Antibodies to canine interleukin-4 receptor alpha

Abstract

The present invention provides antibodies against canine IL-4 receptor alpha which have a high binding affinity to canine IL-4 receptor alpha and can block the binding of canine IL4 and / or IL13 to canine IL-4 receptor alpha. The present invention also relates to an epitope of canine IL-4 receptor alpha which is bound by the antibodies against canine IL4 receptor alpha. The present invention also provides the use of the antibodies for the treatment of atopic dermatitis in dogs.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 63 / 015,209, filed April 24, 2020, U.S. Provisional Patent Application Serial No. 63 / 015,220, filed April 24, 2020, U.S. Provisional Patent Application Serial No. 62 / 951,778, filed December 20, 2019, and U.S. Provisional Patent Application Serial No. 62 / 951,793, filed December 20, 2019, the contents of all of the above applications are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present invention relates to antibodies to canine IL-4 receptor alpha that have high binding affinity to canine IL-4 receptor alpha and that can block the binding of canine IL-4 and / or IL-13 to canine IL-4 receptor alpha. The present invention also relates to the use of the antibodies of the present invention in the treatment of atopic dermatitis in dogs. BACKGROUND

[0004] The immune system comprises a network of resident and recirculating specialized cells that act in concert to protect the host from infectious diseases and cancer. The ability of the immune system to perform this function depends in large part on the biological activities of a group of proteins secreted by white blood cells, collectively known as interleukins. Among the well-studied interleukins, four important molecules have been identified as interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-31 (IL-31), and interleukin-22 (IL-22). IL-4 and IL-13 are closely related proteins that can be secreted by many cell types, including CD4 +Th2 cells, natural killer T cells (NKT), macrophages, mast cells, and basophils. IL-4 and IL-13 show many overlapping functions and are critical for the development of T cell-dependent humoral immune responses. IL-4 is known to bind two receptors, type I and type II IL-4 receptors, with high affinity. Type I IL-4 receptor is composed of the IL-4 receptor alpha chain and the common gamma C chain. Type II IL-4 receptor is composed of the IL-4 receptor alpha chain and the IL13 receptor alpha 1 chain. IL-13 binds to the type II IL-4 receptor and to a distinct receptor called IL-13 receptor alpha 2. Binding of IL-13 to IL-13 receptor alpha 2 does not transduce a signal, and this receptor is also secreted in a soluble form. Thus, IL-13 receptor alpha 2 is often referred to as a decoy receptor. While IL-4, IL-31, and IL-22 are key cytokines for the development of immune responses necessary to protect against extracellular pathogens (e.g., tissue- or lumen- parasitizing parasites), these cytokines are also implicated in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

[0005] Atopic dermatitis (AD) is a relapsing pruritic and chronic inflammatory skin disease characterized by dysregulation of the immune system and abnormalities in the epidermal barrier in humans. The pathological and immunological attributes of atopic dermatitis have been the subject of extensive study [reviewed in Rahman et al. Inflammation & Allergy-drug target 10:486-496 (2011) and Harskamp et al. Seminar in Cutaneous Medicine and Surgery 32:132-139 (2013)]. Atopic dermatitis is also a common disease in companion animals, particularly dogs, with an estimated prevalence of approximately 10-15% of the canine population. The pathogenesis of atopic dermatitis in dogs and cats [reviewed in Nuttall et al. Veterinary Records 172(8):201-207 (2013)] is remarkably similar to that in humans, including skin infiltration of multiple immune cells and a predominance of CD4 + Th2 polarized cytokine environment. In addition, IL-22 is associated with excessive epithelial proliferation leading to epidermal hyperplasia, a feature of atopic dermatitis.

[0006] For example, antibodies against canine IL-31 have been shown to have a significant effect on pruritus associated with atopic dermatitis in dogs [US 8,790,651 B2; US 10,093,731 B2]. In addition, antibodies against human IL-31 receptor alpha (IL31RA) have been tested and found to have a significant effect on atopic dermatitis-associated pruritus in humans [Ruzicka et al., New England Journal of Medicine, 376(9), 826-835 (2017)]. Thus, blocking the binding of IL-31 to its receptor IL31RA results in the relief of pruritus associated with atopic dermatitis.

[0007] Monoclonal antibodies against human IL-4 receptor alpha (IL-4R α ) have been developed and some of these antibodies have been extensively tested for their therapeutic effect on treating atopic dermatitis in humans [see, e.g., US2015 / 0017176 Al]. Recently, caninized antibodies against canine IL-4R α that block the binding of canine IL-4 to canine IL-4R α have also been disclosed [US2018 / 0346580 Al, which is incorporated herein by reference in its entirety]. Since the type II IL-4 receptor is composed of the IL-4 receptor alpha chain and the IL-13 receptor alpha 1 chain, antibodies against canine IL-4R α have been obtained that can block the binding of canine IL-4 and canine IL-13 to the type II canine IL-4 receptor, thereby serving to help block inflammation associated with atopic dermatitis [US2018 / 0346580 Al].

[0008] Interleukin 22 (IL-22), also known as IL-10-related T-cell-derived inducible factor (IL-TIF), belongs to the IL-10 cytokine family. IL-22 is produced by normal T cells after anti-CD3 stimulation in humans. IL-22 expression is also induced in various organs in mice after injection of lipopolysaccharide, suggesting that IL-22 can be involved in inflammatory responses. IL-22 specifically binds to a receptor complex composed of a heterodimeric complex of IL-10R2 (also known as IL-10Rβ) and interleukin 22 receptor (IL-22R) and signals through this receptor complex [see Lee et al., Pharmacology Research & Perspectives, pages 1-13 (2018: e00434)]. The interleukin 22 receptor is also known as interleukin 22R, alpha 1; IL-22RA1; IL-22R1; zcytor11; and CRF2-9 [Xu et al., Proc. Nat. Acad. Sci. 98(17) 9511-9516 (2001); Gelebart and Lai, Atlas of Genetics and Cytogenetics 14(12): 1106-1110 (2010)]. IL-22 induces epithelial cell proliferation during wound healing, and its absence can lead to uncontrolled proliferation and enhanced tumor development [Huber et al., Nature 491: 259263 (2012)]. IL-22 has been shown to activate STAT-1 and STAT-3 and upregulate production of acute phase proteins in several hepatocarcinoma cell lines. Antibodies to IL-22 and IL-22R act as anti-proliferative agents by blocking the interaction of IL-22 with IL-22R and thus blocking the associated signaling pathway that leads to epithelial proliferation.

[0009] However, despite recent successes in treating atopic dermatitis, none of the currently employed therapies produce a rapid onset of anti-pruritic effects with a significant effect on skin inflammation and improvement in skin barrier function. Thus, there is a need to design better therapies that can address one or more symptoms of atopic dermatitis.

