Canine interleukin-4 receptor alpha antibody

Abstract

The present invention provides antibodies against canine IL-4 receptor alpha that have high binding affinity for 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 epitopes of IL-4 receptor alpha that bind to antibodies against canine IL4 receptor alpha. The present invention also provides the use of the antibodies for treating 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 / 015,220 filed on April 24, 2020, U.S. Provisional Patent Application Serial No. 63 / 015,209 filed on April 24, 2020, U.S. Provisional Patent Application Serial No. 62 / 951,778 filed on December 20, 2019, and U.S. Provisional Patent Application Serial No. 62 / 951,793 filed on 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 against canine IL-4 receptor α that have high binding affinity for 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 also relates to epitopes bound by antibodies against canine IL-4 receptor α. The present invention also relates to the use of the antibodies of the present invention for treating atopic dermatitis in dogs. Background Art

[0004] The immune system comprises a network of resident and recirculating specialized cells that work together to protect the host from infectious diseases and cancer. The ability of the immune system to perform this function depends largely on the biological activity 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 are 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 key to the development of T cell-dependent humoral immune responses. It is known that IL-4 binds to two receptors with high affinity, namely type I and type II IL-4 receptors. Type I IL-4 receptors are composed of IL-4 receptor α chain and common γ C chain. Type II IL-4 receptors are composed of IL-4 receptor α chain and IL13 receptor α1 chain. IL-13 binds to type II IL-4 receptors and binds to a unique receptor called IL-13 receptor α2. The combination of IL-13 and IL-13 receptor α2 does not transduce signals, and the receptor is also secreted in a soluble form. Therefore, IL-13 receptor α2 is commonly referred to as a decoy receptor. While IL-4, IL-31, and IL-22 are key cytokines in the development of immune responses necessary for protection against extracellular pathogens (e.g., tissue- or cavity-resident parasites), these cytokines have also been implicated in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

[0005] Atopic dermatitis (AD) is a recurrent, pruritic, and chronic inflammatory skin disease characterized by dysregulation of the human immune system and abnormalities in the epidermal barrier. The pathological and immunological properties of atopic dermatitis have been the subject of extensive research [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 T cells, including IL-4, IL-13, and IL-31. + Th2-polarizing cytokine milieu. Furthermore, IL-22 has been implicated in the excessive epithelial proliferation that leads to epidermal hyperplasia, a characteristic 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 α (IL31RA) 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)]. Thus, blocking the binding of IL-31 to its receptor IL31RA leads to relief of pruritus associated with atopic dermatitis.

[0007] IL-4R α ) and some of these antibodies have been extensively tested for their therapeutic effects in treating human atopic dermatitis [see, e.g., US2015 / 0017176 A1]. Recently, a method for blocking the interaction of canine IL-4 with canine IL-4R was also disclosed. α Combined with canine IL-4R α Since the type II IL-4 receptor is composed of the IL-4 receptor α chain and the IL-13 receptor α1 chain, a canine antibody against canine IL-4R has been obtained. α An antibody that blocks the binding of canine IL-4 and canine IL-13 to the type II canine IL-4 receptor is used to help block inflammation associated with atopic dermatitis [US2018 / 0346580A1].

[0008] Interleukin 22 (IL-22), also known as IL-10 related T cell derived inducing factor (IL-TIF), belongs to the IL-10 cytokine family. IL-22 is produced by normal T cells after anti-CD3 stimulation in the human body. After injection of lipopolysaccharide, mouse IL-22 expression is also induced in various organs, indicating that IL-22 may be involved in inflammatory response. IL-22 specifically binds to a receptor complex composed of a heterodimer complex of IL-10R2 (also known as IL-10Rβ) and interleukin 22 receptor (IL-22R), and signals are sent through the receptor complex [see Lee et al., Pharmacology Research & Perspectives, pp. 1-13 (2018: e00434)]. 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 lead to uncontrolled proliferation and enhanced 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 liver cancer cell lines. Antibodies to interleukin-22 and IL-22R act as antiproliferative agents by blocking the interaction of IL-22 with IL-22R and thereby blocking the associated signaling pathways leading to epithelial proliferation.

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

[0010] Citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" to the present application. Summary of the Invention

[0011] The present invention provides a method for targeting canine IL4Rα (IL4R αThe invention provides novel caninized antibodies, which in specific embodiments are isolated, that have superior properties to those of the prior art, such as tighter binding than prior art anti-canine IL-4 receptor alpha antibodies. In specific embodiments, the invention provides mammalian antibodies or antigen-binding fragments thereof that specifically bind to canine interleukin-4 receptor alpha, comprising a heavy chain comprising a set of three heavy chain complementarity determining regions (CDRs): heavy chain CDR 1 (HCDR1), heavy chain CDR 2 (HCDR2), and heavy chain CDR 3 (HCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, HCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 28. In other embodiments, the present invention provides a mammalian antibody or antigen-binding fragment thereof that specifically binds to canine interleukin-4 receptor alpha, comprising a heavy chain comprising a set of three heavy chain complementarity determining regions (CDRs): heavy chain CDR 1 (HCDR1), heavy chain CDR 2 (HCDR2), and heavy chain CDR 3 (HCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, HCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49.

[0012] In a related embodiment, the canine interleukin-4 receptor alpha (IL4R α ) further comprises a light chain comprising a set of three light chain CDRs: CDR light chain 1 (LCDR1), CDR light chain 2 (LCDR2), and CDR light chain 3 (LCDR3), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 30, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 34. In a preferred embodiment, the mammalian antibody or antigen-binding fragment thereof binds to canine IL4R α and block canine IL4R α Binding to canine interleukin-4 (cIL-4). In a related embodiment, the mammalian antibody or antigen-binding fragment thereof binds to canine IL-4R α and block canine IL-4R α Binding to canine interleukin-13 (cIL-13). In other embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine IL-4R α and block canine IL-4R α Binding to cIL4 and to cIL-13.

