Antibody to canine interleukin-4 receptor α

Abstract

The present invention provides antibodies against canine IL-4 receptor α that have high binding affinity to canine IL-4 receptor α and can block the binding of canine IL-4 and / or IL-13 to canine IL-4 receptor α. The present invention further relates to epitopes on canine IL-4 receptor α that bind to antibodies against canine IL-4 receptor α. The present invention further provides use of the antibodies for the treatment of atopic dermatitis in dogs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 015,209, filed April 24, 2020, U.S. Patent Application No. 63 / 015,220, filed April 24, 2020, U.S. Patent Application No. 62 / 951,778, filed December 20, 2019, and U.S. Patent Application No. 62 / 951,793, filed December 20, 2019, the contents of all of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to antibodies against canine IL-4 receptor α that have high binding affinity to canine IL-4 receptor α and are capable of blocking the binding of canine IL-4 and / or IL-13 to canine IL-4 receptor α. The present invention also relates to the use of the antibodies of the present invention in the treatment of atopic dermatitis in dogs. [Background technology]

[0003] The immune system comprises a network of resident and recirculating specialized cells that function in concert to protect the host from infectious diseases and cancer. The immune system's ability to perform this function depends largely on the biological activity of a group of proteins secreted by white blood cells and collectively known as interleukins. There are four key molecules among the well-studied interleukins 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 involved in the regulation of immune responses by CD4 +IL-4 and IL-13 are closely related proteins that can be secreted by many cell types, including Th2 cells, natural killer T cells (NKT), macrophages, mast cells, and basophils. IL-4 and IL-13 exhibit many overlapping functions and are crucial for the development of T cell-dependent humoral immune responses. IL-4 is known to bind with high affinity to two receptors, type I and type II IL-4 receptors. Type I IL-4 receptor consists of the IL-4 receptor α chain and the common γC chain. Type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain. IL-13 binds to the type II IL-4 receptor and a unique receptor called IL-13 receptor α2. IL-13 binding to IL-13 receptor α2 does not signal, and this receptor is also secreted in a soluble form. Therefore, IL-13 receptor α2 has often been referred to as a decoy receptor. IL-4, IL-13, IL-22, and IL-31 are key cytokines for generating the immune responses required for protection against extracellular pathogens (e.g., parasites present in tissues or lumens), but these cytokines are also involved in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

[0004] Atopic dermatitis (AD) is a recurrent, pruritic, and chronic inflammatory skin disease characterized by immune system dysregulation and epidermal barrier abnormalities in humans. The pathological and immunological characteristics of atopic dermatitis have been the subject of extensive investigation (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 condition 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)). ], skin infiltration by various immune cells and CD4 including IL-4, IL-13 and IL-31 predominance. + It shows striking similarities to the pathogenesis of human atopic dermatitis, including a Th2-polarized cytokine environment. Furthermore, IL-22 is involved in the excessive epidermal proliferation that leads to the epidermal thickening characteristic of atopic dermatitis.

[0005] For example, antibodies against canine IL-31 have been shown to have a significant effect on pruritus associated with atopic dermatitis in dogs [U.S. Patent No. 8,790,651; U.S. Patent No. 10,093,731]. Furthermore, antibodies against human IL-31 receptor alpha (IL-31RA) have been tested and found to have a significant effect on pruritus associated with atopic dermatitis in humans [Ruzicka, et al., New England Journal of Medicine, 376(9), 826-835(2017)]. Therefore, blocking IL-31 binding to its receptor IL-31RA reduces pruritus associated with atopic dermatitis.

[0006] Human IL-4 receptor α (IL-4R αMonoclonal antibodies raised against canine IL-4R have been developed, and some of these antibodies have been extensively tested for their therapeutic efficacy in treating atopic dermatitis in humans (see, e.g., U.S. Patent Application Publication No. 2015 / 0017176). More recently, the canine IL-4R of canine IL-4 has been identified. α Blocks binding to canine IL-4R α A caninized antibody against canine IL-4R has also been disclosed (U.S. Patent Application Publication No. 2018 / 0346580, the entire contents of which are incorporated herein by reference). Because type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain, canine IL-4R can block the binding of both canine IL-4 and canine IL-13 to type II canine IL-4 receptor. α Antibodies against α-glucan have been obtained that help block inflammation associated with atopic dermatitis [US Patent Application Publication No. 2018 / 0346580].

[0007] Interleukin-22 (IL-22), also known as IL-10-related T cell-derived inducer factor (IL-TIF), belongs to the IL-10 cytokine family. IL-22 is produced by normal T cells in humans upon anti-CD3 stimulation. Murine IL-22 expression is also induced in various organs upon lipopolysaccharide injection, suggesting that IL-22 may be involved in inflammatory responses. IL-22 specifically binds to and signals through a receptor complex consisting of a heterodimeric complex of IL-10R2 (also known as IL-10Rβ) and the interleukin-22 receptor (IL-22R) [see Lee et al., Pharmacology Research & Perspectives, Pages 1-13 (2018: e00434)]. The interleukin-22 receptor is also known as interleukin-22R, α1; 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 deficiency can allow uncontrolled proliferation and promote tumor development [Huber et al., Nature 491:259-263 (2012)]. IL-22 has been shown to activate STAT-1 and STAT-3 and upregulate the production of acute phase proteins in several hepatoma cell lines. Antibodies against interleukin-22 and IL-22R act as antiproliferative agents by blocking the interaction of IL-22 with IL-22R, thereby blocking the associated signaling pathways that lead to epithelial proliferation.

[0008] However, despite recent successes in treating atopic dermatitis, none of the current therapies in use provide significant effects on skin inflammation and rapid onset of antipruritic effects, while also improving skin barrier function. Therefore, there is a need to design better therapies that can address one or more of the symptoms of atopic dermatitis.