[0010] The citation of any reference in this document should not be construed as an admission that such reference is available as "prior art" to the present application. SUMMARY

[0011] The present application provides antibodies directed against canine IL4Rα (IL4R αnewly caninized antibodies to canine IL-4 receptor alpha that, in particular embodiments, are isolated, that have superior properties to those in the prior art, such as tighter binding than prior art anti-canine IL-4 receptor alpha antibodies. In particular embodiments, the present application provides a mammalian antibody or antigen-binding fragment thereof that specifically binds canine interleukin-4 receptor alpha comprising a heavy chain comprising a set of three heavy chain complementarity determining regions (CDRs): CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2), and CDR heavy 3 (HCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 16. In related embodiments, the mammalian antibody or antigen-binding fragment thereof that specifically binds canine interleukin-4 receptor alpha (IL4R α ) further comprises a light chain comprising a set of three light chain CDRs: CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 22. In preferred embodiments, the mammalian antibody or antigen-binding fragment thereof binds canine IL4R α and blocks canine IL4R α binding to canine interleukin-4 (cIL-4). In related embodiments, the mammalian antibody or antigen-binding fragment thereof binds canine IL-4R α and blocks canine IL-4R α binding to canine interleukin-13 (cIL-13). In other embodiments, the mammalian antibody or antigen-binding fragment thereof binds canine IL-4R α and blocks canine IL-4R α binding to both cIL4 and to cIL-13.

[0012] In particular embodiments, the mammalian antibody to canine IL4Rα is a murine antibody. In related embodiments, the mammalian antibody to canine IL4Rα is a caninized murine antibody. In specific embodiments, the caninized antibody comprises a heavy chain comprising IgG-D cFc, but the naturally occurring IgG1-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO: 6. In other embodiments, the caninized antibody comprises a heavy chain comprising IgG-D cFc, but the naturally occurring IgG1-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO: 7. In other embodiments, the caninized antibody comprises a heavy chain comprising IgG-D cFc, but the naturally occurring IgG1-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO: 8. In other embodiments, the caninized antibody comprises a heavy chain comprising IgG-D cFc, but the naturally occurring IgG1-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO: 9.

[0013] In particular embodiments, the caninized antibody comprises a heavy chain comprising a modified canine IgG-B (IgG1bm) comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 40. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 42. In particular embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 39. In alternative embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0014] In specific embodiments, the caninized antibody or antigen-binding fragment thereof binds to one, two, or all three of the following amino acid residues of SEQ ID NO: 46: K α , H 97 , T 112 , and S 113 . In related embodiments, the caninized antibody or antigen-binding fragment thereof binds to SEQ ID NO: 47. In more specific embodiments, the caninized antibody or antigen-binding fragment thereof binds to one, two, three, four, or all five of the following amino acid residues of canine IL4R α : S 164 , T 165 , S171 , Y 172 , S 173 , and R 175 . In more specific embodiments, the caninized antibody or antigen-binding fragment thereof binds to SEQ ID NO: 46 and SEQ ID NO: 47. In more specific embodiments of this type, the caninized antibody or antigen-binding fragment thereof binds to one, two, or all three of the following amino acid residues of canine IL4R α : K 97 , H 112 , T 113 , and / or one, two, three, four, or all five of the following amino acid residues of canine IL4R α : S 164 , T 165 , S 171 , Y 172 , S 173 , and R 175 .

[0015] The present application also provides nucleic acids encoding the heavy chain of the CDR, caninized antibody or antigen-binding fragment thereof and / or the light chain of the caninized antibody or antigen-binding fragment thereof, including isolated nucleic acids. In addition, the present application provides expression vectors comprising such nucleic acids, and host cells comprising such expression vectors.

[0016] Further, the present application provides pharmaceutical compositions comprising the caninized antibodies and antigen-binding fragments thereof of the present application, and a pharmaceutically acceptable carrier and / or diluent. The present application also provides methods of treating atopic dermatitis, comprising administering one of the above compositions to a canine having atopic dermatitis. In specific embodiments, the present application provides methods of helping to block inflammation associated with atopic dermatitis, comprising administering to a canine in need thereof a therapeutically effective amount of a pharmaceutical composition of the present application.

[0017] These and other aspects of the present application will be better appreciated by reference to the following Figures and detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Two different caninized monoclonal anti-canine interleukin-4 receptor alpha antibodies, designated c4H3 [see, WO2016 / 156588] and c146E2-H3L3, were shown to inhibit STAT-6 phosphorylation. The data show that both antibodies resulted in dose-dependent inhibition of STAT-6 phosphorylation in the presence of canine interleukin-4. IL-4 control in the absence of IL-4R alpha (IL-4R α ) antibody is shown in the upper right portion of the figure.

[0019] Figure 2 Two different caninized monoclonal anti-canine interleukin-4 receptor alpha antibodies, designated c4H3 [see, WO 2016 / 156588] and c146E2-H3L3, were shown to inhibit STAT-6 phosphorylation. The data show that both antibodies resulted in dose-dependent inhibition of STAT-6 phosphorylation in the presence of canine interleukin-13. IL-13 control in the absence of IL-4R α ) antibody is shown in the upper right portion of the figure.

[0020] Figure 3 Binding of caninized anti-canine IL4R α antibodies containing lambda or kappa light chains was assessed by ELISA. The results show that caninized anti-canine IL4R α antibodies containing lambda light chains (c146C1L1-H1, c146C1L1-H2, and c146C1L1-H3) bound to canine IL4R α and caninized anti-canine IL4R α antibodies containing the same CDRs but with kappa light chains (c146E2-H3L3) bound. 146mc is a mouse-canine chimeric antibody positive control, and Iso-Ctr (negative control) is an irrelevant caninized antibody.

[0021] Figure 4 Epitopes on canine IL-4R α bound by the c146E2-H3L3 antibody contain the amino acid sequences SEQ ID NO: 46 and SEQ ID NO: 47. DETAILED DESCRIPTION

[0022] In response to the need for better therapies for atopic dermatitis, the present invention provides caninized antibodies, formulations having caninized antibodies, and methods that can achieve a significant effect on skin inflammation associated with atopic dermatitis.

[0023] ABBREVIATIONS

[0024] Throughout the DETAILED DESCRIPTION and examples of the invention, the following abbreviations will be used:

[0025] ADCC antibody-dependent cellular cytotoxicity

[0026] CDC complement-dependent cellular cytotoxicity

[0027] CDR complementarity determining region in an immunoglobulin variable region, defined using the Kabat numbering system

[0028] EC50 concentration resulting in 50% potency or binding

[0029] ELISA enzyme-linked immunosorbent assay

[0030] FR antibody framework region: immunoglobulin variable region not including CDR regions

[0031] IC50concentration that results in 50% inhibition

[0032] IgG immunoglobulin G

[0033] Kabat immunoglobulin alignment and numbering system initiated by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]

[0034] mAb monoclonal antibody (also referred to as Mab or MAb)

[0035] V region segment of an IgG chain that varies in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain.