[0013] In specific 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 an IgG-D cFc, but the naturally occurring IgG1-D hinge region is 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 an IgG-D cFc, but the naturally occurring IgG1-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 7. In other embodiments, the caninized antibody comprises a heavy chain comprising an IgG-D cFc, but the naturally occurring IgG1-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 8. In other embodiments, the caninized antibody comprises a heavy chain comprising an IgG-D cFc, but the naturally occurring IgG1-D hinge region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO: 9.

[0014] In specific 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: 36. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 37. In other embodiments, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38. In specific embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 35. In alternative embodiments, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 43.

[0015] In a specific embodiment, the caninized antibody or antigen-binding fragment thereof binds to SEQ ID NO: 46. In a more specific embodiment, the caninized antibody or antigen-binding fragment thereof binds to canine IL-4R α One, two, three, four, five, six or all seven of the following amino acid residues: S of SEQ ID NO: 5 111 、H 112 、T 113 、T 119 、Y 122 、T 124 and H 127 In a related embodiment, the caninized antibody or antigen-binding fragment thereof binds to SEQ ID NO: 47. In a more specific embodiment, the caninized antibody or antigen-binding fragment thereof binds to canine IL-4R αOne, two, three, four, five, six, seven, eight, nine, ten or all eleven of the following amino acid residues: Y of SEQ ID NO: 5 150 、T 153 、Y 154 、T 158 、R 160 、S 164 、T 165 、S 168 、S 171 、Y 172 、S 173 In a more specific embodiment, the caninized antibody or antigen-binding fragment thereof binds to SEQ ID NO: 46 and SEQ ID NO: 47. In a more specific embodiment of this type, the caninized antibody or antigen-binding fragment thereof binds to canine IL-4R α One, two, three, four, five, six or all seven of the following amino acid residues: S of SEQ ID NO: 5 111 、H 112 、T 113 、T 119 、Y 122 、T 124 and H 127 and / or binding to canine IL-4R α One, two, three, four, five, six, seven, eight, nine, ten or all eleven of the following amino acid residues: Y of SEQ ID NO: 5 150 、T 153 、Y 154 、T 158 、R 160 、S 164 、T 165 、S 168 、S 171 、Y 172 、S 173 .

[0016] The present invention also provides nucleic acids encoding CDRs, heavy chains of caninized antibodies or antigen-binding fragments thereof, and / or light chains of caninized antibodies or antigen-binding fragments thereof, including isolated nucleic acids. Furthermore, the present invention provides expression vectors comprising such nucleic acids, and host cells comprising such expression vectors.

[0017] In addition, the present invention provides pharmaceutical compositions comprising the caninized antibodies and antigen-binding fragments thereof of the present invention and a pharmaceutically acceptable carrier and / or diluent. The present invention also provides methods for treating atopic dermatitis, comprising administering one of the above-described compositions to a canine suffering from atopic dermatitis. In specific embodiments, the present invention provides methods 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 canine in need thereof.

[0018] These and other aspects of the invention will be better understood with reference to the following description of the drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Is to show IL-4Rα (IL-4R α ) Antibodies inhibit IL-4-mediated STAT-6 phosphorylation. Two different caninized monoclonal anti-canine IL-4R α The ability of the antibodies, termed c4H3 [see, WO2016 / 156588] and c152H11-H3L3, to inhibit STAT-6 phosphorylation. The data showed that both antibodies resulted in a dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4. In the absence of IL-4Rα (IL-4R α ) antibody and the IL-4 control are shown in the upper right part of the figure.

[0020] Figure 2 Figure 2 is a graph showing the inhibition of IL-13-mediated STAT-6 phosphorylation by IL-4Rα antibodies. α The antibodies, designated c4H3 [see, WO2016 / 156588] and c152H11-H3L3, inhibited STAT-6 phosphorylation. The data showed that both antibodies resulted in a dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13. In the absence of IL-4Rα (IL-4R α ) antibody is shown in the upper right part of the figure.

[0021] Figure 3 Caninized anti-canine IL-4R containing λ or κ light chain assessed by ELISA α The results showed that the caninized anti-canine IL-4R containing the λ light chain α Antibodies (c152ClL1-H1, c152ClL1-H2, and c152ClL1-H3) to canine IL-4R α and a caninized anti-canine IL-4R containing the same CDRs but with a kappa light chain αAntibody (c152H11-H3L3) binds. 152mc is a mouse-canine chimeric antibody positive control, and Iso-Ctr (negative control) is an irrelevant caninized antibody.

[0022] Figure 4 Shown in canine IL-4R α The epitope of the c152H11-H3L3 antibody comprises the amino acid sequences SEQ ID NO: 46 and SEQ ID NO: 47. DETAILED DESCRIPTION

[0023] In response to the need for better treatments for atopic dermatitis, the present invention provides caninized antibodies, formulations having caninized antibodies, and methods that can achieve significant effects on skin inflammation associated with atopic dermatitis.

[0024] abbreviation

[0025] Throughout the detailed description and examples of the present invention, the following abbreviations will be used:

[0026] ADCC Antibody-dependent cellular cytotoxicity

[0027] CDC Complement-dependent cytotoxicity

[0028] CDR Complementarity Determining Regions in immunoglobulin variable regions, using the Kabat numbering system

[0029] General definition

[0030] EC50 Concentration resulting in 50% efficacy or binding

[0031] ELISA enzyme-linked immunosorbent assay

[0032] FR Antibody framework region: The immunoglobulin variable region excluding the CDR region

[0033] IC50 concentration that causes 50% inhibition

[0034] IgG immunoglobulin G

[0035] Kabat Sequences of immunoglobulins, a system of alignment and numbering pioneered by Elvin A. Kabat Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]

[0036] mAb Monoclonal Antibody (also called Mab or MAb)

[0037] V region A segment of the IgG chain whose sequence varies among different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain.

