Canine interleukin-4 receptor α antibodies

Abstract

To provide an improved therapy capable of addressing one or more symptoms of atopic dermatitis in dogs.SOLUTION: The present invention provides an antibody to canine IL-4 receptor α that has a 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 further relates to epitopes of IL-4 receptor α that bind to the antibody to canine IL-4 receptor α. The present invention further provides the use of the antibody for the treatment of atopic dermatitis in dogs.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed under 35 U.S.C. § 119(e) on April 24, 2020. U.S. Provisional Patent Application No. 63 / 015,220, filed April 24, 2020, U.S. Patent Application No. 63 / 015,209, filed December 20, 2019, U.S. Patent Application No. 6 No. 2 / 951,778, and U.S. patent application Ser. No. 62, filed December 20, 2019. / 951,793, the entire contents of which are incorporated herein by reference in their entirety. Incorporated into the specification.

[0002] The present invention provides a method for producing a canine IL-4 receptor α-binding protein having high binding affinity to the canine IL-4 receptor α. Canine IL-4 and / or IL-13 binding to IL-α can be blocked. The present invention further relates to an antibody against canine IL-4 receptor alpha. The present invention also relates to epitopes that bind to the α-glucan derivative of the present invention for the treatment of atopic dermatitis in dogs. The present invention relates to the use of the antibodies of the present invention in [Background technology]

[0003] The immune system is a resident defense mechanism that works in concert to protect the host from infectious diseases and cancer. The immune system's ability to perform this function includes a network of specialized cells that transport and recirculate. The immune system is secreted by white blood cells and is a member of a group of proteins collectively known as interleukins. The biological activity of interleukins is highly dependent on their activity. Interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin- four identified as interleukin-31 (IL-31) and interleukin-22 (IL-22) IL-4 and IL-13 are important molecules in the + Th2 cells, natural killer cells Many cell types, including NKT cells, macrophages, mast cells, and basophils IL-4 and IL-13 are closely related proteins that can be secreted by multiple They exhibit many overlapping functions and are crucial for the generation of T cell-dependent humoral immune responses. IL-4 binds with high affinity to two receptors, type I and type II IL-4 receptors. The type I IL-4 receptor is known to bind to the IL-4 receptor α chain and the common γ chain. The type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α chain. IL-13 binds to the type II IL-4 receptor and binds to the IL-13 receptor α2. IL-13 binds to a specific receptor called IL-13 receptor α2. This receptor also transmits IL-13 in a soluble form. Receptor α2 has often been called a decoy receptor. and IL-31 act against extracellular pathogens (e.g., parasites present in tissues or lumina). It is a key cytokine for generating the immune response required for protection against These cytokines are involved in the pathogenesis of allergies in humans and animals, including atopic dermatitis. It is also involved in the pathogenesis of sexually transmitted diseases.

[0004] Atopic dermatitis (AD) is a recurrent, pruritic, and chronic inflammatory skin disease affecting humans. Atopic dermatitis is characterized by immune system dysregulation and epidermal barrier abnormalities in the skin. The physiological and immunological characteristics have been the subject of extensive investigation [Rahman et al., nflammation&allergy-drug target 10:486-4 96 (2011) and Harskamp et al., Seminar in Cu Taneous Medicine and Surgery 32:132-139( Atopic dermatitis is also a common skin condition in pets, especially dogs. It is a common condition affecting the canine population, with an estimated prevalence of approximately 10-15%. Pathogenesis of atopic dermatitis in dogs and cats [Nuttall et al. ,Veterinary Records 172(8):201-207(2013) [Reviewed in ]. The study investigated the skin infiltration by various immune cells and the expression of IL-4, IL-13, and and CD4, including IL-31 dominance. + Th2-polarized cytokine environment The pathogenesis of atopic dermatitis in humans is remarkably similar to that of IL-1. -22 is involved in the excessive epidermal proliferation that leads to the thickening of the epidermis characteristic of atopic dermatitis. There are.