[0009] 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. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 8,790,651 [Patent Document 2] U.S. Patent No. 10,093,731 [Patent Document 3] U.S. Patent Application Publication No. 2015 / 0017176 [Patent Document 4] U.S. Patent Application Publication No. 2018 / 0346580 [Non-patent literature]

[0011] [Non-Patent Document 1] Rahman et al., Inflammation & Allergy-drug target 10:486-496(2011) [Non-patent document 2] Harskamp et al.,Seminar in Cutaneous Medicine and Surgery 32:132-139(2013) [Non-patent document 3] Nuttall et al., Veterinary Records 172(8):201-207(2013) [Non-patent document 4] Ruzicka,et al.,New England Journal of Medicine,376(9),826-835(2017) [Non-patent document 5] Lee et al.,Pharmacology Research & Perspectives,Pages 1-13(2018:e00434) [Non-patent document 6] Xu et al.,Proc.Nat.Acad.Sci.98(17)9511-9516(2001) [Non-Patent Document 7] Gelebart and Lai, Atlas of Genetics and Cytogenetics 14(12):1106-1110(2010) [Non-patent document 8] Huber et al.,Nature 491:259-263(2012) Summary of the Invention [Means for solving the problem]

[0012] In a specific embodiment, the present invention provides canine IL-4Rα (IL-4Rα) isolated from a human IL-4 receptor α antibody, which has superior properties to those of the prior art, for example, stronger binding than the anti-canine IL-4 receptor α antibodies of the prior art. α In a particular embodiment, the present invention provides a novel caninized antibody against canine interleukin-4 receptor alpha (IL-4R). In a particular embodiment, the present invention provides a mammalian antibody or antigen-binding fragment thereof that binds with specificity to canine interleukin-4 receptor alpha, the mammalian antibody or antigen-binding fragment thereof 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 a related embodiment, the present invention provides a mammalian antibody or antigen-binding fragment thereof that binds with specificity to canine interleukin-4 receptor alpha (IL-4R). 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 a preferred embodiment, the mammalian antibody or antigen-binding fragment thereof binds to canine IL-4R. α binds to canine IL-4R α In a related embodiment, the mammalian antibody or antigen-binding fragment thereof blocks the binding of canine IL-4R to canine interleukin-4 (cIL-4). α binds to canine IL-4R α In yet another embodiment, the mammalian antibody or antigen-binding fragment thereof blocks binding of canine cIL-4R to canine interleukin-13 (cIL-13). α Binds to canine cIL-4R α Blocks the binding of cIL-4 and cIL-13.

[0013] In certain embodiments, the mammalian antibody against canine IL-4Rα is a murine antibody. In a related embodiment, the mammalian antibody against canine IL-4Rα is a caninized murine antibody. In certain embodiments, the caninized antibody comprises a heavy chain comprising IgG-D cFc, but the naturally occurring IgG-D hinge region has been replaced by 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 IgG-D hinge region has been replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 7. In yet other embodiments, the caninized antibody comprises a heavy chain comprising IgG-D cFc, but the naturally occurring IgG-D hinge region has been replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 8. In yet other embodiments, the caninized antibody comprises a heavy chain comprising IgG-D cFc, but the naturally occurring IgG-D hinge region has been replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 9.

[0014] In certain embodiments, the caninized antibody comprises a heavy chain comprising a modified canine IgG-B (IgG-Bm) 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 yet other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 39. In an alternative embodiment, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0015] In a particular embodiment, the caninized antibody or antigen-binding fragment thereof binds to SEQ ID NO: 46. In a more particular embodiment, the caninized antibody or antigen-binding fragment thereof binds to canine IL-4R α the following amino acid residue: K of SEQ ID NO: 5 97 , H 112 , T 113 In a related embodiment, the caninized antibody or antigen-binding fragment thereof binds to SEQ ID NO: 47. In a more particular embodiment, the caninized antibody or antigen-binding fragment thereof binds to canine IL-4R. α the following amino acid residues: S of SEQ ID NO:5 164 , T 165 , S 171 , Y 172 , S 173 and R 175 In an even more particular embodiment, the caninized antibody or antigen-binding fragment thereof binds to both SEQ ID NO: 46 and SEQ ID NO: 47. In an even further particular embodiment of such, the caninized antibody or antigen-binding fragment thereof binds to canine IL-4R. α the following amino acid residue: K of SEQ ID NO: 5 97 , H 112 , T 113 and / or canine IL-4R α the following amino acid residues: S of SEQ ID NO:5 164 , T 165 , S 171 , Y 172 , S173 and R 175 It binds to one, two, three, four or all five of the following:

[0016] The present invention also provides nucleic acids, including isolated nucleic acids, encoding CDRs, the heavy chain of a caninized antibody or antigen-binding fragment thereof, and / or the light chain of a caninized antibody or antigen-binding fragment thereof. Additionally, the present invention provides expression vectors comprising such nucleic acids and host cells comprising such expression vectors.

[0017] The present invention further provides pharmaceutical compositions comprising the caninized antibodies and antigen-binding fragments thereof of the present invention together with a pharmaceutically acceptable carrier and / or diluent. The present invention further provides methods for treating atopic dermatitis, comprising administering one of the compositions to a dog with atopic dermatitis. In certain embodiments, the present invention provides a method for helping to block inflammation associated with atopic dermatitis, comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a dog in need thereof.

[0018] These and other aspects of the present invention will be better understood by reference to the following brief description and detailed description of the drawings. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows two different caninized monoclonal anti-canine interleukin-4 receptor alpha antibodies, designated c4H3 [see WO 2016 / 156588] and c146E2-H3L3, that were evaluated for their ability to inhibit STAT-6 phosphorylation. The data show that both antibodies result in dose-dependent inhibition of STAT-6 phosphorylation in the presence of canine interleukin-4. An IL-4 control in the absence of IL-4Rα (IL-4Rα) antibody is shown in the upper right portion of the graph. [Figure 2]Figure 2 shows two different caninized monoclonal anti-canine interleukin-4 receptor alpha antibodies, designated c4H3 [see WO 2016 / 156588] and c146E2-H3L3, that were evaluated for their ability to inhibit STAT-6 phosphorylation. The data show that both antibodies result in dose-dependent inhibition of STAT-6 phosphorylation in the presence of canine interleukin-13. The IL-13 control in the absence of IL-4Rα (IL-4Rα) antibody is shown in the upper right portion of the graph. [Figure 3] Figure 3 shows the binding of caninized anti-canine IL-4Rα antibodies containing either lambda or kappa light chains, as assessed by ELISA. The results show that caninized anti-canine IL-4Rα antibodies containing lambda light chains (c146ClL1-H1, c146ClL1-H2, and c146ClL1-H3) bind to canine IL-4Rα as well as to a caninized anti-canine IL-4Rα antibody containing the same CDRs but with a kappa light chain (c146E2-H3L3). 146mc is a mouse-canine chimeric antibody positive control, and Iso-Ctr, a negative control, is an irrelevant caninized antibody. [Figure 4] FIG. 4 shows the epitopes comprising the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 47 on canine IL-4Rα for the c146E2-H3L3 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0020] In response to the need for better treatments for atopic dermatitis, the present invention provides caninized antibodies, formulations comprising caninized antibodies, and methodologies that can achieve significant effects on skin inflammation associated with atopic dermatitis.