[0038] VH immunoglobulin heavy chain variable region

[0039] VL immunoglobulin light chain variable region

[0040] VK immunoglobulin kappa light chain variable region

[0041] definition

[0042] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0043] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0044] "Administering" and "treating," as applied to an animal (e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid), refers to contacting an exogenous drug, therapeutic, diagnostic agent, or composition with an animal (e.g., a canine subject, a cell, a tissue, an organ, or a biological fluid). Treatment of cells includes contacting an agent with cells, as well as contacting an agent with a fluid, wherein the fluid is contacted with cells.

[0045] "Administering" and "treating" also mean in vitro and ex vivo treatment, for example, by an agent, a diagnostic, a binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a canine, a feline, or a human) and most preferably a canine.

[0046] "Treatment" or "treatment" refers to the internal or external administration of a therapeutic agent (e.g., a composition containing any of the antibodies of the present invention) to a canine subject or patient, e.g., having one or more symptoms or suspected of having a condition for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate and / or improve one or more disease / condition symptoms in the subject or population being treated, whether by inducing regression of such symptoms or inhibiting the progression of such symptoms by any clinically measurable measure. The amount of a therapeutic agent that is effective to alleviate the symptoms of any particular disease / condition (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 the disease / condition symptoms have been alleviated or improved can be assessed by any clinical assessment commonly used by a veterinarian or other skilled healthcare provider to assess the severity or progression of the symptoms. Although an embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease / disorder in every subject, it should alleviate the symptoms of the target disease / disorder in a statistically significant number of subjects, as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0047] "Treatment," as applied to humans, veterinary (e.g., canines), or research subjects, refers to therapeutic treatment as well as research and diagnostic applications. "Treatment," as applied to humans, veterinary (e.g., canines), or research subjects, or cells, tissues, or organs, includes contacting an antibody of the invention with, for example, a canine or other animal subject, cell, tissue, physiological compartment, or physiological fluid.

[0048] As used herein, unless otherwise indicated, the term "canine" includes all domestic dogs, Canis lupus familiaris, or Canis familiaris.

[0049] As used herein, the term "feline" refers to any member of the family Felidae. Members of this family include wild, zoo, and domesticated members, including house cats, purebred and / or mongrels companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

[0050] As used herein, the term "canine framework" refers to the amino acid sequence of the heavy and light chains of a canine antibody, excluding the hypervariable region residues defined herein as CDR residues. With regard to caninized antibodies, in most embodiments, the amino acid sequence of the native canine CDR is replaced by the corresponding exogenous CDRs (e.g., CDRs from a mouse antibody) in both chains. Optionally, the heavy and / or light chains of the canine antibody may include some exogenous non-CDR residues, for example, to maintain the conformation of the exogenous CDRs in the canine antibody, and / or to alter Fc function, as exemplified below and / or disclosed in US Pat. No. 10,106,607B2, which is hereby incorporated herein by reference in its entirety.

[0051] The "Fragment crystallizable region" abbreviated as "Fc" corresponds to the region called Fc receptors cell surface receptors that interact with Antibody The CH3-CH2 portion of the canine fragment crystallizable region (cFc) of each of 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].

[0052] 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 without a 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 an alanine residue (A) in IgG-Bm. These two amino acid residue substitutions serve to significantly reduce the 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 herein by reference in their entirety]. Further amino acid substitutions of IgG-Bm are also contemplated, which are similar to those that can be made in IgG-B and may include amino acid substitutions that favor heterodimer formation in bispecific antibodies. The amino acid sequence of IgG-B, SEQ ID NO: 45, is:

[0053]

[0054] The amino acid sequence of IgG-Bm is provided below, SEQ ID NO: 10.

[0055] LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARG QAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

[0056] As used herein, for example, "replacing 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 represents that the specific amino acid residue at a specific position in the amino acid sequence has been replaced (or substituted) by a different amino acid residue. Such replacements can be specifically designed, i.e., purposefully replacing alanine with serine at a specific position in the amino acid sequence by, for example, recombinant DNA technology. Alternatively, the specific amino acid residue or amino acid residue string of an antibody can be replaced with one or more amino acid residues through a more natural selection process, such as the ability of a cell-based antibody to bind to a given region (e.g., a region containing an epitope or a portion thereof) on the antigen, and / or making the antibody contain a specific CDR that retains the same canonical structure as the CDR it replaces. Such replacements / substitutions can result in "variant" CDRs and / or variant antibodies.

[0057] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Antibodies can be monomers, dimers, or larger multimers. Therefore, 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 "parent antibody" is an antibody obtained by exposing the immune system to an antigen, which is then modified for its intended use, such as caninizing an antibody for use as a canine therapeutic antibody.

[0058] As used herein, as in a standard binding assay (e.g., An antibody of the invention that "blocks" or "prevents" or "blocks" the binding of, for example, a canine receptor to its binding partner (ligand), as determined by ELISA or flow cytometry, is one that (partially or completely) blocks the binding of a canine receptor to its canine ligand, or vice versa.

[0059] Typically, when canine antigen binding activity is expressed on a molar basis, the antibodies or antigen-binding fragments of the present invention retain at least 10% of their activity (when compared to the parent antibody). Preferably, the antibodies or antigen-binding fragments of the present invention retain 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 antibodies or antigen-binding fragments of the present invention may include conservative or non-conservative amino acid substitutions that do not significantly change their biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody).

[0060] An "isolated antibody" refers to a purified state, and in this case, refers to a molecule that is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other substances such as cell debris and growth medium. In general, the term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compounds described herein.