[0036] VH immunoglobulin heavy chain variable region

[0037] VL immunoglobulin light chain variable region

[0038] VK immunoglobulin kappa light chain variable region

[0039] Definitions

[0040] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used have the meanings that are commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] As used herein, including the appended claims, the singular forms "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0042] "Administering" and "treatment," when applied to an animal (e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid), means contacting the animal (e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid) with an exogenous agent, therapeutic, diagnostic reagent, or composition. Treatment of a cell includes contacting the reagent with the cell, as well as contacting the reagent with a fluid that is in contact with the cell.

[0043] "Administering" and "treatment" also mean in vitro and ex vivo treatment, for example, by an agent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a canine, feline, or human) and most preferably a canine.

[0044] "Treatment" or "treatment" refers to the internal or external administration of a therapeutic agent, such as a composition containing any of the antibodies of the present application, to, for example, a canine subject or patient having one or more symptoms of, or suspected of having, a condition for which the agent has therapeutic activity.

[0045] Generally, an agent is administered in an amount effective to reduce and / or ameliorate one or more symptoms of a disease / condition in a treated subject or population, whether by inducing regression of such symptoms or by inhibiting progression of such symptoms, as measured by any clinically measurable metric. The amount of a therapeutic agent that is effective to alleviate any particular disease / condition symptom (also referred to as a "therapeutically effective amount") can vary according to factors such as the disease / condition state, age, and weight of the patient (e.g., canine), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Whether a disease / condition symptom has been reduced or ameliorated can be assessed by any clinical assessment typically used by a veterinarian or other skilled healthcare provider to assess the severity or progression state of the symptom. While embodiments of the present application (e.g., a method of treatment or article of manufacture) can not be effective to reduce the target disease / condition symptom in every subject, it should reduce the target disease / condition symptom in a statistically significant number of subjects, as determined by any statistical test known in the art, such as Student's t-test, chi-squared test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0046] "Treatment" when applied to humans, veterinary subjects (e.g., canines), or research subjects, refers to therapeutic treatment as well as research and diagnostic applications. "Treatment" when applied to humans, veterinary subjects (e.g., canines), or research subjects, or cells, tissues, or organs, includes contact of an antibody of the present application with, for example, a canine or other animal subject, cell, tissue, physiological compartment, or physiological fluid.

[0047] As used herein, the term "canine" includes all domestic dogs, shepherd dogs (Canis lupus familiaris) or domestic dogs (Canis familiaris), unless otherwise indicated.

[0048] As used herein, the term “feline” refers to any member of the family Felidae. Members of this family include wild, zoo, and domestic members, including domestic cats, purebred and / or hybrid companion cats, show cats, laboratory cats, cloned cats, and wild cats or feral cats.

[0049] As used herein, the term “canine framework” refers to the amino acid sequences of the heavy and light chains of a canine antibody other than the hypervariable region residues defined herein as CDR residues. With respect to caninized antibodies, in most embodiments, the amino acid sequences of native canine CDRs are replaced by corresponding exogenous CDRs (e.g., CDRs from a mouse antibody) in both chains. Optionally, the heavy and / or light chains of a canine antibody can comprise some exogenous non-CDR residues, e.g., to maintain the conformation of the exogenous CDRs within the canine antibody, and / or to alter Fc function, as exemplified below and / or disclosed in US 10,106,607 B2, the entire contents of which are hereby incorporated herein by reference.

[0050] The “Fragment crystallizable region” abbreviated “Fc” corresponds to the CH3-CH2 portion of the Fc region that interacts with the cell surface receptor known as Fc receptor (FcR). Fc receptors Antibodies The canine Fragment crystallizable region (cFc) of each of the four canine IgGs was first described by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001); see also, Bergeron et al., Vet. Immunol. Immunopathol. 157: 31-41 (2014) and US 10,106,607 B2].

[0051] ​As used herein, canine Fc (cFc) “IgG-Bm” is a canine IgG-B Fc comprising two (2) amino acid residue substitutions, D31A and N63A (see below), in the amino acid sequence of SEQ ID NO: 10 of IgG-B and no c-terminal lysine (“K”). The aspartic acid residue (D) at position 31 of SEQ ID NO: 10 and the asparagine residue (N) at position 63 of SEQ ID NO: 10 are substituted with alanine residues (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B [see US 10,106,607 B2, the contents of which are incorporated by reference in their entirety herein]. Further amino acid substitutions to IgG-Bm are also contemplated, similar to those that can be made in IgG-B, and can include amino acid substitutions that favor the formation of heterodimers in bispecific antibodies. The amino acid sequence of IgG-B, SEQ ID NO: 45 is:

[0052]

[0053] The amino acid sequence of IgG-Bm, SEQ ID NO: 10, is provided below.

[0054] LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0055] As used herein, for example, "substituting an amino acid residue" with another amino acid residue in the amino acid sequence of an antibody is equivalent to "replacing an amino acid residue" with another amino acid residue and means that the particular amino acid residue at a particular position in the amino acid sequence has been replaced (or substituted) with a different amino acid residue. Such substitutions can be specifically designed, i.e., by replacing an alanine with a serine at a particular position in the amino acid sequence by, for example, recombinant DNA technology. Alternatively, a particular amino acid residue or string of amino acid residues of an antibody can be replaced with one or more amino acid residues by a more natural selection process, for example, based on the ability of a cell-produced antibody to bind a given region on the antigen (e.g., a region containing an epitope or portion thereof), and / or to cause the antibody to comprise a particular CDR that retains the same canonical structure as the CDR it is replacing. Such substitutions / replacements can result in a "variant" CDR and / or a variant antibody.

[0056] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. The antibody can be a monomer, a dimer, or a larger multimer. Thus, it is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies, and camelized single-domain antibodies. A "parental antibody" is an antibody obtained by exposing the immune system to an antigen, which is then modified for the intended use, for example, caninizing the antibody for use as a canine therapeutic antibody.

[0057] As used herein, "blocks" or "prevents" or "precludes" the binding of, for example, a canine receptor to its binding partner (ligand) is an antibody of the application that (partially or completely) blocks the binding of the canine receptor to its canine ligand, and vice versa, as determined in a standard binding assay (e.g., ELISA or flow cytometry).

[0058] Generally, the antibodies or antigen-binding fragments of the application retain at least 10% of their activity (when compared to the parental antibody) when canine antigen binding activity is expressed on a molar basis. Preferably, the antibodies or antigen-binding fragments of the application retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the canine antigen binding affinity of the parental antibody. It is also intended that the antibodies or antigen-binding fragments of the application can include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).