[0005] For example, antibodies against canine IL-31 have been shown to reduce scratching associated with atopic dermatitis in dogs. It has been shown to have a significant effect on pruritus [U.S. Patent No. 8,790,651 ; U.S. Patent No. 10,093,731]. Furthermore, human IL-31 receptor alpha (IL-31 Antibodies against rheumatoid arthritis (RA) have been tested for the pruritus associated with atopic dermatitis in humans. It was found to have a significant effect [Ruzicka, et al., New Eng land Journal of Medicine,376(9),826-835( 2017)], thus blocking IL-31 from binding to its receptor, IL-31RA. This reduces the itching associated with atopic dermatitis.

[0006] Human IL-4 receptor α (IL-4R α ) was developed. Some of these antibodies have been used to treat atopic dermatitis in humans. have been widely tested for their therapeutic efficacy [see, e.g., U.S. Patent Application Publication No. 2015 More recently, canine IL-4R has been reported to be a soluble form of canine IL-4. α Blocks binding to canine IL-4R α A caninized antibody against the No. 2018 / 0346580, the entire contents of which are incorporated herein by reference. The type II IL-4 receptor consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain. Therefore, both canine IL-4 and canine IL-13 bind to the type II canine IL-4 receptor. Canine IL-4R can be blocked α Antibodies against Helps prevent inflammation associated with atopic dermatitis [U.S. Patent Application Publication No. 2018 / No. 0346580].

[0007] Interleukin-10, also known as IL-10-related T cell-derived inducible factor (IL-TIF), Interleukin-22 (IL-22) belongs to the IL-10 cytokine family. , which is produced by normal T cells upon anti-CD3 stimulation in humans. It was also induced in various organs upon lipopolysaccharide injection, and IL-22 was involved in the inflammatory response. These results suggest that IL-22 may be involved in the regulation of IL-10R2 (also known as IL-10Rβ). It consists of a heterodimeric complex of the interleukin-22 receptor (IL-22R) and the IL-22 receptor (IL-22R). It specifically binds to the receptor complex and transmits signals through the receptor complex [Lee et al. t al.,Pharmacology Research&Perspectives ,Pages 1-13(2018:e00434)]. Interleukin-22 The receptors are interleukin-22R, α1; IL-22RA1; IL-22R1; zc ytor11; and CRF2-9 [Xu et al., Pro c.Nat.Acad.Sci.98(17)9511-9516(2001);Gel ebart and Lai, Atlas of Genetics and Cyto genetics 14(12):1106-1110(2010)]. IL-22 is It induces epithelial cell proliferation during wound healing, and its deficiency allows uncontrolled proliferation and tumorigenesis. Huber et al., Nature 491:259-263(20 12]. IL-22 inhibits STAT-1 and STAT-2 expression in several hepatoma cell lines. It has been shown to activate IL-3 and upregulate the production of acute phase proteins. Antibodies against IL-22 and IL-22R were used to investigate the interaction of IL-22 with IL-22R. by blocking the action of ATP and related signaling pathways that lead to epithelial proliferation. It therefore acts as an antiproliferative agent.

[0008] However, despite recent success in treating atopic dermatitis, All of the current treatments available have only a limited effect on skin inflammation while also improving the skin barrier function. It does not provide significant efficacy and rapid onset of antipruritic action. There is a need to design better treatments that can address one or more of the symptoms of inflammatory dermatitis. do.

[0009] The citation of any reference herein does not imply that such reference is a "subject matter" to this application. This disclosure should not be construed as an admission that the technology is available as "previous art." [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] The present invention is isolated in certain embodiments and has properties, e.g., superior to those in the prior art. For example, canine IL-4Rα antibodies having stronger binding than prior art anti-canine IL-4 receptor α antibodies (IL-4R α In certain embodiments, the present invention provides novel caninized antibodies against a mammalian antibody or antibody thereof that specifically binds to canine interleukin-4 receptor alpha; An antigen-binding fragment comprising a set of three heavy chain complementarity-determining regions (CDRs), CDR heavy 1 (H heavy chain containing CDR1, CDR2 (HCDR2) and CDR3 (HCDR3) HCDR1 comprises the amino acid sequence of SEQ ID NO:24, and HCDR2 comprises the amino acid sequence of SEQ ID NO:26. a mammalian antibody comprising the amino acid sequence of SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 28; or In another aspect, the present invention provides an antigen-binding fragment thereof. A mammalian antibody or antigen-binding fragment thereof that binds to leukin-4 receptor alpha, comprising three a heavy chain comprising a set of heavy CDRs, HCDR1, HCDR2 and HCDR3, DR1 comprises the amino acid sequence of SEQ ID NO: 24, and HCDR2 comprises the amino acid sequence of SEQ ID NO: 26 and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49. Provide a binding fragment.