[0061] As used herein, a "chimeric antibody" is an antibody that has a variable domain from a first antibody and a constant domain 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 (parent antibody) from an experimental animal (e.g., a rodent), while the constant domain sequences are obtained from an animal subject antibody (e.g., human or canine), thereby producing a chimeric antibody that is less likely to elicit an adverse immune response in a human or canine subject, respectively, than the parent (e.g., rodent) antibody.

[0062] As used herein, the term "caninized antibody" refers to an antibody form comprising sequences from canine and non-canine (e.g., murine) antibodies. In general, a caninized antibody will comprise substantially all of at least one or more, typically two, variable domains (wherein all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., comprising the 6 CDRs 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 three heavy chain CDRs and three light chain CDRs from a murine anti-canine antigen antibody and a canine framework or a modified canine framework. The modified canine framework comprises 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 binding of the canine antigen to its natural binding partner.

[0063] The variable region pairing of each light / heavy chain pair forms an antibody binding site. Therefore, in general, a complete antibody has two binding sites. Except in bifunctional or bispecific antibodies, these two binding sites are usually the same. Typically, the variable domains of the heavy and light chains comprise three hypervariable regions, also referred to as complementary determining regions (CDRs), which are located within relatively conserved framework regions (FRs). CDRs are typically aligned by framework regions so as to be able to bind to specific epitopes. Typically, from N-terminal to C-terminal, the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Generally, the assignment of amino acids to each domain is according to the definitions in [Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; 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)].

[0064] As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from "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 Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which define the CDR regions of antibodies by sequence; see also Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which define the CDR regions of antibodies by structure]. As used herein, the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0065] There are four known IgG heavy chain subtypes of dog IgG, designated IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chain subtypes are designated λ and κ. In specific embodiments of the present invention, in addition to binding and activation of canine immune cells, the canine or caninized antibodies of the present invention directed against their antigens optimally possess two properties:

[0066] 1. Lack of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

[0067] 2. Easy to purify on a large scale using industry standard techniques such as those based on Protein A chromatography.

[0068] Naturally occurring canine IgG isotypes do not meet these two criteria. For example, IgG-B can be purified using Protein A, but has high levels of ADCC activity. On the other hand, IgG-A binds weakly to Protein A, but also exhibits ADCC activity. In addition, neither IgG-C nor IgG-D can be purified on a Protein A column, although IgG-D does not exhibit ADCC activity. (IgG-C exhibits considerable ADCC activity). One way the present invention addresses these issues is by providing modified canine IgG-B antibodies of the present invention that are specific for the antigens of the present invention, which lack effector functions such as ADCC and can be easily purified using industry standard Protein A chromatography.

[0069] 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, canines, and / or felines, particularly with respect to atopic dermatitis. In certain embodiments, the anti-inflammatory antibody binds to a specific protein in the IL-4 / IL-3 signaling pathway, such as IL-4 or the receptor IL-4R. α Anti-inflammatory antibodies and their corresponding antigens (e.g., IL-4 or IL-4R α ) inhibits the binding of IL-4 to IL 4R α and interfere with and / or block the signaling of this pathway, thereby interfering with or preventing the chronic inflammation associated with atopic dermatitis.

[0070] As used herein, "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences when they are optimally aligned. When one position in both of the two compared sequences is occupied by the same base or amino acid residue, for example, if one position in each of the two DNA molecules is occupied by adenine, then 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 × 100. For example, if 6 out of 10 positions of the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Typically, two sequences are compared when they are aligned to obtain the maximum percentage homology. Sequence identity refers to the extent to which the amino acids of the two polypeptides are identical at equivalent positions when the two sequences are optimally aligned.

[0071] 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 identical. Thus, an amino acid sequence is 50% "identical" to the second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparisons are performed over a continuous stretch of amino acid residues comprised by a given protein (e.g., a portion of the protein or polypeptide being compared). In specific embodiments, selected deletions or insertions that might 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 have similar properties and can be used interchangeably.

[0072] "Conservatively modified variants" or "conservative substitutions" refer to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), such that the change can generally be made without altering the biological activity of the protein. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do 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)]. In addition, substitutions of amino acids with similar structure or function are less likely to destroy biological activity. Exemplary conservative substitutions are listed in Table A immediately below.

[0073] Table A

[0074] Exemplary conservative amino acid substitutions

[0075] Original residue Conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr;Met;Leu Pro(P) Ala; Gly Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

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

[0077] An "isolated nucleic acid molecule" refers to a genomic DNA or RNA, mRNA, cDNA or synthetic source, or some combination thereof, that is not associated with all or part of a polynucleotide to which the isolated polynucleotide is found in nature, or is attached to a polynucleotide to which it is not naturally attached. For the purposes of this disclosure, it will be understood that a "nucleic acid molecule comprising (a specific nucleotide sequence)" does not include an entire chromosome. An isolated nucleic acid molecule that "comprising" a specified nucleic acid sequence may include, in addition to the specified sequence, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the nucleic acid sequence, and / or may include vector sequences.

[0078] The present invention provides caninized antibodies of the present invention that are isolated and used for treating diseases, such as methods for treating atopic dermatitis in dogs. In canines, there are four IgG heavy chains, referred to as A, B, C, and D. These heavy chains represent the four different subclasses of canine IgG, referred to as 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 a variable domain (VH) and three constant domains, referred to as CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain by an amino acid sequence referred to as 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 can also be obtained from the GenBank database. For example, the amino acid sequence of the IgGA heavy chain is accession number AAL35301.1, the accession number of IgGB is AAL35302.1, the accession number of IgGC is AAL35303.1, and the accession number of IgGD is (AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequences of these light chains can be obtained from the GenBank database. For example, the accession number of the kappa light chain amino acid sequence is ABY 57289.1, and the accession number of the lambda light chain is ABY 55569.1.