[0059] An "isolated antibody" refers to the state of purification and, in this case, the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris and growth media. In general, the term "isolated" does not mean completely free from such materials or from water, buffers, or salts, unless they are present in an amount that substantially interferes with the experimental or therapeutic use of the binding compound described herein.

[0060] As used herein, a "chimeric antibody" is an antibody having variable domains from a first antibody and constant domains from a second antibody, wherein the first and second antibodies are from different species. [US 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)]. Typically, the variable domains are obtained from an antibody from an experimental animal (e.g., a rodent) (parental antibody), while the constant domain sequences are obtained from an animal subject antibody (e.g., human or canine), thus producing a chimeric antibody that is less likely to elicit an adverse immune response in a human or canine subject, respectively, compared to the parental (e.g., rodent) antibody.

[0061] As used herein, the term "caninized antibody" refers to a form of antibody comprising sequences from both canine and non-canine (e.g., murine) antibodies. Generally, a caninized antibody will comprise substantially all of at least one or more, usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., comprising the six CDRs as exemplified below) and all or substantially all of the framework (FR) regions (and generally all or substantially all of the remaining framework) are those of a canine immunoglobulin sequence. As exemplified herein, a caninized antibody comprises three heavy chain CDRs and three light chain CDRs from a murine anti-canine antigen antibody and canine frameworks or modified canine frameworks. The modified canine frameworks comprise one or more amino acid changes as exemplified herein that further optimize the effectiveness of the caninized antibody, e.g., increase its binding to its canine antigen and / or its ability to block the canine antigen from binding to its natural binding partner.

[0062] The variable regions of each light / heavy chain pair interact to form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, these two binding sites are typically the same. Generally, the variable domains of the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), within the overall framework region (FR). The CDRs are primarily responsible for binding to an epitope of a particular antigen. Generally, both light and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from N- to C-terminus. Generally, the assignment of amino acids to each domain is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Ed. (1991); Kabat, Adv. Prot. Chem. 32: 1-75 (1978); Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Chothia et al., J. Mol. Biol. 196: 901-917 (1987) or Chothia et al., Nature 342: 878-883 (1989)].

[0063] As used herein, the term "hypervariable region" refers to 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., LCDR1, LCDR2, and LCDR3 in the light chain variable domain and HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain) [see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), for the definitions of the CDR regions of an antibody by sequence; see also Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987), for the definitions of the CDR regions of an antibody by structure]. As used herein, the term "framework" or "FR" residues refer to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0064] There are four known IgG heavy chain subtypes of dog IgG and are referred to as IgG-A, IgG-B, IgG-C, and IgG-D. There are two known light chain subtypes referred to as lambda and kappa. In particular embodiments of the invention, in addition to binding and activation of canine immune cells, the canine or caninized antibodies of the invention against their antigens optimally have two attributes:

[0065] 1. lack of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

[0066] 2. ease of large scale purification using industry standard techniques such as protein A chromatography based techniques.

[0067] None of the naturally occurring canine IgG isotypes meet both criteria. For example, IgG-B can be purified using protein A, but has high levels of ADCC activity. IgG-A, on the other hand, binds weakly to protein A, but also shows ADCC activity. In addition, neither IgG-C nor IgG-D can be purified on a protein A column, although IgG-D shows no ADCC activity. (There is considerable ADCC activity with IgG-C). One way in which the present invention addresses these problems is by providing a modified canine IgG-B antibody of the invention specific for the antigens of the invention, which lacks effector functions such as ADCC and can be easily purified using industry standard protein A chromatography.

[0068] As used herein, an "anti-inflammatory antibody" is an antibody that can act as an anti-inflammatory agent in an animal, including a mammal such as a human, a canine, and / or a feline, particularly with respect to atopic dermatitis. In particular embodiments, the anti-inflammatory antibody binds to a particular protein in the IL-4 / IL-3 signaling pathway, such as IL-4 or the receptor IL-4R α . Binding of the anti-inflammatory antibody to its corresponding antigen (e.g., IL-4 or IL-4R α ) inhibits, for example, the binding of IL-4 to IL 4R α , and interferes with and / or prevents signaling of the pathway, thereby interfering with or preventing chronic inflammation associated with atopic dermatitis.

[0069] As used herein, "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences when aligned for maximum correspondence. Two molecules are homologous at a position if they are occupied by the same base or amino acid residue, e.g., if one position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at this position. The percent homology is the number of homologous positions shared by two sequences divided by the total number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Typically, the comparison is made when the two sequences are aligned for maximum percent homology. Sequence identity refers to the extent to which two polypeptides are identical at equivalent positions when the two sequences are optimally aligned.

[0070] As used herein, an amino acid sequence is 100% "identical" to a second amino acid sequence when the amino acid residues of the two sequences are the same at each corresponding position. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence when 50% of the amino acid residues of the two sequences are the same. Sequence comparisons are performed on segments of consecutive amino acid residues encompassed by a given protein (e.g., a portion of the protein or polypeptide being compared). In particular embodiments, selected deletions or insertions are taken into account that might otherwise alter the correspondence between two amino acid sequences. Sequence similarity includes identical residues and non-identical, biochemically related amino acids. Biochemically related amino acids have similar properties and can be used interchangeably.

[0071] A "conservatively modified variant" or "conservative substitution" refers to the substitution of one amino acid for another amino acid in a protein with similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.) such that the change can typically be made without altering the biological activity of the protein. Generally, one of skill in the art recognizes that a single amino acid substitution in a non-essential region of a polypeptide will not significantly alter biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. Moreover, substitutions of amino acids with similar structures or functions are less likely to disrupt biological activity. Exemplary conservative substitutions are listed in Table A, immediately below.

[0072] Table A

[0073] Exemplary conservative amino acid substitutions

[0074]

[0075]

[0076] The present application also encompasses function-conservative variants of the antibodies of the present application. As used herein, "function-conservative variants" refer to antibodies or fragments in which one or more amino acid residues have been altered without altering the desired properties (e.g., antigen affinity and / or specificity). Such variants include, but are not limited to, substitution of an amino acid with one having similar properties, e.g., the conservative amino acid substitutions of Table A above.

[0077] "Isolated nucleic acid molecule" refers to a DNA or RNA, mRNA, cDNA, or synthetic origin, or some combination thereof, of a genome, which is not associated with all or a portion of the polynucleotide as it exists in nature, or linked to polynucleotides with which it is not linked in nature. For purposes of the present disclosure, it is understood that "nucleic acid molecule comprising (a specified nucleotide sequence)" does not include an entire chromosome. An isolated nucleic acid molecule comprising a specified nucleotide sequence can include, in addition to the specified sequence, up to ten or even up to twenty or more additional coding sequences of proteins or portions or fragments thereof, or can include operably linked regulatory sequences that control expression of the coding region of the nucleic acid sequence, and / or can include vector sequences.