[0021] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention: ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity CDR Complementarity determining region in an immunoglobulin variable region, defined using the Kabat numbering system EC50 Concentration that produces 50% efficacy or binding ELISA enzyme-linked immunosorbent assay FR Antibody framework region: immunoglobulin variable region excluding the CDR regions. IC50: Concentration that produces 50% inhibition IgG immunoglobulin G The immunoglobulin alignment and numbering system pioneered by Elvin 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) V region The segment of an IgG chain whose sequence is variable among different antibodies. The V region spans Kabat residue 109 in the light chain and Kabat residue 113 in the heavy chain. VH immunoglobulin heavy chain variable region VL immunoglobulin light chain variable region VK immunoglobulin kappa light chain variable region

[0022] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0023] As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0024] "Administration" and "treatment," as applied to an animal, e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the animal, e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid. Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid in contact with the cell.

[0025] "Administration" and "treatment" also refer to in vitro and ex vivo treatments, e.g., of a cell, with a reagent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a dog, cat, or human), most preferably a dog.

[0026] "Treat" or "treating" refers to administering, internally or externally, a therapeutic agent, such as a composition containing any of the antibodies of the present invention, to, for example, a canine subject or patient having or suspected of having one or more symptoms for which the therapeutic agent has therapeutic activity. Typically, the therapeutic agent is administered in an amount effective to reduce and / or ameliorate one or more disease / condition symptoms in the treated subject or population by inducing regression or inhibiting progression of such symptom(s) by any clinically measurable degree. The amount of therapeutic agent that is effective to reduce any particular disease / condition symptom (also referred to as a "therapeutically effective amount") may vary depending on factors such as the disease / condition state, age, and weight of the patient (e.g., dog), and the ability of the pharmaceutical composition to elicit a desired response in the subject. Reduction or improvement of disease / condition symptoms can be assessed by any clinical measurement routinely used by veterinarians or other skilled medical personnel to assess the severity or progression of the symptoms. An embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the target disease / condition symptom(s) in every subject, but should alleviate the target disease / condition symptom(s) 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, Mann-Whitney U test, Kruskal-Wallis test (H test), Joncke-Terpstra test, and Wilcoxon test.

[0027] "Treatment," when applied to a human, veterinary subject (e.g., a dog), or research subject, refers to therapeutic treatment as well as research and diagnostic uses. When applied to a human, veterinary subject (e.g., a dog), or research subject, or to a cell, tissue, or organ, "treatment" encompasses contacting an antibody of the invention with, for example, a dog or other animal subject, cell, tissue, physiological compartment, or physiological fluid.

[0028] As used herein, the term "dog" includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise specified.

[0029] As used herein, the term "cat" refers to any member of the Felidae family, including wild, zoo, and domestic members, including domestic cats, purebred and / or mixed breed pet cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

[0030] As used herein, the term "canine framework" refers to the amino acid sequences of the heavy and light chains of a canine antibody, excluding the hypervariable region residues defined herein as CDR residues. For caninized antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains with the corresponding foreign CDRs (e.g., from a mouse antibody). The heavy and / or light chains of the canine antibody may contain some foreign non-CDR residues to preserve the conformation of the foreign CDRs in the canine antibody and / or to modify Fc function, for example, as exemplified below and / or as disclosed in U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference.

[0031] The "fragment crystallizable region," abbreviated as "Fc," corresponds to the CH3-CH2 portion of an antibody that interacts with cell surface receptors called Fc receptors. 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 U.S. Patent No. 10,106,607].

[0032] As used herein, canine Fc (cFc) "IgG-Bm" refers to canine IgG-B Fc containing two amino acid residue substitutions, D31A and N63A (see below), in the amino acid sequence of SEQ ID NO: 10 of IgG-B, and lacking the 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 both replaced by alanine residues (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly attenuate the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B (see U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference). Additional amino acid substitutions in IgG-Bm, similar to those that can be made in IgG-B and which may include amino acid substitutions that promote heterodimer formation in bispecific antibodies, are also contemplated. The amino acid sequence of IgG-B, SEQ ID NO: 45, is as follows: [Table 1]

[0033] The amino acid sequence of IgG-Bm, SEQ ID NO: 10, is provided below. LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARG QAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0034] As used herein, "substitution of an amino acid residue" in the amino acid sequence of an antibody with another amino acid residue is equivalent to, for example, "replacing an amino acid residue" with another amino acid residue, and indicates that a 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., an alanine at a particular position in the amino acid sequence can be intentionally replaced with a serine, for example, by recombinant DNA technology. Alternatively, a particular amino acid residue or series of amino acid residues in an antibody can be replaced with one or more amino acid residues through a more natural selection process, for example, based on the ability of an antibody produced by a cell to bind to a given region on its antigen, e.g., a region containing an epitope or portion thereof, and / or such that the antibody contains a particular CDR that retains the same canonical structure as the CDR it replaces. Such substitutions / replacements can result in "variant" CDRs and / or mutant antibodies.

[0035] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Antibodies can be monomeric, dimeric, or larger multimeric. Thus, the term "antibody" 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 "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as caninizing an antibody for use as a canine therapeutic antibody.

[0036] As used herein, an antibody of the invention that "blocks" or "blocking" or "blocking binding" of, for example, a canine receptor to its binding partner (ligand) is an antibody that blocks (partially or completely) the binding of a canine receptor to its canine ligand, and vice versa, as determined in a standard binding assay (e.g., BIACore®, ELISA, or flow cytometry).

[0037] Typically, an antibody or antigen-binding fragment of the present invention retains at least 10% of its canine antigen-binding activity (compared to the parent antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the canine antigen-binding affinity of the parent antibody. It is also intended that the antibody or antigen-binding fragment of the present invention may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not substantially alter its biological activity.