[0080] In the present invention, the amino acid sequence of each of the four canine IgG Fc fragments is based on the defined boundaries of the CH1 and CH2 domains determined by Tang et al., supra. Binding to canine IL-4R α Caninized mouse anti-dog antibodies include, but are not limited to, antibodies of the present invention comprising canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains and / or canine κ or λ light chains and mouse anti-canine IL-4R α Therefore, the present invention provides an isolated caninized murine anti-dog antibody of the present invention, which binds to canine IL-4R α Binds to and blocks canine IL-4R α Binding to its natural binding partners canine IL-4 and / or IL-13.

[0081] Thus, the present invention further provides caninized murine antibodies and methods of using the antibodies of the invention to treat disorders, for example, to treat atopic dermatitis in dogs.

[0082] The present invention further provides full-length canine heavy chains that can be paired with corresponding light chains to prepare caninized antibodies. Accordingly, the present invention further provides caninized murine anti-canine antigen antibodies of the present invention (including isolated caninized murine anti-canine antibodies), as well as methods of using the antibodies of the present invention in treating conditions, such as atopic dermatitis in dogs.

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

[0084] To generate variants of canine IgG lacking effector function, a number of mutant canine IgGB heavy chains were generated. These variants can include one or more of the following substitutions, either alone or in combination, 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 expression plasmids and transfected into HEK 293 cells along with a plasmid containing genes encoding the light chain. The intact antibodies expressed and purified from HEK 293 cells were evaluated for binding to FcγRI and C1q to assess their potential to mediate immune effector function. [See US 10,106,607 B2, the contents of which are incorporated herein by reference in their entirety.]

[0085] The present invention also provides modified canine IgG-D which, instead of its native IgG-D hinge region, comprises 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 serine residues with proline residues, i.e. SEQ ID NO: 9 (the proline residue (P) replacing the naturally occurring serine residue is underlined and in bold). 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 variants, a nucleic acid encoding the amino acid sequence of 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 may comprise a canine antibody κ 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 Acids

[0092] The invention also includes nucleic acids encoding the antibodies of the invention (see, eg, the Examples below).

[0093] The present invention 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 the caninized antibody provided herein, when compared by the BLAST algorithm, except for the unchanged CDRs, wherein the parameters of the algorithm are selected to give the maximum match of the corresponding sequence over the entire length of the corresponding reference sequence. The present invention 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 reference amino acid sequence when compared using the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match of the corresponding sequence over the entire length of the corresponding reference sequence, which are also included in the present invention.

[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 default parameters for alignment 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, an advanced Blast search under default filtering conditions can be used, for example, using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program with default parameters.

[0095] The following references are related to the BLAST algorithm commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., J. Mol. Biol. 215:403-410 (1990); Gish, W. et al., Nature Genet. 3:266-272 (1993); Madden, TL et al., Meth. Enzymol. 266:131-141 (1996); Altschul, SF et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J. et al., Genome Res. 7:649-656 (1997); Wootton, JC et al., Comput. Chem. 17: 149-163 (1993); Hancock, JM et 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 Sequenceand Structure, vol.5, suppl.3.MODayhoff (ed.), pp.345-352, (1978); Natl.Biomed.Res.Found., Washington, DC; Schwartz, RM et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3." (1978), MO Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, J. Mol. Biol. 219: 555-565 (1991); States, DJ et 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); and Altschul, SF "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] The antibodies of the present invention can be recombinantly produced by methods known in the art. Mammalian cell lines that can be used as hosts for expressing 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 hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, 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 heavy chains, or antigen-binding portions thereof, or fragments thereof, light chains and / or antigen-binding fragments thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibodies in the host cells, or more preferably, to allow secretion of the antibodies into the culture medium in which the host cells are grown.

[0097] The antibodies can be recovered from the culture medium using standard protein purification methods. In addition, many known techniques can be used to enhance expression of the antibodies of the invention (or other portions thereof) from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions. The GS system is discussed in whole or in part in conjunction with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.

[0098] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic of the glycoprotein produced in the cell line or transgenic animal. Thus, the specific glycosylation pattern of an antibody will depend on the specific 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 that the antibody may have. Similarly, in certain embodiments, antibodies having a glycosylation pattern that comprises only non-fucosylated N-glycans may be advantageous because these antibodies have been shown to generally exhibit greater 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] A cDNA encoding the predicted full-length canine IL-4 receptor α chain (SEQ ID NO: 1) was identified by searching the Genbank database (Accession No. XM_547077.4; see also US Pat. No. 7,208,579B2). This predicted cDNA encodes 823 amino acids (SEQ ID NO: 2), including a 25-amino acid leader sequence, and was identified as Accession No. XP_547077.3. The mature predicted canine IL-4 receptor α chain protein (SEQ ID NO: 4) shares 65% identity with the human IL-4 receptor α chain (Accession No. NP_000409.1) and 70% identity with the porcine IL-4 receptor α chain (Accession No. NP_999505.1). The mature predicted canine IL-4 receptor α chain protein is encoded by the nucleotide sequence identified as SEQ ID NO: 3. Comparison of the predicted mature IL-4 receptor α chain with the known sequence of the human IL-4 receptor α chain identified the extracellular domain (ECD) of the mature canine IL-4 receptor α chain protein and designated SEQ ID NO: 5. This has been previously described in its entirety in [US2018 / 0346580; the entire contents of which are incorporated herein by reference].

[0101] Canine IL-4 receptor α 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]

[0108] Canine IL-4 receptor alpha chain extracellular domain [SEQ ID NO: 5]

[0109]

[0110] Antibody protein engineering

[0111] The antibody may further comprise a light chain constant region, such as a canine light chain constant region, such as 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 may be from IgG-B or a modified cFc, such as IgG-Bm as used herein [see US 10,106,607 B2, incorporated herein by reference in its entirety], and the canine light chain constant region may be from kappa.