[0078] The present application provides isolated caninized antibodies of the present application for use in treating a disorder, e.g., a method of use for treating atopic dermatitis in a dog. In canines, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgG-A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC), and IgG-D (or IgGD). Each of the two heavy chains consists of one variable domain (VH) and three constant domains designated CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain by an amino acid sequence designated a "hinge" or alternatively a "hinge region."

[0079] The nucleic acid and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001)]. The amino acid and nucleic acid sequences of these heavy chains are also available from the GenBank database. For example, the accession number for the amino acid sequence of the IgGA heavy chain is AAL35301.1, for IgGB is AAL35302.1, for IgGC is AAL35303.1, and for IgGD is (AAL35304.1). Canine antibodies also comprise two types of light chains, kappa and lambda. The DNA and amino acid sequences of these light chains are available from the GenBank database. For example, the accession number for the kappa light chain amino acid sequence is ABY57289.1, and for the lambda light chain is ABY 55569.1.

[0080] In the present application, the amino acid sequence of each of the four canine IgG Fc fragments is based on the determined boundaries of the CH1 and CH2 domains determined by Tang et al., supra. Caninized murine anti-canine antibodies that bind canine IL-4R α include, but are not limited to, antibodies of the present application comprising canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine kappa or lambda light chains and murine anti-canine IL-4R α CDRs. Accordingly, the present application provides isolated caninized murine anti-canine antibodies of the present application that bind to canine IL-4R α and block canine IL-4R α binding to its natural binding partners canine IL-4 and / or IL-13.

[0081] Accordingly, the present application further provides caninized murine antibodies and methods of using the antibodies of the present application to treat a disorder, for example, atopic dermatitis in a canine.

[0082] The present application further provides full-length canine heavy chains that can be matched with corresponding light chains to make caninized antibodies. Accordingly, the present application further provides caninized murine anti-canine antigen antibodies of the present application (including isolated caninized murine anti-canine antibodies) and methods of using the antibodies of the present application to treat a disorder, for example, atopic dermatitis in a canine.

[0083] The present application also provides antibodies of the present application comprising a canine fragment crystallizable region (cFc region), wherein the cFc is genetically modified to enhance, decrease, or eliminate one or more effector functions. In one aspect of the present application, the genetically modified cFc reduces or eliminates one or more effector functions. In another aspect of the present application, the genetically modified cFc enhances one or more effector functions. In certain embodiments, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a specific embodiment, the effector function that is enhanced, decreased, or eliminated is antibody-dependent cellular cytotoxicity (ADCC). In another embodiment, the effector function that is enhanced, decreased, or eliminated is complement-dependent cytotoxicity (CDC). In yet another embodiment, the cFc region is genetically modified to increase, decrease, or eliminate both ADCC and CDC.

[0084] To generate variants of canine IgG that lack effector function, a number of mutated canine IgG B heavy chains were generated. These variants can include one or more of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The variant heavy chains (i.e., comprising such amino acid substitutions) were cloned into an expression plasmid and transfected into HEK 293 cells along with a plasmid comprising a gene encoding a light chain. The intact antibodies expressed and purified from the HEK 293 cells were evaluated for binding to Fc γ RI and Clq to assess their potential to mediate immune effector functions. [See US 10,106,607 B2, the contents of which are incorporated by reference in their entirety.]

[0085] The present disclosure also provides modified canine IgG-Ds that, in place of their native IgG-D hinge regions, they comprise a hinge region from:

[0086] IgG-A: FNECRCTDTPPCPVPEP SEQ ID NO: 6

[0087] IgG-B: PKRENGRVPRPPDCPKCPAPEM SEQ ID NO: 7; or

[0088] IgG-C: AKECECKCNCNNCPCPGCGL SEQ ID NO: 8.

[0089] Alternatively, the IgG D hinge region can be genetically modified by replacing a serine residue with a proline residue, i.e., (proline residue (P) in place of the naturally occurring serine residue is underlined and in bold). Such modifications can result in canine IgG-Ds that lack Fab arm exchange. Standard methods of recombinant DNA technology can be used to construct the modified canine IgG-Ds [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these variants, the nucleic acid encoding the amino acid sequence of the canine IgG-D can be modified so that it encodes the modified IgG-D. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0090] The six complementarity determining regions (CDRs) of the caninized murine anti-canine antibody as described herein can comprise a canine antibody kappa light chain comprising murine light chain LCDR1, LCDR2, and LCDR3, and a canine antibody heavy chain IgG comprising murine heavy chain HCDR1, HCDR2, and HCDR3.

[0091] Nucleic acid

[0092] The present application also includes nucleic acids encoding the antibodies of the present application (see, e.g., the Examples below).

[0093] The present application also includes nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence that is 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 sequence of a caninized antibody provided herein, except for unaltered CDRs, when compared using the BLAST algorithm, with parameters of the algorithm selected to give the maximum match over the entire length of the respective sequence to the corresponding reference sequence. The present application further provides nucleic acids encoding immunoglobulin polypeptides comprising an amino acid sequence that is at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences, when compared using the BLAST algorithm, with parameters of the algorithm selected to give the maximum match over the entire length of the respective sequence to the corresponding reference sequence, are also included in the present application.

[0094] As used herein, percent nucleotide and amino acid sequence 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 similar default parameters. Alternatively, high advanced Blast searches under default filter conditions can be used, e.g., the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program using default parameters.

[0095] The following references relate to the BLAST algorithm commonly 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).

[0096] Antibodies of the present application can be recombinantly produced by methods known in the art. Mammalian cell lines useful as hosts for the 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). In particular, these cell lines include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocarcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many others. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, equine, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When a recombinant expression vector encoding a heavy chain or antigen binding portion thereof or fragment thereof, a light chain and / or antigen binding fragment thereof is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or more, preferably, for the antibody to be secreted into the culture medium in which the host cells are grown.

[0097] Antibodies can be recovered from the culture medium using standard protein purification methods. Furthermore, a number of known techniques can be used to enhance expression of antibodies of the present application (or other portions therein) from a production cell line. For example, the glutamine synthetase gene expression system (GS system) is a commonly used method to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997, and European Patent Application No. 89303964.4.

[0098] In general, a glycoprotein produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic of glycoproteins produced in the cell line or transgenic animal. Thus, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein constitute the invention, regardless of the glycosylation pattern the antibodies can have. Similarly, in certain embodiments, antibodies having a glycosylation pattern comprising only non-fucosylated N-glycans can be advantageous, as these antibodies have been shown to generally exhibit stronger efficacy in vitro and in vivo than their fucosylated counterparts [see, e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775].