[0038] "Isolated antibody" refers to a purified state, and in this context means that the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with experimental or therapeutic uses of the binding compounds described herein.

[0039] As used herein, a "chimeric antibody" is an antibody having variable domains derived from a first antibody and constant domains derived from a second antibody, wherein the first and second antibodies are derived from different species. [U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)]. Typically, the variable domains are derived from an antibody derived from an experimental animal such as a rodent (the "parent antibody"), and the constant domain sequences are derived from an animal subject antibody, e.g., human or canine, so that the resulting chimeric antibody is less likely to provoke an adverse immune response in a human or canine subject, respectively, than the parent (e.g., rodent) antibody.

[0040] As used herein, the term "caninized antibody" refers to a form of antibody that contains sequences from both canine and non-canine (e.g., murine) antibodies. Generally, caninized antibodies comprise at least one or more, typically substantially all of two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., including six CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining framework) are those of a canine immunoglobulin sequence. As exemplified herein, a caninized antibody comprises both three heavy chain CDRs and three light chain CDRs from a murine anti-canine antigen antibody, together with a canine frame or a modified canine frame. For example, the modified canine frame comprises one or more amino acid changes, as exemplified herein, that further optimize the effectiveness of the caninized antibody to increase binding of the caninized antibody to its canine antigen and / or its ability to block binding of the canine antigen to its natural binding partner.

[0041] The variable regions of each light / heavy chain pair form the antibody binding site. Therefore, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same. Typically, both the heavy and light chain variable domains contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned with the framework regions, enabling binding to specific epitopes. Generally, from N- to C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is generally based on the Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md., 5 th ed.,NIH Publ.No.91-3242(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)].

[0042] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. Hypervariable regions include amino acid residues from the "complementarity-determining regions" or "CDRs" (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), which defines antibody CDR regions by sequence; see also Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which defines antibody CDR regions by structure.) As used herein, the term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0043] There are four known IgG heavy chain subtypes of canine IgG, termed IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chain subtypes are termed λ and κ. In certain embodiments of the present invention, aside from binding and activating canine immune cells, a canine or caninized antibody of the present invention directed against its antigen optimally possesses the following two attributes: 1. Lack of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and 2. Easily purified on a large scale using industry standard techniques such as those based on Protein A chromatography.

[0044] None of the naturally occurring canine IgG isotypes meets 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 exhibits ADCC activity. Furthermore, neither IgG-C nor IgG-D can be purified on a Protein A column, yet IgG-D does not exhibit ADCC activity. (IgG-C has significant ADCC activity.) One way the present invention addresses these problems is by providing modified canine IgG-B antibodies of the present invention specific for the antigens of the present invention that lack effector functions such as ADCC and can be easily purified using industry-standard Protein A chromatography.

[0045] As used herein, an "anti-inflammatory antibody" is an antibody that can act as an anti-inflammatory agent in animals, including mammals such as humans, dogs, and / or cats, particularly with respect to atopic dermatitis. In certain embodiments, the anti-inflammatory antibody is an antibody that inhibits IL-4 or the receptor IL-4R. α Anti-inflammatory antibodies bind to specific proteins in the IL-4 / IL-13 signaling pathway, such as IL-4 or IL-4R. α ) is, for example, the binding of IL-4 to IL-4R α This inhibits binding to and disrupts and / or prevents signaling of this pathway, thereby disrupting or preventing the chronic inflammation associated with atopic dermatitis.

[0046] As used herein, "homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences when the two sequences are optimally aligned. If a position in both compared sequences is occupied by the same base or amino acid residue, for example, if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared x 100. For example, if 6 out of 10 positions in two sequences are identical or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Generally, comparison is performed when the two sequences are aligned to give the maximum percentage homology. 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.

[0047] As used herein, an amino acid sequence is 100% "identical" to a second amino acid sequence if the amino acid residues of both sequences are identical. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparison is performed over a contiguous block of amino acid residues contained by a given protein, e.g., a portion of the protein or polypeptide being compared. In certain embodiments, selected deletions or insertions that would otherwise alter the correspondence between the two amino acid sequences are taken into account. Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids share similar properties and may be interchangeable.

[0048] "Conservatively modified variants" or "conservative substitutions" refer to the substitution 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 changes can frequently be made without altering the biological activity of the protein. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.; 1987)). Furthermore, substitution of structurally or functionally similar amino acids is less likely to destroy biological activity. Exemplary conservative substitutions are shown in Table A immediately below.

[0049] [Table 2]

[0050] Function-conservative variants of the antibodies of the present invention are also contemplated by the present invention. As used herein, the term "function-conservative variant" refers to an antibody or fragment in which one or more amino acid residues have been altered without altering desired properties such as antigen affinity and / or specificity. Such variants include, but are not limited to, replacing an amino acid with an amino acid having similar properties, such as the conservative amino acid substitutions in Table A above.

[0051] An "isolated nucleic acid molecule" refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, wherein the isolated polynucleotide is unaccompanied by all or part of polynucleotides with which it is found in nature, or wherein the isolated nucleic acid molecule is linked to polynucleotides with which it is not naturally linked. In this disclosure, it is understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences for up to 10 or up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences.

[0052] The present invention provides isolated caninized antibodies of the present invention and methods for using the antibodies in the treatment of conditions, such as atopic dermatitis in dogs. In dogs, four IgG heavy chains exist, designated A, B, C, and D. These heavy chains represent four distinct 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 via an amino acid sequence called the "hinge" or "hinge region."

[0053] 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 for these heavy chains are also available from the GenBank database. For example, the amino acid sequence of the IgGA heavy chain has accession number AAL35301.1, the IgGB has accession number AAL35302.1, the IgGC has accession number AAL35303.1, and the IgGD has accession number AAL35304.1. Canine antibodies also contain 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 kappa light chain amino acid sequence has accession number ABY57289.1, and the lambda light chain has accession number ABY55569.1.