[0112] Antibodies can be engineered to include modifications to the canine framework and / or canine framework residues within the variable domain of the parent (i.e., mouse) monoclonal antibody, for example to improve the properties of the antibody. In particular, a single CDR can be modified, as exemplified below.

[0113] Pharmaceutical compositions and administration

[0114] To prepare pharmaceutical or sterile compositions comprising the antibodies of the invention, these antibodies can be mixed 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)].

[0115] Formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions [see, e.g., Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the antibodies of the invention are diluted to an appropriate concentration in sodium acetate solution at pH 5-6, and NaCl or sucrose is added to achieve tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to enhance stability.

[0116] Toxicity and therapeutic efficacy of the antibody composition, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., to determine LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 / ED 50 In certain aspects, antibodies that exhibit high therapeutic indices are desired. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range for canines. The dosage of such compounds is preferably within the range that includes the ED with little or no toxicity. 50The dosage may vary within this range depending upon the dosage form employed and the route of administration employed.

[0117] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, directly intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transcutaneous or intraarterial. In specific embodiments, the antibodies of the present invention can be administered by invasive routes, such as by injection. In further embodiments of the present invention, the antibodies of the present invention or their pharmaceutical compositions are administered intravenously, subcutaneously, intramuscularly, intraarterially or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., oral; e.g., in pills, capsules or tablets) is also within the scope of the present invention.

[0118] The compositions can be administered using medical devices known in the art. For example, the pharmaceutical compositions of the present invention can be administered by injection using a hypodermic needle (including, for example, a prefilled syringe or an autoinjector). The pharmaceutical compositions disclosed herein can also be administered using a needle-free hypodermic injection device; 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.

[0119] The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0120] Alternatively, antibodies of the invention may be administered in a local rather than systemic manner, typically in a depot or sustained-release formulation.

[0121] 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 target cells in the biological matrix. Preferably, the administration regimen delivers enough therapeutic antibody to improve the target disease / condition state while minimizing adverse 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 is provided for selecting appropriate doses of therapeutic antibodies [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. 342:1966-1973 (1999); Slamon et al. New Engl. J. Med. 343:1974-1983 (1999); and 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)].

[0122] Determination of the appropriate dosage is performed by a veterinarian, for example, using parameters or factors known in the art or suspected to affect treatment. Typically, the dosage is started at an amount slightly lower than the optimal dose and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measurements include those of symptoms.

[0123] The antibodies provided herein can be provided by continuous infusion or by dosages administered, for example, daily, 1-7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, annually, etc. Dosages can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is typically at least 0.05 μg / kg body weight, more typically 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 / 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)]. Dosages can also be provided to achieve a predetermined target concentration of an antibody of the invention in the serum of the dog, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. 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 4 weeks," monthly, bimonthly, or quarterly.

[0124] As used herein, "inhibit" or "treat" or "treating" includes delaying the onset of symptoms associated with a disorder and / or reducing the severity of the symptoms of such disorder. The terms also include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of these symptoms. These terms also include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of these symptoms. Thus, these terms indicate that a beneficial result is produced in a vertebrate subject (e.g., a canine) having a disease, condition, and / or symptom, or at risk of developing such a disease, condition, or symptom.

[0125] 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 is effective to cause a measurable improvement in one or more symptoms of a disease or condition, or the progression of such a disease or condition, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject (e.g., a canine). A therapeutically effective dose further refers to an amount of an antibody sufficient to result in at least partial symptom improvement, such as treatment, cure, prevention, or amelioration of a related medical condition, or to increase the rate of treatment, cure, prevention, or improvement 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, serially, or simultaneously. An effective amount of a therapeutic agent will produce an improvement in a diagnostic measure or parameter of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%. In cases where subjective indicators are used to assess the severity of the condition, an effective amount may also produce an improvement in the subjective indicator.

[0126] Example

[0127] Example 1

[0128] Anti-IL-4 receptor alpha antibody

[0129] General Materials and Methods:

[0130] Recombinant proteins are obtained by providing the amino acid sequence of a selected protein to a commercial manufacturer (ATUM, Newark, California), which in turn selects the appropriate nucleotide sequence encoding that amino acid sequence. Nucleotide sequences can also be obtained from publicly available DNA databases, such as The nucleic acid is then chemically synthesized by a commercial manufacturer and then cloned into an expression plasmid (pD2610-v10; available from AUTM) by ATUM to produce the corresponding recombinant protein. The plasmid is placed in HEK-293 cells or CHO cells to express the recombinant protein, which is then isolated by conventional methods.

[0131] Balb / c mice were immunized multiple times over a 17-day period (10 μg each time). The immunizing antigen was a canine IL-4Rα 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 α chain ECD HIS-labeled protein. Mouse splenocytes with the highest serum anti-IL-4 receptor α chain ECD titer were fused with the myeloma P3X63Ag8.653 cell line. Approximately 2 weeks after fusion, supernatants from putative hybridoma cells were tested for reactivity to IL-4 receptor α chain ECD HIS-labeled protein by ELISA. Hybridomas that produced strong positive signals in the ELISA were subcloned by limiting dilution and retested for reactivity with canine IL-4 receptor α chain ECD HIS-labeled protein. Anti-canine IL-4 receptor alpha antibodies include antibody c152H11VL3-cCLk-s / c152H11VH3-cIgGBm and antibody c146E2VL3-cCLk-s / c146E2VH3-cIgGBm.