[0099] Canine IL-4 Receptor Alpha Receptor

[0100] The cDNA encoding the predicted full-length canine IL-4 receptor alpha chain (SEQ ID NO: 1) was determined by searching the Genbank database (Accession No. XM_547077.4; see also US 7,208,579 B2). This predicted cDNA encodes 823 amino acids (SEQ ID NO: 2), including a 25 amino acid leader sequence, and is identified as Accession No. XP_547077.3. The mature predicted canine IL-4 receptor alpha chain protein (SEQ ID NO: 4) shares 65% identity with the human IL-4 receptor alpha chain (Accession No. NP_000409.1) and 70% identity with the porcine IL-4 receptor alpha chain (Accession No. NP_999505.1). The mature predicted canine IL-4 receptor alpha chain protein is encoded by the nucleotide sequence identified as SEQ ID NO: 3. Comparison of the predicted mature IL-4 receptor alpha chain with the known sequence of the human IL-4 receptor alpha chain identified the extracellular domain (ECD) of the mature canine IL-4 receptor alpha chain protein and is designated SEQ ID NO: 5. This has been previously described in its entirety in [US 2018 / 0346580; incorporated herein by reference in its entirety].

[0101] Canine IL-4 Receptor Alpha Chain Full-Length DNA with Signal Sequence [SEQ ID NO: 1]

[0102]

[0103] Canine IL-4 Receptor Alpha Full-Length Protein [SEQ ID NO: 2], Signal Sequence in Bold

[0104]

[0105] Canine IL-4 receptor alpha mature full length protein without signal sequence [SEQ ID NO: 4]

[0106]

[0107] Canine IL-4 receptor alpha mature full length DNA without signal sequence [SEQ ID NO: 3]

[0108]

[0109] Canine IL-4 receptor alpha chain extracellular domain [SEQ ID NO: 5]

[0110]

[0111] Antibody protein engineering

[0112] The antibody can also comprise a light chain constant region, e.g., a canine light chain constant region, e.g., a lambda or kappa canine light chain constant region or a variant thereof. By way of example and not limitation, the canine heavy chain constant region can be from IgG-B or a modified cFc, e.g., IgG-Bm as used herein [see US 10,106,607 B2, hereby incorporated by reference in its entirety], and the canine light chain constant region can be from kappa.

[0113] The antibody can be engineered to include modifications to canine framework and / or canine framework residues within the parent (i.e., mouse) monoclonal antibody variable domains, e.g., to improve properties of the antibody.

[0114] Pharmaceutical compositions and administration

[0115] To prepare a pharmaceutical or sterile composition comprising an antibody of the present application, the antibodies 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)].

[0116] Formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, for example, a lyophilized powder, a syrup, a solution, or a suspension [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, the antibodies of the application are diluted to the appropriate concentration in a sodium acetate solution at pH 5-6, and NaCl or sucrose is added to obtain tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, can be added to enhance stability.

[0117] Toxicity and therapeutic efficacy of the antibody compositions, 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 LD 50 (the dose lethal to 50% of the population) and ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 / ED 50 ). In particular aspects, antibodies exhibiting a high therapeutic index are desired. The data obtained from these cell culture tests and animal studies can be used in formulating a range of dosage for use in canine animals. The dosage of such compounds lies preferably within a range of ED 50Within the circulating concentration range. The dosage can be varied depending upon the dosage form employed and the route of administration utilized.

[0118] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial. In particular embodiments, the antibodies of the application can be administered by invasive routes, for example, by injection. In further embodiments of the application, the antibodies of the application or pharmaceutical compositions thereof are administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; e.g., in a pill, capsule, or tablet) are also within the scope of the application.

[0119] The compositions can be administered with medical devices known in the art. For example, the pharmaceutical compositions of the application can be administered by injection with a hypodermic needle, including, for example, a pre-filled syringe or an auto-injector. The pharmaceutical compositions disclosed herein can also be administered with needle-free hypodermic injection devices; for example, 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.

[0120] The pharmaceutical compositions disclosed herein can also be administered by infusion. Well-known examples of implants and modules for 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 drug 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.

[0121] Alternatively, the antibodies of the application can be administered in a local rather than systemic manner, often being presented in a depot or sustained release formulation.

[0122] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to ameliorate the status of the disease / condition of interest while minimizing adverse side effects. Thus, the amount of biologic delivered depends in part on the particular therapeutic antibody as well as the severity of the condition being treated. Guidance for selecting appropriate dosages of therapeutic antibodies is provided [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)].

[0123] Determining appropriate dosages is performed by a veterinarian, e.g., using parameters or factors known in the art or suspected of affecting treatment. Usually, the dosage is started at an amount somewhat less than the optimal dose and it is increased by small increments until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those that measure the symptoms.

[0124] The antibodies provided herein can be provided by continuous infusion or by doses administered, e.g., daily, 1-7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semiannually, annually, etc. The doses can be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is typically at least 0.05 pg / kg body weight, more typically 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 can also be provided to achieve predetermined target concentrations of the antibodies of the application in the serum of the canine, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 pg / ml, or more. In other embodiments, the antibodies of the application are administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg per subject per week, biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly.

[0125] As used herein, “inhibit” or “treat” or “treating” includes delaying the onset and / or reducing the severity of symptoms associated with a disorder. The term also includes ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of these symptoms. The terms also include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of these symptoms. Thus, the terms denote a beneficial result to a vertebrate subject (e.g., a canine) that is suffering from a disease, condition, and / or symptom or that is at risk of developing such a disease, condition, or symptom.

[0126] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to the amount of an antibody of the application that, when administered to a cell, tissue, or subject (e.g., a canine) either alone or in combination with another therapeutic agent, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or in the progression of such disease or condition. A therapeutically effective dose further refers to an amount of an antibody that is sufficient to result in at least a partial improvement in symptoms, e.g., treatment, cure, prevention, or amelioration of a relevant medical condition, or an increase in the rate of treatment, cure, prevention, or amelioration of such condition. When applied to combination therapy, a therapeutically effective dose refers to combined amounts of the active ingredients that produce the therapeutic effect whether administered jointly, sequentially, or simultaneously. An effective amount of a therapeutic agent will produce an improvement in a diagnostic measure or parameter of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. Where subjective parameters are used to assess severity of illness, an effective amount can also produce an improvement in subjective parameters.

[0127] Examples

[0128] Example 1

[0129] Anti-IL-4 Receptor Alpha Antibodies

[0130] General Materials and Methods:

[0131] Recombinant proteins were obtained by providing the amino acid sequence of the selected protein to a commercial manufacturer (ATUM, Newark, California), who in turn selected an appropriate nucleotide sequence that encodes the amino acid sequence. Nucleotide sequences were also obtained from publicly available DNA databases, such as GenBank®. The commercial manufacturer then chemically synthesized the nucleic acid, which was then cloned by ATUM into an expression plasmid (pD2610-v10; available from AUTM) to produce the corresponding recombinant protein. The plasmid was placed into HEK-293 cells or CHO cells to express the recombinant protein, which was then isolated by conventional methods.