[0054] In the present invention, the amino acid sequences for each of the four canine IgG Fc fragments are based on the identified boundaries of the CH1 and CH2 domains determined by Tang et al., supra. α Caninized mouse anti-dog antibodies that bind to, but are not limited to, canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine κ or λ light chains can be used to bind to mouse anti-dog IL-4R. α The present invention also provides an antibody of the present invention comprising canine IL-4R along with its CDRs. α and binds to its natural binding partners, canine IL-4 and / or canine IL-13. α The present invention provides an isolated caninized mouse anti-dog antibody that blocks binding of

[0055] Accordingly, the invention further provides caninized murine antibodies and methods of using the antibodies of the invention in treating conditions, such as atopic dermatitis in dogs.

[0056] The present invention further provides full-length canine heavy chains that can be matched with corresponding light chains to create caninized antibodies. Accordingly, the present invention further provides caninized murine anti-canine antigen antibodies of the invention (including isolated caninized murine anti-canine antibodies), as well as methods of using the antibodies of the invention in treating conditions, such as atopic dermatitis in dogs.

[0057] The present invention also provides antibodies of the present invention comprising a canine fragment crystallizable region (cFc region), wherein the cFc has been genetically modified to enhance, reduce, or eliminate one or more effector functions. In one embodiment of the present invention, the genetically modified cFc reduces or eliminates one or more effector functions. In another embodiment of the present invention, the genetically modified cFc region 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 certain embodiments, the effector function is enhanced, reduced, or eliminated antibody-dependent cellular cytotoxicity (ADCC). In another embodiment, the effector function is enhanced, reduced, or eliminated complement-dependent cytotoxicity (CDC). In yet another embodiment, the cFc region has been genetically modified to enhance, reduce, or eliminate both ADCC and CDC.

[0058] To generate mutant forms of canine IgG lacking effector functions, a number of mutant canine IgGB heavy chains were generated. These mutant forms can contain 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 mutant heavy chains (i.e., containing such amino acid substitutions) were cloned into expression plasmids and transfected into HEK293 cells along with a plasmid containing a gene encoding the light chain. To evaluate their potential for mediating immune effector functions, the Fc γIntact antibodies expressed and purified from HEK293 cells were evaluated for binding to RI and C1q (see U.S. Pat. No. 10,106,607, the entire contents of which are incorporated herein by reference).

[0059] The present invention also provides a modified canine IgG-D that comprises, in place of its native IgG-D hinge region, a hinge region from: IgG-A: FNECRCTDTPPCPVPEP SEQ ID NO: 6 IgG-B:PKRENGRVPRPPDCPKCPAPEM SEQ ID NO:7; or IgG-C:AKECECKCNCNNCPCPGCGL SEQ ID NO:8.

[0060] Alternatively, the IgG-D hinge region can be genetically modified by substituting serine residues with proline residues, i.e., PKESTCKCI. P PCPVPES, SEQ ID NO: 9 (the proline residue (P) is underlined and shown in bold, replacing the naturally occurring serine residue). Such modifications can result in a canine IgG-D lacking Fab arm exchange. Modified canine IgG-D can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these mutants, a nucleic acid encoding the amino acid sequence of canine IgG-D can be modified to encode the modified IgG-D. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0061] The six complementarity determining regions (CDRs) of the caninized mouse anti-canine antibodies described herein can comprise a canine antibody kappa light chain comprising mouse light chains LCDR1, LCDR2, and LCDR3, and a canine antibody IgG heavy chain comprising mouse heavy chains HCDR1, HCDR2, and HCDR3.

[0062] nucleic acid The present invention further includes nucleic acids encoding the antibodies of the invention (see, eg, the Examples below).

[0063] Also included in the present invention are 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, excluding the unchanged CDRs, when the comparison is performed using a BLAST algorithm in which the algorithm parameters are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. The present invention further provides and is included in the present invention 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 the comparison is performed using a BLAST algorithm in which the algorithm parameters are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.

[0064] As used herein, the percent identity of nucleotide and amino acid sequences can be determined using the C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and Clustal W algorithms with default alignment and identity parameters. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, for example, Advanced Blast search can be performed under default filter conditions using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program with default parameters.

[0065] The following references relate to the BLAST algorithm, which is 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. Met al.,Comput.Appl.Biosci.10:67-70(1994);ALIGNMENT SCORING SYSTEMS:Dayhoff,MO,et al.,“A model of evolutionary change in proteins.”in Atlas of Protein Sequence and Structure, vol. 5, sup. Structure, vol. 5, suppl. 3.” (1978), MODayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, J. Mol. Biol. al.,Methods 3:66-70(1991);Henikoff,S.,et al.,Proc.Natl.Acad.Sci.USA 89:10915-10919(1992);Altschul,SF,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);および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)。.

[0066] The antibodies of the present invention can be recombinantly produced by methods known in the art. Mammalian cell lines available as hosts for expressing 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, among others, 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., HepG2), A549 cells, 3T3 cells, HEK-293 cells, and numerous other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, 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 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 antibody is 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.

[0067] The antibody can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibody of the present invention (or other moieties thereof) from the production cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, and 0323997, as well as European Patent Application No. 89303964.4.

[0068] Generally, glycoproteins produced in a particular cell line or transgenic animal have a glycosylation pattern characteristic of the glycoprotein produced in that cell line or transgenic animal. Thus, the specific glycosylation pattern of an antibody depends 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 present invention, regardless of the glycosylation pattern the antibody may have. Similarly, in certain embodiments, antibodies with a glycosylation pattern containing only nonfucosylated N-glycans may be advantageous, as these antibodies have typically been shown to exhibit stronger efficacy both 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).

[0069] Canine IL-4 receptor α receptor Through a search of the GenBank database (accession number XM_547077.4; see also U.S. Patent No. 7,208,579), a cDNA encoding a predicted full-length canine IL-4 receptor alpha chain (SEQ ID NO: 1) was identified. This predicted cDNA encodes 823 amino acids (SEQ ID NO: 2), including a 25-amino acid leader sequence, and is identified as accession number 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 number NP_000409.1) and 70% identity with the porcine IL-4 receptor alpha chain (accession number 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, designated SEQ ID NO: 5. This has all been previously described [U.S. Patent Application Publication No. 2018 / 0346580; incorporated herein in its entirety].