[0132] The sets of six (6) CDRs (three individual light chain (LC) and three heavy chain (HC) sequences) for these two antibody families are provided below in Table 1A (nucleic acid sequence) and Table 1B (amino acid sequence). Table 1B includes the modified murine HCDR3 of 152H11, which comprises the amino acid sequence of SEQ ID NO:28, while the remaining are unmodified murine CDRs. Table 1A provides the nucleic acids encoding the 12 CDRs listed in Table 1B. Table 1C provides the amino acid sequence of the unmodified murine HCDR3 of 152H11 (SEQ ID NO:49) and the nucleic acid sequence encoding this unmodified murine HCDR3 (SEQ ID NO:48). The amino acid sequence of the modified murine HCDR3 of the 152H11 antibody family (SEQ ID NO:28) differs from the amino acid sequence of the corresponding unmodified murine HCDR3 in that the C-terminal cysteine ​​residue of the amino acid sequence of SEQ ID NO:49 is replaced by a serine residue. The unmodified murine HCDR3 (SEQ ID NO: 49) of the 152H11 antibody family is found in c152H11VH1-cIgGBm (SEQ ID NO: 36) and c152H11VH2-cIgGBm (SEQ ID NO: 37), while c152H11VH3-cIgGBm (SEQ ID NO: 38) contains a modified murine HCDR3 (SEQ ID NO: 28). The amino acid sequences of the full-length light and heavy chains of the caninized antibodies are provided immediately below in Table 1C.

[0133] IL-4Rα antibody CDR nucleic acid and amino acid sequences

[0134] Table 1A

[0135]

[0136]

[0137] #See footnote to Table 1B below.

[0138] Table 1B

[0139]

[0140] #As described above, the C-terminal cysteine ​​residue of HCDR3 of 152H11 was replaced by a serine residue.

[0141]

[0142] c152H11VL3-cCLk-s (κ light chain): [SEQ ID NO: 35]

[0143]

[0144] c152H11VH1-cIgGBm (heavy chain): [SEQ ID NO: 36]

[0145]

[0146] c152H11VH2-cIgGBm (heavy chain): [SEQ ID NO: 37]

[0147]

[0148] c152H11VH3-cIgGBm (heavy chain): [SEQ ID NO: 38]

[0149]

[0150] c146E2VL3-cCLk-s (kappa light chain): [SEQ ID NO: 39]

[0151]

[0152] c146E2VH1-cIgGBm (heavy chain): [SEQ ID NO: 40]

[0153]

[0154] c146E2VH2-cIgGBm (heavy chain): [SEQ ID NO: 41]

[0155]

[0156] c146E2VH3-cIgGBm (heavy chain): [SEQ ID NO: 42]

[0157]

[0158] In addition, the light chain of the canine IL-4 receptor alpha antibody was also constructed using the lambda light chain provided below.

[0159] c152H11LV1-cCl (λ light chain) [SEQ ID NO: 43]

[0160]

[0161] c146E2LV1-cCl (λ light chain) [SEQ ID NO: 44]

[0162]

[0163] Example 2

[0164] STAT-6 inhibition

[0165] The ability of antibodies directed against canine IL-4 receptor alpha to inhibit STAT-6 phosphorylation in DH82 cells was tested as follows:

[0166] Material

[0167] 1. Actively growing DH82 cells

[0168] 2. DH82 cell growth medium ( 302003 TM , Eagle's Minimum Essential Medium, providing heat-inactivated fetal bovine serum to a final concentration of 15% w / v)

[0169] 3. AlphaLISA p-STAT6 (Tyr641) detection kit: Perkin Elmer catalog number: ALSU-PST6-A-HV

[0170] 4. Recombinant canine IL-4: R&D Systems, catalog number: 752-CL / CF

[0171] 5. Recombinant canine IL-13: R&D Systems, catalog number: 5894-CL / CF

[0172] 6.Perkin Elmer Envision

[0173] a. Canine anti-canine IL-4R α Monoclonal antibodies

[0174] b.c152H11-H3L3

[0175] c. 4H3 caninized antibody from US2018 / 0346580

[0176] Antibodies against canine IL-4 receptor alpha were tested for their ability to inhibit STAT-6 phosphorylation in DH82 cells as follows:

[0177] method

[0178] 1. Inoculate 8x10 cells per well of two tissue culture plates. 4 DH82 cells (200 μL, density of 4 × 10 5 cells / mL) and incubated at 37°C overnight.

[0179] 2. Pre-dilute the test antibody to 500 μg / mL and then serially dilute 3-fold in DH82 cell growth medium. Remove the medium from the cell culture plate and transfer 50 μL / well of the serially diluted test sample to each plate.

[0180] 3. Dilute canine IL-4 to 5 ng / mL in DH82 cell growth medium and add 50 μL to each well of one plate. Dilute canine IL-13 to 10 ng / mL in DH82 cell growth medium and add 50 μL to each well of a second plate. Incubate the plates at 37°C for 15 minutes.

[0181] 4. Remove the culture medium from the plate and add 100 μL of freshly prepared 1× lysis buffer from the AlphaLISA p-STAT-6 detection kit to each well of the plate. Agitate the plate at 350 rpm on a plate shaker at room temperature for 10 minutes.

[0182] 5. Prepare the Acceptor Mix using the AlphaLISA p-STAT6 Detection Kit and add 15 μL per well to 30 μL of cell lysate in 96-well 1 / 2 Area Plates. Seal the plate, agitate at 350 rpm for 2 minutes, and then incubate at room temperature for 2 hours.

[0183] 6. Prepare Donor Mix using the AlphaLISA p-STAT6 Detection Kit under subdued laboratory lighting and add 15 μL per well to each plate. Seal the plate, cover with foil, stir at 350 rpm for 2 minutes, and then incubate at room temperature for 2 hours.

[0184] 7. Set up the reading plate using the AlphaScreen on a Perkin Elmer EnVision.