[0132] Balb / c mice were immunized multiple times (10 pg each) over a 17 day period. The immunizing antigen was a canine IL-4R alpha chain extracellular domain (ECD)-human Fc fusion protein. After immunization, serum was collected from each mouse and tested for reactivity with canine IL-4 receptor alpha chain ECD HIS-labeled protein. About 2 weeks after fusion, supernatants from putative hybridoma cells were tested for reactivity with IL-4 receptor alpha chain ECD HIS-labeled protein by ELISA. Hybridomas that produced strong positive signals in the ELISA were subcloned by limiting dilution and tested again for reactivity with canine IL-4 receptor alpha chain ECD HIS-labeled protein.

[0133] Anti-canine IL-4 receptor alpha antibodies include antibody c152H11 VL3-cCLk-s / c152H11 VH3-cIgGBm and antibody c146E2 VL3-cCLk-s / c146E2 VH3-cIgGBm. The sets of six (6) CDRs (three separate light chain (LC) and three heavy chain (HC) sequences) for these two antibodies are provided below in Tables 1A and 1B. Table 1A provides the nucleic acids encoding the amino acid sequences of the twelve CDRs listed in Table 1B. The amino acid sequences of the full-length light and heavy chains of these caninized antibodies are provided immediately below in Table 1B.

[0134] IL-4R alpha antibody CDR nucleic acid and amino acid sequences

[0135] Table 1A

[0136]

[0137]

[0138] Table 1B

[0139]

[0140] c152H11 VL3-cCLk-s (kappa light chain): [SEQ ID NO: 35]

[0141]

[0142] c152H11 VH1 -cIgGBm (heavy chain): [SEQ ID NO: 36]

[0143]

[0144] c152H11 VH2-cIgGBm (heavy chain): [SEQ ID NO: 37]

[0145]

[0146] c152H11 VH3-cIgGBm (heavy chain): [SEQ ID NO: 38]

[0147]

[0148] c146E2 VL3-cCLk-s (kappa light chain): [SEQ ID NO: 39]

[0149]

[0150] c146E2VH1 -cIgGBm (heavy chain): [SEQ ID NO: 40]

[0151]

[0152] c146E2VH2 -cIgGBm (heavy chain): [SEQ ID NO: 41]

[0153]

[0154] c146E2VH3 -cIgGBm (heavy chain): [SEQ ID NO: 42]

[0155]

[0156] In addition, the light chain of the canine IL-4 receptor alpha antibody was also constructed using lambda light chains provided as follows.

[0157] c152H11 LV1 -cCl (lambda light chain) [SEQ ID NO: 43]

[0158]

[0159] c146E2 LV1 -cCl (lambda light chain) [SEQ ID NO: 44]

[0160]

[0161] Example 2

[0162] STAT-6 Inhibition

[0163] Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STAT-6 phosphorylation in DH82 cells as follows:

[0164] Materials

[0165] 1. Actively growing DH82 cells

[0166] 2. DH82 cell growth medium (DMEM, Eagle's Minimum Essential Medium, provided with heat-inactivated fetal bovine serum to a final concentration of 15% w / v) 302003 TM

[0167] 3. AlphaLISA p-STAT6 (Tyr641) assay kit: Perkin Elmer Cat# ALSU-PST6-A-HV

[0168] 4. Recombinant canine IL-4: R&D Systems, Cat# 752-CL / CF​

[0169] 5. Recombinant canine IL-13: R&D Systems, Catalog # 5894-CL / CF

[0170] 6. Perkin Elmer Envision

[0171] a. Caninized anti-canine IL-4R α Monoclonal Antibodies

[0172] b. c146E2-H3L3

[0173] c. 4H3 caninized antibody from US2018 / 0346580

[0174] Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STAT-6 phosphorylation in DH82 cells as follows:

[0175] Methods

[0176] 1. Two tissue culture plates were seeded with 8 x 10 4 DH82 cells per well (200 μL at a density of 4 x 10 5 cells / mL) and incubated at 37°C overnight.

[0177] 2. Test antibodies were pre-diluted to 500 μg / mL and then serially 3-fold diluted in DH82 cell growth media. The media was removed from the cell culture plates and 50 μL / well of the serially diluted test samples were transferred to each plate.

[0178] 3. Canine IL-4 was diluted to 5 ng / mL in DH82 cell growth media and 50 μL was added to each well of one plate. Canine IL-13 was diluted to 10 ng / mL in DH82 cell growth media and 50 μL was added to each well of the second plate. The plates were incubated at 37°C for 15 minutes.

[0179] 4. The media was removed from the plates and 100 μL / well of freshly prepared lx lysis buffer from the AlphaLISA p-STAT-6 assay kit was added to the plates. The plates were agitated on a plate shaker at 350 rpm for 10 minutes at room temperature.

[0180] 5. The Acceptor Mix was prepared from the AlphaLISA p-STAT6 assay kit and 15 μL per well was added to 30 μL of cell lysate in a 96-well ½ Area Plates. The plates were sealed, agitated at 350 rpm for 2 minutes, and then incubated at room temperature for 2 hours.

[0181] 6. Donor Mix was prepared using the AlphaLISA p-STAT6 assay kit under soft laboratory lighting, with 15 μL added to each well of each plate. The plates were sealed, covered with foil, stirred at 350 rpm for 2 minutes, and then incubated at room temperature for 2 hours.

[0182] 7. Set up the reading board using AlphaScreen on Perkin Elmer EnVision.

[0183] Two different canine-derived anti-canine IL-4Rs, named c4H3 [WO2016 / 156588; US2018 / 0346580] and c146E2-H3L3, were evaluated. α Monoclonal antibodies work by blocking canine IL-4 or canine IL-13 from canine IL-4R. α The ability of binding to inhibit αSTAT-6 phosphorylation. Figure 1 The data shown indicate that both antibodies resulted in dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4, but surprisingly, c146E2-H3L3 bound more tightly than the prior art anti-canine IL-4 receptor α antibody c4H3 [WO2016 / 156588]. IL-4Rα (IL-4R) was not present. α The IL-4 control with antibody is shown in the upper right part of the figure. Figure 2 The data also show that, in the presence of IL-13, both antibodies resulted in dose-dependent inhibition of STAT-6 phosphorylation, with c146E2-H3L3 binding more tightly than the prior art anti-canine IL-4 receptor α antibody c4H3. In the absence of IL-4Rα (IL-4R... α The IL-13 control with antibody is shown in the upper right part of the figure. Figure 3 The results showed that replacing the κ light chain with the λ light chain had no effect on the binding of c146E2-H3L3 to IL-4Rα.