[0013] In a related embodiment, the canine interleukin-4 receptor alpha (IL-4 R α ), a mammalian antibody or antigen-binding fragment thereof, which binds to the IgG1A-G1A gene, has three light chain CDR sets: CDR light 1 (LCDR1), CDR light 2 (LCDR2) and CDR light 3 (LCDR3) and a light chain comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO: 30; and LCDR2 comprising the amino acid sequence of SEQ ID NO: 31. comprises the amino acid sequence of SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 34 In a preferred embodiment, the mammalian antibody or antigen-binding fragment thereof is a canine IL-4R α to Binds to canine IL-4R α Blocks the binding of IL-4 to canine interleukin-4 (cIL-4) In a related embodiment, the mammalian antibody or antigen-binding fragment thereof is α binds to canine IL-4R α Binding of IL-13 to canine interleukin-13 (cIL-13) In yet another embodiment, the mammalian antibody or antigen-binding fragment thereof is a canine cI L-4R α Binds to canine cIL-4R α Blocks the binding of cIL-4 and cIL-13 I refuse.

[0014] In a particular embodiment, 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 not a naturally occurring The IgG-D hinge region present in In other embodiments, the caninized antibody has a heavy chain comprising IgG-D cFc. wherein the naturally occurring IgG-D hinge region comprises the amino acid sequence of SEQ ID NO: 7. In yet another embodiment, the caninized antibody is an IgG- D cFc containing heavy chains, but the naturally occurring IgG-D hinge region is the amino acid sequence of SEQ ID NO: 8. In yet another embodiment, the hinge region is replaced by a hinge region comprising the amino acid sequence Nucleotidic antibodies contain heavy chains containing IgG-D cFc, but lack the naturally occurring IgG-D hinge. The region is replaced by a hinge region comprising the amino acid sequence of SEQ ID NO:9.

[0015] In certain embodiments, the caninized antibody comprises a modified caninized antibody comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the caninized antibody comprises a heavy chain comprising a caninized IgG-B (IgG-Bm). comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. In other embodiments, the caninized antibody comprises In yet another embodiment, the caninized antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 37. , and a heavy chain comprising the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the caninized antibody comprises and further comprising a light chain comprising the amino acid sequence of SEQ ID NO: 35. In an alternative embodiment, the caninized antibody further comprises a light chain comprising the amino acid sequence of SEQ ID NO:43.

[0016] In certain embodiments, 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 is canine IL-4R α of The following amino acid residue: S of SEQ ID NO:5 111 , H 112 , T 113 , T 119 , Y 122 , T 124 and H 127 Binds to 1, 2, 3, 4, 5, 6 or all 7 of 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 is canine IL-4R α the following amino acid residue: Y of SEQ ID NO: 5 150 , T 153 , Y 154 , T 158 , R 16 0, S 164 , T 165 , S 168 , S 171 , Y 172 , S 173 1, 2, 3 of them In an even more particular embodiment, it binds to all 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids. wherein the caninized antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 47. In still further particular embodiments of this type, the caninized antibody or its antigen-binding The fragment is a canine IL-4R α The following amino acid residues of SEQ ID NO:5: 111 , H 112 , T 113 , T 119 , Y 122 , T 124 and H 127 If 1, 2, 3, 4, 5, 6 In many cases, all seven and / or canine IL-4R α The following amino acid residues: SEQ ID NO: 5 Y150 , T 153 , Y 154 , T 158 , R 160 , S 164 , T 165 , S 16 8. S 171 , Y 172 , S 173 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 all combine.