[0185] Two different caninized anti-canine IL-4R antibodies, designated c4H3 [WO2016 / 156588; US2018 / 0346580] and c152H11-H3L3, were evaluated. α Monoclonal antibodies block the interaction of canine IL-4 or canine IL-13 with canine IL-4R α The ability of αSTAT-6 phosphorylation to be inhibited by binding. Figure 1 The data presented in WO2016 / 156588 show that both antibodies resulted in dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4, but surprisingly, c152H11-H3L3 bound more tightly than the prior art anti-canine IL-4 receptor α antibody c4H3 [WO2016 / 156588]. α ) antibody and the IL-4 control are shown in the upper right part of the figure. Figure 2 The data presented in also show that in the presence of IL-13, both antibodies resulted in a dose-dependent inhibition of STAT-6 phosphorylation, with c152H11-H3L3 again binding more tightly than the prior art anti-canine IL-4 receptor α antibody c4H3. α ) antibody is shown in the upper right part of the figure. Figure 3 The results showed that replacing the kappa light chain with the lambda light chain had an important effect on the binding of c152H11-H3L3 to IL-4R. α The combination has no effect.

[0186] Example 3

[0187] Epitope mapping

[0188] The interaction between an antibody and its cognate protein antigen is mediated by 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 from an immunoglobulin. An epitope consists of a group of amino acids on the surface of an antigen. A protein of interest may contain several epitopes that are recognized by different antibodies. Epitopes recognized by antibodies are classified as linear or conformational epitopes. Linear epitopes are formed by stretches of continuous sequences of amino acids in a protein, while conformational epitopes are composed of amino acids that are discontinuous (e.g., far apart) in the primary amino acid sequence but come together when the protein folds in three dimensions.

[0189] Epitope mapping refers to the process of identifying the amino acid sequence (i.e., epitope) recognized by an antibody on its target antigen. Identifying the epitope recognized by a monoclonal antibody (mAb) on a target antigen has important applications. For example, it can help develop new therapeutic agents, diagnostic agents, and vaccines. Epitope mapping can also help select optimized therapeutic mAbs and help clarify their mechanism of action. Epitope information about IL-4 receptor α can also clarify 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 the identified peptide epitopes to carrier proteins or other immunostimulants.

[0190] Epitope mapping can be carried out using polyclonal or monoclonal antibodies, and according to the suspected nature of the epitope (i.e. linear vs conformation), several methods are used to identify the epitope. Linear epitope mapping is more direct and relatively easier to carry out. For this reason, commercial services for linear epitope mapping generally use peptide scanning. In this case, a group of overlapping short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind the antibody of interest. This strategy is fast, high throughput, and relatively cheap to perform. On the other hand, the mapping of discontinuous epitopes is technically more challenging and requires more specialized techniques, such as X-ray co-crystallography of monoclonal antibodies and their target proteins, hydrogen-deuterium (H / D) exchange, mass spectrometry coupled with enzyme digestion, and several other methods well known to those skilled in the art.

[0191] Mapping of the Canine IL-4 Receptor Alpha Epitope Using Mass Spectrometry:

[0192] A method based on chemical cross-linking, mass spectrometric detection, and covalent labeling for identifying the epitope recognized by anti-canine IL-4 receptor alpha mAb [CovalX Instruments Incorporated, 999 Broadway, Suite 305, Saugus, MA 01906-4510 USA].

[0193] In a previous study, this technique was applied to epitope mapping of the canine IL-4 receptor α chain and demonstrated that the mAb recognized a specific peptide epitope present within the extracellular domain of canine IL-4 receptor α [US2018 / 0346580]. Figure 4 Similar analysis performed with the c152H11-H3L3 antibody against canine IL-4Rα identified amino acid sequences SEQ ID NO: 46 and SEQ ID NO: 47 as epitopes with reasonable similarity to previously identified epitopes. Figure 4 As shown, amino acid residue S 111 、H 112 、T 113 、T 119 、Y 122 、T 124 、H127 、Y 150 、T 153 、Y 154 、T 158 、R 160 、S 164 、T 165 、S 168 、S 171 、Y 172 and S 173 Specific contact points were identified [see, e.g., SEQ ID NO: 5 for amino acid residue numbers].

[0194] Sequence Listing Table

[0195]

[0196]

[0197] # The C-terminal cysteine ​​residue of HCDR3 of 152H11 was replaced by a serine residue.

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 comprises a heavy chain comprising a heavy chain variable region (VH) and a light chain comprising a light chain variable region (VL), wherein the VH comprises three complementarity determining regions (CDRs): heavy chain CDR 1 (HCDR1), heavy chain CDR 2 (HCDR2), and heavy chain CDR 3 (HCDR3), and wherein the VL comprises three CDRs: light chain CDR 1 (LCDR1), light chain CDR 2 (LCDR2), and light chain CDR 3 (LCDR3); (i) the HCDR1 consists of the amino acid sequence of SEQ ID NO: 24; (ii) the HCDR2 consists of the amino acid sequence of SEQ ID NO: 26; (iii) the HCDR3 consists of the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence of SEQ ID NO: 49; (iv) the LCDR1 consists of the amino acid sequence of SEQ ID NO: 30; (v) the LCDR2 consists of the amino acid sequence of SEQ ID NO: 32; and (vi) The LCDR3 consists of the amino acid sequence of SEQ ID NO:

34.

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-4R α and block canine IL-4R α Binding to canine interleukin-4.

3. The isolated mammalian antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a caninized antibody or a caninized antigen-binding fragment thereof. 4 . The caninized antibody or antigen-binding fragment thereof according to 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. The caninized antibody or antigen-binding fragment thereof according to claim 3 , comprising a heavy chain containing 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 according to claim 5 , comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO:

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

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

43.

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

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

12. A host cell comprising the expression vector according to claim 10 or the expression vector system according to claim 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. Use of the pharmaceutical composition of claim 13 in the preparation of a medicament for helping to block inflammation associated with atopic dermatitis.

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