[0184] Example 3

[0185] Epitope plotting

[0186] The interaction between an antibody and its homologous protein antigen is mediated by the binding of a specific amino acid (complementary site) of the antibody to a specific amino acid (epitaxe) of the target antigen. An epitope is an antigenic determinant that elicits a specific immunoglobulin response. An epitope consists of a set of amino acids on the surface of an antigen. A protein of interest may contain several epitopes recognized by different antibodies. Epitopes recognized by antibodies are classified as linear or conformational epitopes. Linear epitopes are formed by the extension of a continuous amino acid sequence in a protein, while conformational epitopes consist of amino acids that are discontinuous (e.g., far apart) in the primary amino acid sequence but aggregate together during three-dimensional protein folding.

[0187] Epitope mapping refers to the process of identifying the amino acid sequence (i.e., epitope) on its target antigen recognized by an antibody. Identifying the epitope on a target antigen recognized by a monoclonal antibody (mAb) has important applications. For example, it can help develop new therapeutic, diagnostic, and vaccine agents. Epitope mapping can also help select optimized therapeutic mAbs and help elucidate their mechanism of action. Epitope information on IL-4 receptor alpha can also elucidate unique epitopes and define the protective or pathogenic role of a vaccine. Epitope identification can also lead to the development of subunit vaccines based on the identified peptide epitope chemically or genetically coupled to a carrier protein or other immunostimulatory agent.

[0188] Epitope mapping can be performed using polyclonal or monoclonal antibodies, and epitope identification is performed using several methods depending on the suspected nature of the epitope (i.e., linear vs. conformational). Linear epitope mapping is more straightforward and relatively easier to perform. For this reason, commercial services for linear epitope mapping typically employ peptide scanning. In this case, a set of overlapping short peptide sequences of a target protein are chemically synthesized and tested for their ability to bind the antibody of interest. This strategy is fast, high-throughput, and relatively inexpensive to perform. On the other hand, mapping of discontinuous epitopes is technically more challenging and requires more specialized techniques, such as X-ray co-crystallography of monoclonal antibodies with their target protein, hydrogen-deuterium (H / D) exchange, mass spectrometry coupled with enzymatic digestion, and several other methods known to those skilled in the art.

[0189] Mapping of canine IL-4 receptor alpha epitopes using mass spectrometry:

[0190] A method for identifying epitopes recognized by anti-canine IL-4 receptor alpha mAbs based on chemical cross-linking, mass spectrometric detection, and covalent labeling [CovalX Instruments Incorporated, located at 999 Broadway, Suite 305, Saugus, MA 01906-4510 USA].

[0191] Application of this technology to epitope mapping of the canine IL-4 receptor alpha chain in previous studies indicated that the mAbs recognized specific peptide epitopes present within the extracellular domain of canine IL-4 receptor alpha [US 2018 / 0346580]. The analysis of the c146E2-H3L3 antibody shown below identified amino acid sequences SEQ ID NO: 46 and SEQ ID NO: 47 as epitopes with reasonable similarity to previously identified epitopes. In addition, as shown below, amino acid residues K Figure 4 Figure 4 K 97 , H 112 , T 113 , S 164 , T 165 ​, S 171 , Y 172 , S 173 , and R 175 are identified as specific contact points [amino acid residue numbering see, e.g., SEQ ID NO: 5].

[0192] Table of Sequences

[0193]

[0194]

[0195]

Claims

1. An isolated mammalian antibody or antigen-binding fragment thereof that binds to canine interleukin-4 receptor alpha (IL4R α ), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises three complementarity determining regions (CDRs): CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2), and CDR heavy 3 (HCDR3), and wherein the VL comprises three CDRs: CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3); wherein: the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

6. (i) the HCDR1 consists of the amino acid sequence of SEQ ID NO: 12; (ii) the HCDR2 consists of the amino acid sequence of SEQ ID NO: 14; (iii) the HCDR3 consists of the amino acid sequence of SEQ ID NO: 16; (iv) the LCDR1 consists of the amino acid sequence of SEQ ID NO: 18; (v) the LCDR2 consists of the amino acid sequence of SEQ ID NO: 20; and (vi) the LCDR3 consists of the amino acid sequence of SEQ ID NO:

22.

2. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds canine IL4R α and blocks canine IL4R α binding to canine interleukin-4.

3. The isolated mammalian antibody or antigen-binding fragment thereof of claim 1 or 2, which is a caninized antibody or caninized antigen-binding fragment thereof.

4. The caninized antibody or antigen-binding fragment thereof of claim 3, which comprises a hinge region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:

9.

5. The caninized antibody or antigen-binding fragment thereof of claim 3, which comprises a heavy chain comprising a modified canine IgG-B (IgG-Bm), wherein the IgG-Bm comprises the amino acid sequence of SEQ ID NO:

10.

6. The caninized antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO:

42.

7. The caninized antibody or antigen-binding fragment thereof of claim 6, which comprises a light chain comprising the amino acid sequence of SEQ ID NO:

39.

8. The caninized antibody or antigen-binding fragment thereof of claim 7, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

44.

9. A nucleic acid composition comprising: (a) a first nucleotide sequence encoding a heavy chain of the caninized antibody or antigen-binding fragment thereof of any one of claims 3, 4, 5, 6, 7, and 8; and (b) a second nucleotide sequence encoding a light chain of the caninized antibody or antigen-binding fragment thereof of any one of claims 3, 4, 5, 6, 7, and 8.

10. An expression vector comprising a first nucleotide sequence encoding a heavy chain of the caninized antibody or antigen-binding fragment thereof of any one of claims 3, 4, 5, 6, 7, and 8; and a second nucleotide sequence encoding a light chain of the caninized antibody or antigen-binding fragment thereof of any one of claims 3, 4, 5, 6, 7, and 8.

11. An expression vector system comprising: (a) a first expression vector comprising a first nucleotide sequence encoding a heavy chain of the caninized antibody or antigen-binding fragment thereof of any one of claims 3, 4, 5, 6, 7, and 8; and (b) a second expression vector comprising a second nucleotide sequence encoding a light chain of the caninized antibody or antigen-binding fragment thereof of any one of claims 3, 4, 5, 6, 7, and 8.

12. A host cell comprising the expression vector of claim 10 or the expression vector system of claim 11.

13. A pharmaceutical composition comprising the caninized antibody or antigen binding fragment thereof of any one of claims 3, 4, 5, 6, 7, and 8 and a pharmaceutically acceptable carrier or diluent.

14. Use of the pharmaceutical composition of claim 13 in the manufacture of a medicament for aiding in blocking inflammation associated with atopic dermatitis.

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