[0017] The present invention also provides CDRs, heavy chains and / or caninized antibodies or antigen-binding fragments thereof. Nucleic acid comprising an isolated nucleic acid encoding a light chain of a nucleoside antibody or antigen-binding fragment thereof. Furthermore, the present invention provides an expression vector containing such a nucleic acid and a method for expressing such a nucleic acid. A host cell containing the vector is provided.

[0018] Furthermore, the present invention provides caninized antibodies and antigen-binding fragments thereof, which can be administered in pharmaceutically acceptable carriers. The present invention further provides a pharmaceutical composition comprising the compound of formula (I) and a carrier and / or diluent. atopic dermatitis, comprising administering one of the compositions to a dog having atopic dermatitis. In certain embodiments, the present invention provides a method for treating rheumatoid arthritis in a dog in need thereof. and administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a patient suffering from atopic dermatitis. The present invention provides a method for helping block inflammation caused by steroids.

[0019] These and other aspects of the present invention are described in detail below with reference to the following Brief Description and Detailed Description of the Drawings. It will be better understood by [Brief explanation of the drawings]

[0020] [Figure 1]Figure 1 is a graph showing the inhibition of IL-4-mediated STAT-6 phosphorylation by IL-4Rα (IL-4Rα) antibodies. Two different caninized monoclonal anti-canine IL-4Rα antibodies, designated c4H3 [see WO 2016 / 156588] and c152H11-H3L3, 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 IL-4. The IL-4 control in the absence of IL-4Rα (IL-4Rα) antibodies is shown in the upper right portion of the graph. [Figure 2] Figure 2 is a graph showing the inhibition of IL-13-mediated STAT-6 phosphorylation by IL-4Rα antibodies. Two different caninized monoclonal anti-canine IL-4Rα antibodies, designated c4H3 [see WO 2016 / 156588] and c152H11-H3L3, 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 IL-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 (c152ClL1-H1, c152ClL1-H2, and c152ClL1-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 (c152H11-H3L3). 152mc 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 on canine IL-4Rα for the c152H11-H3L3 antibody, including the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 47. DETAILED DESCRIPTION OF THE INVENTION

[0021] In response to the need for better treatments for atopic dermatitis, the present invention provides Caninized antibodies, formulations containing caninized antibodies, and compositions for treating skin inflammation associated with atopic dermatitis The present invention provides a methodology that can achieve significant results.

[0022] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention: ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity CDRs are immunoglobulin variable fragments, defined using the Kabat numbering system. Complementarity-determining regions in the region EC50 Concentration that results in 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 Immunoglobulins pioneered by Elvin A. Kabat Alignment and numbering system of Sequences of Protein ins of Immunological Interest,5th Ed.Pub lic Health Service, National Institutes o f 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. It spans Kabat residue 109 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

[0023] definition In order that the present invention may be more readily understood, certain technical and scientific terms will be specifically defined below. As used herein, unless specifically defined elsewhere in this specification, All other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. do.

[0024] As used in this specification, including the appended claims, "a," "an," and "t" refer to The singular form of a word such as "he" is used unless the context clearly dictates otherwise. Including corresponding multiple references to et al.

[0025] "Administration" and "treatment" refer to the administration of an animal, e.g., a canine subject, a cell, tissue, organ, or biological When applied to a fluid, the exogenous medicine, therapeutic agent, diagnostic agent or composition is delivered to an animal, e.g., a dog. Treatment of cells refers to the application of a test substance to a cell. This includes contact of drugs as well as contact of reagents with fluids in contact with the cells.

[0026] "Administration" and "treatment" also include administering, e.g., administering, administering to a child ... The term "subject" refers to the in vitro and ex vivo treatment of cells with a cell. Any organism, preferably an animal, more preferably a mammal (e.g., a dog, cat, or human) ), most preferably including dogs.

