to extending the in vivo half-life of the IL-31 RA antibody, whereas other amino acid residues at position 252 can decrease half-life of the I L-31 RA antibody in vivo. Additionally, adding one or two additional substitutions at positions 256, or at 254 and at position 256 to an antibody with a phenylalanine (F) at amino acid position 252, still had superior half-life in vivo as compared to the control 218D9 antibody (unsubstituted c218D9VH4-clgGBm antibody).

[0453] EXAMPLE 6

[0454] ASSESSMENT OF ANTIBODY EFFICACY IN TREATMENT OF ATOPIC DERMATITIS

[0455] The efficacy of the canine IL-31 receptor alpha (cl L-31 RA) antibodies variants comprising modified cFc as disclosed herein in treatment of atopic dermatitis can be assessed in animals diagnosed with atopic dermatitis. For this purpose, groups of dogs can be administered antibodies of the present invention by subcutaneous route for example at doses from 0.1 to 10 mg / kg. For comparison, a group of dogs is administered a placebo also via subcutaneous route. Animals are then monitored at regular intervals (for example weekly) for manifestation of key clinical signs of disease over 30 or 60-day period or longer. The success of treatment is typically determined by assessment of the impact of antibody treatment on extent of pruritus using so called Pruritus Visual Analog Scale (P AS; a scale of 0-10 indicating the itch severity) and assessment of the impact of the antibody treatment on extent of skin lesion scores referred to as CADESI (canine atopic dermatitis extent and severity index) in treated dogs versus dogs treated with a placebo. Successful treatment outcome is indicated by a reduction of at least 20% in PAVS score and a reduction of at least 50% in CADESI scores; relative to baseline measurements. It is expected that in this study the antibodies according to the present invention will result in any one or more of the desired beneficial effects in the dogs that relate to an increased half-life of the antibody, e.g., less frequent dosing and / or lower dose of the antibody drug, improved patient compliance, lowered concentration-dependent cytotoxicity (CDC) or reduced occurrence of adverse events, etc.

[0456] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0457] Table 10: SEQUENCE LISTING TABLE

[0458]

[0459]

Claims

CLAIMS1. A caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody comprising a canine fragment crystallizable region (cFc) that comprises at least four of:(i) an amino acid other than an asparagine residue at position 434,(ii) an amino acid other than a glutamine residue at position 438,(iii) an amino acid other than a serine residue at position 440, and(iv) an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436. wherein the numbering of amino acid residue positions is according to the EU Index as in Kabat.

2. A caninized antibody or antigen binding fragment thereof that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody comprising a canine fragment crystallizable region (cFc), the cFc selected from the group consisting of: a) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). b) a cFc comprising: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). c) a cFc comprising: an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E). d) a cFc comprising: a leucine amino acid residue at position 428 (M428L),a serine amino acid residue at position 434 (N434S) wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

3. The caninized antibody of any of claim 1 or 2, wherein the cFc comprises one or more residues selected from the group consisting of: a leucine amino acid residue at position 252, an alanine amino acid residue at position 254, a threonine amino acid residue at position 256, a glutamine amino acid residue at position 311 , and wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

4. The caninized antibody of claims 1 or 2, wherein the cFc further comprises one or more residues selected from the group consisting of: an amino acid other than a leucine residue at position 252, an amino acid other than an alanine residue at position 254, an amino acid other than a threonine residue at position 256, and an amino acid other than a glutamine residue at position 311 , wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

5. The caninized antibody of claim 4, wherein the cFc comprises the one or more residues selected from the group consisting of:(i) an amino acid selected from a phenylalanine (F), or tyrosine (Y) or tryptophan (W) or threonine (T) amino acid residues at position 252, (L252F, L252Y, L252W, L252T),(ii) a threonine (T) amino acid residue at position 254 (A254T),(iii) a glutamic acid (E) or aspartic acid (D) amino acid residue at position 256 (T256D, T256E), and(iv) a serine amino acid residue position 311 (Q311 S), wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

6. The caninized antibody of any one of the previous claims, wherein the cFc comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc comprises at least four of:(i) an amino acid other than an asparagine residue at position 434,(ii) an amino acid other than a glutamine residue at position 438,(iii) an amino acid other than a serine residue at position 440, and(iv) an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436. wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

7. The caninized antibody of claim 6, wherein the cFc comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc is selected from the group consisting of: a. a cFc comprising:(i) a leucine amino acid residue at position 428 (M428L),(ii) an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H),(iii) a threonine amino acid residue at position 436 (Y436T),(iv) an arginine amino acid residue at position 438 (Q438R), and(v) a glutamic acid amino acid residue at position 440 (S440E). b. a cFc comprising:(i) a leucine amino acid residue at position 428 (M428L),(ii) an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H),(iii) an arginine amino acid residue at position 438 (Q438R), and(iv) a glutamic acid amino acid residue at position 440 (S440E); c. a cFc comprising:(i) an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H),(ii) a threonine amino acid residue at position 436 (Y436T),(iii) an arginine amino acid residue at position 438 (Q438R), and(iv) a glutamic acid amino acid residue at position 440 (S440E); d. a cFc comprising:(i) a leucine amino acid residue at position 428 (M428L),(ii) a serine amino acid residue at position 434 (N434S) wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