[0070] Canine IL-4 receptor α chain full-length DNA with signal sequence [SEQ ID NO: 1]

[0071] Canine IL-4 receptor alpha full-length protein with signal sequence in bold [SEQ ID NO: 2] MGRLCSGLTFPVSCLVLVWVASSGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWA QTYNSTWSDWSPSTTWLNYYEPWEQHLPLGVSISCLVILAICLSCYFSIIKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEHGLGREEESPKTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCPSLEGSGGSFQEGREGIVARLTESLFL DLLGGENGGFCPQGLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQPRRPESALQASPTQSAGSSAFPEPPPVVTDNPAYRSFGSFLGQSSDPDGDSDPELADRPGEADPGIPSAPQPPEPPAALQPEPESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSLLPGGATCPGT SGGEAGSGEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHLCGHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLEASLSPASLVPSGVSKEGKSSPFSQPASSSAQSSSQTPKKLAVLSTEPTCMSAS

[0072] Canine IL-4 receptor alpha mature full-length protein without signal sequence [SEQ ID NO: 4] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNY YEPWEQHLPLGVSISCLVILAICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEHGLGREEESPKTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCPSLEGSGGSFQEGREGIVARLTESLFLDLLGGENGGFCPQ GLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQPRRPESALQASPTQSAGSSAFPEPPPVVTDNPAYRSFGSFLGQSSDPDGDSDPELADRPGEADPGIPSAPQPPEPPAALQPEPESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSLLPGGATCPGTSGGEAG SGEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHLCGHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLEASLSPASLVPSGVSKEGKSSPFSQPASSSAQSSSQTPKKLAVLSTEPTCMSAS

[0073] Canine IL-4 receptor α mature full-length DNA without signal sequence [SEQ ID NO: 3]

[0074] Canine IL-4 receptor α chain extracellular domain [SEQ ID NO: 5] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLP

[0075] Antibody Protein Engineering By way of example, and not limitation, the canine heavy chain constant region can be derived from IgG-B or a modified cFc such as IgG-Bm as used herein (see U.S. Pat. No. 10,106,607, incorporated herein by reference in its entirety), and the canine light chain constant region can be derived from Kappa.

[0076] Antibodies can be engineered to contain modifications to the canine framework and / or canine frame residues within the variable domains of a parent (ie, murine) monoclonal antibody, for example, to improve the properties of the antibody.

[0077] Pharmaceutical Compositions and Administration To prepare pharmaceutical or sterile compositions containing the antibodies of the present invention, the antibodies can be mixed with a pharmaceutically acceptable carrier or excipient (see, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)).

[0078] Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing with acceptable carriers, excipients, or stabilizers in the form of 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: Tablets, Marcel Dekker, NY]. Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the antibody of the present invention is diluted to an appropriate concentration in sodium acetate solution, pH 5-6, with NaCl or sucrose added for isotonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

[0079] The toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined, for example, by the LD 50 (a dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of the population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is known as the therapeutic index (LD50 / ED 50 ) In certain aspects, antibodies that exhibit a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in dogs. The dosage of such compounds is preferably such that the ED 50 The dosage may vary within this range depending upon the dosage form used and the route of administration.

[0080] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial. In certain embodiments, the antibody of the present invention can be administered by an invasive route such as injection. In further embodiments of the present invention, the antibody of the present invention or a pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation or aerosol delivery. Administration by non-invasive routes (e.g., oral; e.g., pill, capsule, or tablet) is also within the scope of the present invention.

[0081] Composition can be administered using medical equipment known in the art.For example, pharmaceutical compositions of the present invention can be administered by injection with hypodermic needle, including for example pre-filled syringe or auto-injector.Pharmaceutical compositions disclosed herein can also be administered using needleless hypodermic injection device, such as the device disclosed in United States Patent No. 6,620,135; United States Patent No. 6,096,002; United States Patent No. 5,399,163; United States Patent No. 5,383,851; United States Patent No. 5,312,335; United States Patent No. 5,064,413; United States Patent No. 4,941,880; United States Patent No. 4,790,824 or United States Patent No. 4,596,556.

[0082] The pharmaceutical compositions disclosed herein can also be administered by infusion.The well-known implant and module forms for administering pharmaceutical compositions include: United States Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at controlled speed; United States Patent No. 4,447,233, which discloses an infusion pump for delivering drugs at precise infusion rates; United States Patent No. 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; United States Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments.Many other such implants, delivery systems and modules are well known to those skilled in the art.

[0083] Alternatively, antibodies of the invention can be administered locally rather than systemically, often in a depot or sustained-release formulation.

[0084] The dosage 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 target cells in the biological matrix.Preferably, the dosage regimen delivers enough therapeutic antibody to bring about improvement of the target disease / symptom state while simultaneously minimizing undesirable side effects.Therefore, the amount of biologic delivered depends in part on the specific therapeutic antibody and the severity of the condition being treated. Guidance on selecting appropriate doses of therapeutic antibodies is available [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 See 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)].

[0085] Determination of appropriate dosages will be made by a veterinarian, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, dosages will begin somewhat less than the optimum dose and then be increased by small increments until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include symptomatic diagnostic measures.

[0086] The antibodies provided herein can be provided by continuous infusion or by doses administered, e.g., daily, 1 to 7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, etc. Doses can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0.05 μg / kg body weight, more usually at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, or more [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)]. Dosages for achieving a predetermined target concentration of the antibody of the invention in the serum of a dog, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more, can also be provided. In other embodiments, the antibody of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg / subject weekly, biweekly, "every four weeks," monthly, bimonthly, or quarterly.

[0087] As used herein, "inhibiting" or "treating" or "treatment" includes delaying the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such a disorder. These terms further include ameliorating existing uncontrolled or undesirable symptoms, preventing further symptoms, and ameliorating or preventing the underlying cause of such symptoms. Thus, these terms refer to a beneficial result being imparted to a vertebrate subject (e.g., a dog) having a disorder, condition, and / or symptom, or having a potential for developing such a disorder, disease, or symptom.