[0027] "Treat" or "treating" refers to, for example, the treatment of a disease in which the therapeutic agent has therapeutic activity. Canine subjects or patients with or suspected of having the above symptoms may be given internal or or administering to the exogenous host a therapeutic agent, such as a composition containing any of the antibodies of the present invention. Typically, a therapeutic agent will alleviate such symptoms to any clinically measurable extent. Inducing regression or inhibiting progression of (classes of) in treated subjects or populations The compound is administered in an amount effective to reduce and / or ameliorate one or more disease / condition symptoms. The amount of a therapeutic agent that is effective in alleviating the symptoms of a particular disease / condition of interest (also referred to as a "therapeutically effective amount"). The amount of the dose to be administered depends on the disease / symptom status, age, and weight of the patient (e.g., dog), and the subject's The effectiveness of the pharmaceutical composition may vary depending on factors such as the ability of the pharmaceutical composition to elicit a desired response in the patient. Whether a symptom has been alleviated or improved is an assessment of the severity or progression of the symptom. Any clinical test routinely used by veterinarians or other skilled medical personnel to evaluate Aspects of the Invention (e.g., Methods of Treatment or Articles of Manufacture) may not be effective in alleviating the target disease / symptom(s) in all subjects. There may be no correlation coefficients, but there are Student's t-test, chi-square test, Mann-Whitney test, etc. U test, Kruskal-Wallis test (H test), Jonckheer-Terpstra test, and The case may be determined by any statistical test known in the art, such as the Wilcoxon test. If the compound is a steroid drug, it should alleviate the target disease / symptom(s) in a statistically significant number of subjects. do.

[0028] "Treatment" as applied to humans, veterinary subjects (e.g., dogs), or research subjects means "Treatment" refers to therapeutic treatment as well as research and diagnostic uses. "Treatment" refers to treatment with human, veterinary subjects (e.g., When applied to a human, animal, or research subject, or cell, tissue, or organ, the present invention of antibodies to, for example, a dog or other animal subject, cell, tissue, physiological compartment, or organism. This includes contact with physical fluids.

[0029] As used herein, the term "dog" refers to any domestic dog unless otherwise specified. Includes domestic dogs, Canis lupus familiaris or Canis familiaris.

[0030] As used herein, the term "cat" refers to any member of the Felidae family. Members of this family include domestic cats, purebred and / or mixed breed pet cats, and show cats. , wild, including laboratory cats, cloned cats and wild or feral cats , zoo and household members.

[0031] As used herein, the term "canine frame" refers to the CDR residues and The amino acid sequences of the heavy and light chains of a canine antibody, excluding the hypervariable region residues, are defined as follows: For caninized antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are both are replaced with the corresponding foreign CDRs (for example, from a mouse antibody) in the corresponding chains of the target polypeptide. The heavy and / or light chains of the canine antibody may be, for example, as exemplified below and / or No. 10,106,607, the entirety of which is incorporated herein by reference. to preserve the conformation of the foreign CDRs in the canine antibody, as indicated, and / or Some extraneous non-CDR residues may be included to alter Fc function.

[0032] The "fragment crystallizable region," abbreviated as "Fc," is a cell surface receptor called an Fc receptor. The CH3-CH2 region of the antibody that interacts with the human body corresponds to the CH3-CH2 region of each of the four dog IgGs. The fragment crystallizable region (cFc) was prepared by Tang et al. [Vet. Immunol. First described by Immunopathol. 80:259-270 (2001) ,Bergeron et al.,Vet.Immunol.Immunopathy l.157:31-41 (2014) and U.S. Patent No. 10,106,607. I want to be done that.

[0033] As used herein, canine Fc (cFc) "IgG-Bm" refers to the IgG-B sequence. The amino acid sequence of sequence number 10 contains two amino acid residue substitutions, D31A and N63A. (see below), a canine IgG-B Fc lacking the c-terminal lysine ("K"). SEQ ID NO: 1 The aspartic acid residue (D) at position 31 of SEQ ID NO: 10 and the asparagine residue at position 63 of SEQ ID NO: 10 (N) is replaced by an alanine residue (A) in IgG-Bm These two amino acid residue substitutions are essential for the antibody-dependent cell-mediated cytotoxicity of naturally occurring canine IgG-B. It helps to significantly attenuate ADCC and complement-dependent cytotoxicity (CDC) No. 10,106,607, the entire contents of which are incorporated herein by reference. Similar to the amino acid substitutions that can be made in IgG-B, bispecific antibodies Further modifications to IgG-Bm may include amino acid substitutions that promote heterodimer formation in The amino acid sequence of IgG-B, SEQ ID NO: 45, is: is.