8. The caninized antibody of any one of the previous claims, comprising that the antibody or fragment thereof comprises a heavy chain variable domain (VH) and / or a light chain variable domain (VL), wherein the VH contains the following CDRs:VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 13;VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 14;VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and the VL comprises a light chain variable domain that contains the following CDRs: VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 16;VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 17;VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

9. The caninized antibody of any one of the previous claims, wherein the antibody comprises any one or more of: a. a cFc comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc comprises at least four of:(i) an amino acid other than an asparagine residue at position 434,(ii) an amino acid other than a glutamine residue at position 438,(iii) an amino acid other than a serine residue at position 440, and(iv) an amino acid other than a methionine residue at position 428, and / or an amino acid other than a tyrosine residue at position 436, b. a cFc comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7 and 8, or an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7 and 8, and wherein the cFc is selected from the group consisting of:A. a cFc comprising:(i) a leucine amino acid residue at position 428 (M428L),(ii) an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H),(iii) a threonine amino acid residue at position 436 (Y436T),(iv) an arginine amino acid residue at position 438 (Q438R), and(v) a glutamic acid amino acid residue at position 440 (S440E);B. a cFc comprising:(i) a leucine amino acid residue at position 428 (M428L),(ii) an alanine amino acid residue at position 434 (N434A) or a histidine at position 434 (N434H),(iii) an arginine amino acid residue at position 438 (Q438R), and(iv) a glutamic acid amino acid residue at position 440 (S440E).C. a cFc comprising:(i) an alanine amino acid residue at position 434 (N434A) or a histidine amino acid residue at position 434 (N434H),(ii) a threonine amino acid residue at position 436 (Y436T),(iii) an arginine amino acid residue at position 438 (Q438R), and(iv) a glutamic acid amino acid residue at position 440 (S440E).D. a cFc comprising:(i) a leucine amino acid residue at position 428 (M428L),(ii) a serine amino acid residue at position 434 (N434S)E. a cFc comprising a hinge region that comprises an amino acid sequence that comprises at least 90%, 95%, 97%, 98%, 99% identity sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 or SEQ ID NO: 46, andF. a light chain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 21 , 22 and 23, or has at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID Nos: 21 , 22 and 23.

10. The caninized antibody of any of claims 1-9, wherein the heavy chain comprises an amino acid sequence comprising amino acid residues of SEQ ID NO: 28, 29, 30 or 31 , or comprises at least 90%, 95%, 97%, 98%, 99% identity to SEQ ID NOs: 28, 29, 30 or 31.

11. A caninized antibody that binds canine interleukin-31 receptor alpha (clL31 RA), the antibody selected from the group consisting of:(i) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and wherein the heavy chain comprises an amino acid sequence comprising SEQID NO: 30, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 30,(ii) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: a leucine amino acid residue at position 428 (M428L), an alanine amino acid residue at position 434 (N434A), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and wherein the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 29, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 29,(iii) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: an alanine amino acid residue at position 434 (N434A), a threonine amino acid residue at position 436 (Y436T), an arginine amino acid residue at position 438 (Q438R), and a glutamic acid amino acid residue at position 440 (S440E), and wherein the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 28, or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 28, and(iv) an antibody comprising a heavy chain, the heavy chain comprising a canine fragment crystallizable region (cFc) that comprises: a leucine amino acid residue at position 428 (M428L) and an alanine amino acid residue at position 434 (N434A), and wherein the heavy chain comprises an amino acid sequence comprising SEQ ID NO: 31 , or comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 31 , wherein the numbering of amino acid residue positions is according to the Ell Index as in Kabat.

12. A nucleic acid molecule that encodes the caninized antibody or antigen binding fragment thereof of any one of the previous claims, and optionally, wherein the nucleic acid is present in an expression vector, and optionally, wherein the expression vector is present in a host cell.

13. A pharmaceutical composition comprising the caninized antibody of any one of Claim 1 - 11 , the nucleic acid molecule according to claim 12, the expression vector according to Claim 12 orthe host cell according to Claim 12 or any combination thereof, and a pharmaceutically acceptable carrier.

14. A caninized antibody or antigen binding fragment thereof according to any one of Claim 1 - 11 , the nucleic acid molecule according to claim 12, the expression vector according to claim 12 or the host cell according to claim 12, or any combination thereof, or the pharmaceutical composition according to Claim 13 for use in the treatment of atopic dermatitis in a canine.

15. A method of treating a canine that has atopic dermatitis comprising administering to the canine anyone, or a combination of, caninized antibody or antigen binding fragment thereof according to any one of Claim 1 - 11 , the nucleic acid molecule according to Claim 12, the expression vector according to Claim 12 or the host cell according to Claim 12, or the pharmaceutical composition according to Claim 13.

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