[0088] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an antibody of the present invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, e.g., a dog, is effective in causing a measurable improvement in one or more symptoms of a disease or condition or the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of antibody sufficient to cause at least partial improvement of a symptom, e.g., treatment, cure, prevention, or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or amelioration of such a condition. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will produce an improvement in a diagnostic measure or parameter by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. An effective amount can also produce an improvement in a subjective measure when a subjective measure is used to assess the severity of the condition. [Example]

[0089] [Example 1] Anti-IL-4 receptor α antibody General Materials and Methods Recombinant proteins were obtained by providing the amino acid sequence of a selected protein to a commercial manufacturer (ATUM, Newark, California), which selected an appropriate nucleotide sequence encoding the amino acid sequence. Nucleotide sequences can also be 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 either HEK-293 cells or CHO cells to express the recombinant protein, which was then isolated by conventional methods.

[0090] Balb / c mice were immunized multiple times (10 μg each time) over a 17-day period. The immunizing antigen was a canine IL-4R α chain extracellular domain (ECD)-human Fc fusion protein. After immunization, serum was collected from each mouse and tested for reactivity with the canine IL-4 receptor α chain ECD HIS-tagged protein. Spleen cells from the mouse with the highest serum anti-IL-4 receptor α chain ECD titer were fused to the myeloma P3X63Ag8.653 cell line. Approximately two weeks after fusion, supernatants from putative hybridoma cells were tested for reactivity to the IL-4 receptor α chain ECD HIS-tagged protein by ELISA. Hybridomas that produced a strongly positive signal in ELISA were subcloned by limiting dilution and retested for reactivity to the canine IL-4 receptor α chain ECD HIS-tagged protein.

[0091] Anti-canine IL-4 receptor alpha antibodies include antibody c152H11VL3-cCLk-s / c152H11VH3-cIgG-Bm and antibody c146E2VL3-cCLk-s / c146E2VH3-cIgG-Bm. The sets of six CDRs (three individual light chain (LC) and three heavy chain (HC) sequences) for these two antibodies are shown in Tables 1A and 1B below. Table 1A provides nucleic acids encoding the amino acid sequences of the 12 CDRs listed in Table 1B. The amino acid sequences of the full-length light and heavy chains of these caninized antibodies are provided immediately following Table 1B below.

[0092] IL-4Rα antibody CDR nucleic acid and amino acid sequences [Table 3]

[0093] [Table 4]

[0094] c152H11VL3-cCLk-s (κ light chain): [SEQ ID NO: 35] EIVMTQSPASLSLSQEEKVTITC KASQNVGTNVA WYQQKPGQAPKLLIY SASYRYS GLPDRFSGSGSGTDFSFTISSLEPEDVAEFFC QQYNSYPYT FGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0095] c152H11VH1-cIgGBm (heavy chain): [SEQ ID NO: 36] EVQLVESGGDLVKPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAMYYCAK GTLNNRGFACWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0096] c152H11VH2-cIgGBm (heavy chain): [SEQ ID NO: 37] EVQLVESGGDLVKPGGSLRLSCAASGFTFS SYGMS WVRQAPDKRLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAMYYCAR GTLNNRGFAC WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0097] c152H11VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 38] EVQLVESGGDLVKPGGSLRLSCAASGFTFS SYGMS WVRQAPDKRLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAMYYCAR GTLNNRGFAS WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0098] c146E2VL3-cCLk-s (κ light chain): [SEQ ID NO: 39] DIVLTQTPLSLSVSPGETASIYC RASESVDSYGNSFLN WYQQKPGQPPKLLIY RASNLAS EIPDRFSGSGSRTEFTLKISRVEADDAGVYYC QQNYENPRT FGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0099] c146E2VH1-cIgGBm (heavy chain): [SEQ ID NO: 40] EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WVRQAPGAGLDWMG MIHPDSGNINYNERFKT RVTLTADTSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0100] c146E2VH2-cIgGBm (heavy chain): [SEQ ID NO: 41] EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WMKQAPGAGLDWIG MIHPDSGNINYNERFKT KATLTADTSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDY WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0101] c146E2VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 42] EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WMKQAPGAGLDWIG MIHPDSGNINYNERFKT KATLTVDKSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDY WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0102] Additionally, the light chain of the canine IL-4 receptor α antibody was also constructed with a λ light chain as provided below.

[0103] c152H11LV1-cCl (lambda light chain) [SEQ ID NO: 43] QSVLTQPASVSGSLGQRVTISC KASQNVGTNVA WYQQLPGTSPRTLIY SASYRYS GVPDRFSGSRSGSTATLTISGLQAEDEADYYC QQYNSYPYT FGGGTHLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS

[0104] c146E2LV1-cCl (lambda light chain) [SEQ ID NO: 44] QSVLTQPASVSGSLGQRVTISC RASESVDSYGNSFLN WYQQLPGKAPSLLIY RASNLAS GVPERFSGSKSGSSATLTITGLQAEDEADYYC QQNYENPRTFGGGTHLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS

[0105] [Example 2] STAT-6 inhibition Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STAT-6 phosphorylation in DH82 cells as follows: material 1. Actively growing DH82 cells 2. DH82 Cell Growth Media (ATCC® 302003™, Eagle's Minimum Essential Medium supplemented with heat-inactivated fetal bovine serum to a final concentration of 15% w / v) 3. AlphaLISA p-STAT6 (Tyr641) Assay Kit: Perkin Elmer Catalog: ALSU-PST6-A-HV 4. Recombinant canine IL-4: R&D Systems, Catalog: 752-CL / CF 5. Recombinant canine IL-13: R&D Systems, Catalog: 5894-CL / CF 6. Perkin Elmer Envision a. Caninized anti-canine IL-4R α Monoclonal antibodies b.c146E2-H3L3 c. 4H3 caninized antibody from U.S. Patent Application Publication No. 2018 / 0346580

[0106] Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STAT-6 phosphorylation in DH82 cells as follows: method 1. Two tissue culture plates, 8 x 10 cells per well 4 DH82 cells (4 × 10 5 Cells / mL) were seeded in 200 μL of cells and incubated overnight at 37 °C. 2. Test antibodies were pre-diluted to 500 μg / mL and then serially diluted 3-fold in DH82 Cell Growth Media. The medium was removed from the cell culture plates and 50 μL / well of the serially diluted test sample was transferred to each plate. 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 a second plate. The plates were incubated at 37°C for 15 minutes. 4. Remove the medium from the plate and add 100 μL / well of freshly prepared 1× lysis buffer from the AlphaLISA p-STAT-6 Assay Kit to the plate. The plate was agitated at 350 rpm on a plate shaker at room temperature for 10 minutes. 5. Prepare Acceptor Mix from the AlphaLISA p-STAT6 Assay Kit and add 15 μL / well to 30 μL of cell lysate in 96-well ½ area plates. The plates were sealed and agitated at 350 rpm for 2 minutes, then incubated at room temperature for 2 hours. 6. Under dimmed laboratory lighting, prepare the Donor Mix from the AlphaLISA p-STAT6 Assay kit and add 15 μL / well to each plate. The plates were sealed, covered with foil, and agitated at 350 rpm for 2 minutes, then incubated at room temperature for 2 hours. 7. Plates were read using the AlphaScreen setting on a Perkin Elmer EnVison.