[0034] [Table 1]

[0035] The amino acid sequence of IgG-Bm, SEQ ID NO: 10, is provided below. LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEV QISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDW LKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP SREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKY RTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEA LHNHYTQESLSHSPG

[0036] As used herein, "antibody" refers to a sequence of antibodies that is "antibody" or "antibody-specific" by another amino acid residue in the amino acid sequence of the antibody. "Substitution of an amino acid residue" means, for example, "replacing an amino acid residue" with another amino acid residue. Amino acids that are equivalent and differ in specific amino acid residues at specific positions in the amino acid sequence This indicates that the residue is replaced (or substituted) by a can be specifically designed, i.e., amino acid sequence can be linked, for example, by recombinant DNA techniques. Alanine at a specific position in the amino acid sequence can be deliberately replaced with serine, or , specific amino acid residues or sequences of amino acid residues in antibodies may be selected through more natural selection processes. For example, antibodies produced by a cell may target a given region on the antigen, e.g., an epitope. and / or that the antibody is capable of binding to a region containing the Specific CDRs may contain specific CDRs that have the same canonical structure as the CDRs they replace. Such substitutions / alternatives may be replaced by one or more amino acid residues so as to The shuffling can result in "variant" CDRs and / or variant antibodies.

[0037] As used herein, the term "antibody" refers to an antibody that exhibits a desired biological activity. Antibodies can be monomeric, dimeric, or of higher multimeric form. Therefore, the term "antibody" is used in the broadest sense and includes monoclonal antibodies (full-length monoclonal antibodies). monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies, and camelized single domain antibodies. "Parent antibody" refers to an antibody used as a therapeutic antibody for dogs. Exposing the immune system to the antigen prior to modifying the antibody for its intended use, such as caninizing the antibody for It is an antibody obtained by

[0038] As used herein, the binding of a canine receptor to its binding partner (ligand) Antibodies of the Invention That "Block" or "Blocking" or "Binding-Blocking" The antibody can be detected by standard binding assays (e.g., BIACore®, ELISA or flow cytometry). Binding of the canine receptor to its canine ligand as determined by flow cytometry ( antibodies that block (partially or completely) and vice versa.

[0039] Typically, the antibodies or antigen-binding fragments of the invention are expressed as antibodies or antigen-binding fragments of the invention when their activity is expressed on a molar basis. If the antibody is a canine antigen-binding antibody, it retains at least 10% of its canine antigen-binding activity (when compared to the parent antibody). Preferably, the antibody or antigen-binding fragment of the present invention has the same antigen-binding affinity as the canine antibody as the parent antibody. At least 20%, 50%, 70%, 80%, 90%, 95% or 100% The antibodies or antigen-binding fragments of the invention retain substantially more of their biological activity. Conservative or non-conservative amino acid substitutions that do not alter the function of the antibody ("conservative variants" or "functional variants") It is also intended that the sequences may include "conservative variants" (referred to as "conservative variants").

[0040] "Isolated antibody" refers to a purified state, and in this context, the molecule is not a nucleic acid, tag, or antibody. other biological molecules such as proteins, lipids, carbohydrates, or cell debris and growth medium Generally, the term "isolated" means that the substance is substantially free of other materials such as is present in an amount that substantially prevents the experimental or therapeutic use of the binding compounds described herein. If not present, it indicates the complete absence of such material or the presence of water, buffers or salts. It is not intended to imply non-existence.

[0041] As used herein, a "chimeric antibody" refers to an antibody that contains a variable domain derived from a first antibody and a an antibody having a constant domain derived from a second antibody, the first and second antibodies being from different species; [U.S. Patent No. 4,816,567; and Morrison et al. ,Proc.Natl.Acad.Sci.USA 81:6851-6855(198 4)]. Typically, the resulting chimeric antibody is more specific than the parent (e.g., rodent) antibody. so as to be less likely to elicit a harmful immune response in human or canine subjects. the variable domains are derived from an antibody from a laboratory animal such as a rodent (the "parent antibody"); and The constant domain sequences are obtained from an animal subject antibody, eg, human or canine.