[0107] Canine IL-4R α Two different caninized monoclonal anti-canine IL-4R antibodies, designated c4H3 [WO 2016 / 156588; U.S. Patent Application Publication No. 2018 / 0346580] and c146E2-H3L3, were tested for their ability to inhibit αSTAT-6 phosphorylation by blocking the binding of either canine IL-4 or canine IL-13 to STAT-6. αThe antibodies were evaluated. The data shown in Figure 1 demonstrate that both antibodies produced 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 [WO 2016 / 156588]. IL-4Rα (IL-4R α The IL-4 control in the absence of IL-13 antibody is shown in the upper right portion of the graph. The data shown in Figure 2 also demonstrate that both antibodies produced dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13, and that c146E2-H3L3 also binds more tightly than the prior art anti-canine IL-4 receptor α antibody c4H3. α ) An IL-13 control in the absence of antibody is shown in the upper right portion of the graph. Figure 3 shows that replacing the kappa light chain with a lambda light chain had no effect on the binding of c146E2-H3L3 to IL-4Rα.

[0108] [Example 3] Epitope mapping The interaction of an antibody with its cognate protein antigen is mediated through the binding of specific amino acids (paratope) of the antibody to specific amino acids (epitope) of the target antigen. An epitope is an antigenic determinant that elicits a specific response by immunoglobulins. An epitope consists of a group 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 epitopes or structural epitopes. Linear epitopes are formed by the contiguous sequence of amino acids in a protein, while structural epitopes are composed of amino acids that are discontinuous (e.g., widely separated) in the primary amino acid sequence but come together upon three-dimensional protein folding.

[0109] Epitope mapping refers to the process of identifying the amino acid sequence (i.e., epitope) recognized by an antibody on its target antigen. Identifying epitopes recognized by a monoclonal antibody (mAb) on a target antigen has important applications. For example, identifying epitopes recognized by a monoclonal antibody (mAb) on a target antigen can aid in the development of new therapeutics, diagnostics, and vaccines. Epitope mapping can also aid in the selection of optimized therapeutic mAbs and help elucidate the mechanism of action of optimized therapeutic mAbs. Epitope information on the IL-4 receptor α can also elucidate unique epitopes and define the protective or pathogenic effects of vaccines. Epitope identification can also lead to the development of subunit vaccines based on chemical or genetic coupling of identified peptide epitopes to carrier proteins or other immunostimulatory agents.

[0110] Epitope mapping can be performed using polyclonal or monoclonal antibodies, and several methods have been used for epitope identification, depending on the suspected nature of the epitope (i.e., linear versus structural). Mapping linear epitopes is more straightforward and relatively easy to perform. To this end, commercial services for linear epitope mapping often use peptide scanning. In this case, an overlapping set of short peptide sequences of the target protein is chemically synthesized and tested for their ability to bind to an antibody of interest. This strategy is rapid, high-throughput, and relatively inexpensive to implement. On the other hand, mapping discontinuous epitopes is technically more challenging and requires more specialized techniques, such as X-ray cocrystallography of a monoclonal antibody with its target protein, hydrogen-deuterium (H / D) exchange, mass spectrometry combined with enzymatic digestion, and several other methods known to those skilled in the art.

[0111] Mapping the Canine IL-4 Receptor Alpha Epitope Using Mass Spectrometry: A method based on chemical cross-linking, mass spectrometry detection, and covalent labeling was used to identify the epitope recognized by the anti-canine IL-4 receptor α mAb (CovalX Instruments Incorporated, 999 Broadway, Suite 305, Saugus, MA 01906-4510 USA). Application of this technology to epitope mapping of the canine IL-4 receptor α chain in a previous study demonstrated that the mAb recognizes a specific peptide epitope present within the extracellular domain of canine IL-4 receptor α [U.S. Patent Application Publication No. 2018 / 0346580]. Similar analysis performed on the c146E2-H3L3 antibody against canine IL-4 receptor α, shown in Figure 4, identified the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 47 for epitope(s) that share reasonable similarity with the previously identified epitope. Furthermore, as shown in Figure 4, amino acid residue K 97 , H 112 , T 113 , S 164 , T 165 , S 171 , Y 172 , S 173 and R 175 were identified as specific contact points [see, eg, SEQ ID NO: 5 for amino acid residue numbering].

[0112] [Table 5] TIFF0007727633000006.tif63158

Claims

1. An isolated mammalian antibody or antigen-binding fragment thereof that binds to canine interleukin-4 receptor alpha (IL-4Rα), comprising a heavy chain variable region (VH) and a light chain variable region (VL), 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: (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12; (ii) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 14; (iii) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 16; (iv) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 18; (v) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and (vi) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 22; An isolated mammalian antibody or antigen-binding fragment thereof.

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

3. 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, comprising 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, comprising a heavy chain comprising 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, comprising a heavy chain comprising 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, comprising a light chain comprising the amino acid sequence of SEQ ID NO:

39.

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

44. (a) a first nucleic acid encoding the VH 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 nucleic acid encoding the VL of the caninized antibody or antigen-binding fragment thereof according to any one of claims 3, 4, 5, 6, 7 and 8; A nucleic acid composition comprising:

10. An expression vector composition comprising the nucleic acid composition of claim 9, An expression vector composition, wherein the first nucleic acid is contained in a first expression vector and the second nucleic acid is contained in a second expression vector.

11. 10. An expression vector composition comprising the nucleic acid composition of claim 9, wherein the first nucleic acid and the second nucleic acid are contained in the same expression vector.

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

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

14. A method for assisting in blocking inflammation associated with atopic dermatitis in a canine subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 13 to a canine subject in need thereof.

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