[0042] As used herein, the term "caninized antibody" refers to antibodies that are specifically engineered to be canine and non-canine (e.g., Caninized antibodies generally refer to a form of antibody that contains sequences from both caninized (mouse) and caninized (mouse) antibodies. All or substantially all of the hypervariable loops (e.g., six as exemplified below) corresponding to the hypervariable loops of non-canine immunoglobulins (including CDRs) and framework (FR) All or substantially all of the region (and typically all or substantially all of the remaining frames) At least one of the nucleotide sequences (essentially all of the nucleotide sequences) is a framework (FR) region of a canine immunoglobulin sequence. The antibody may contain substantially all of at least one or more, typically two, variable domains. As shown, the caninized antibody contains three heavy chain CDRs and three CDRs derived from a mouse anti-canine antigen antibody. and the light chain CDRs of both of the canine and modified canine frames. The modified canine frame allows for the binding of the caninized antibody to its canine antigen and / or the binding of the caninized antibody to the canine antigen. Increased ability of caninized antibodies to block binding of their canine antigens to their native binding partners. The caninized antibody may be further optimized to achieve the desired effect by administering one or more amino acids exemplified herein. Contains acid changes.

[0043] The variable regions of each light / heavy chain pair form the antibody binding site. Except for bifunctional or bispecific antibodies, most antibodies have two binding sites. The binding sites are generally the same. Typically, the variable domains of both the heavy and light chains are Complementarity-determining regions (CDRs) located within relatively conserved framework regions (FRs) The CDRs contain three hypervariable regions, also called framework regions. Generally, from the N-terminus to the C-terminus, The heavy chain variable domain and the heavy chain variable domain each comprise FR1, CDR1, FR2, CDR2, F The amino acid assignments for each domain are generally sequences of Proteins of Immunological In terest,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.Bio l.196:901-917(1987) or Chothia, et al., Nat ure 342:878-883(1989)].

[0044] As used herein, the term "hypervariable region" refers to the region of an antibody that is responsible for antigen binding. The hypervariable regions refer to the amino acid residues that make up the "complementarity determining regions" or "CDRs" (i.e., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and LCDR1 in the heavy chain variable domain The sequence contains amino acid residues derived from HCDR1, HCDR2, and HCDR3. Therefore, the sequences of Kabat et al. Proteins of Immunological Interest,5th Ed.Public Health Service,National Institute See utes of Health, Bethesda, Md. (1991); Therefore, the CDR regions of an antibody are defined by Chothia and Lesk, J. Mol. B see also Iol. 196:901-917 (1987). As used herein, " The term "framework" or "FR" residues are defined herein as CDR residues. These residues refer to variable domain residues other than the hypervariable region residues.

[0045] There are four known IgG heavy chain subtypes of canine IgG: IgG-A, IgG-B, The two known light chain subtypes are called IgG-C and IgG-D. In certain aspects of the invention, apart from binding and activating canine immune cells, the present invention The canine or caninized antibody against the antigen of the canine or caninized antibody is optimally one of the following two: It has the following attributes. 1. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) Lack of vector function, and 2. Large-scale purification using industry-standard techniques such as those based on Protein A chromatography. It is easily refined into a similar product.

[0046] No naturally occurring canine IgG isotype meets both criteria. For example, , IgG-B can be purified using Protein A, but not with high levels of ADCC. On the other hand, IgG-A 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. IgG-D does not exhibit ADCC activity. (IgG-C has considerable ADCC activity.) One way in which the present invention addresses these issues is by inhibiting effector functions such as ADCC. lacks ATP and can be easily purified using industry-standard Protein A chromatography and (c) providing a modified canine IgG-B antibody of the present invention specific for an antigen of the present invention. This is due to the following.

[0047] As used herein, "anti-inflammatory antibodies" refers to antibodies that are effective against human atopic dermatitis, particularly in the treatment of atopic dermatitis. Acts as an anti-inflammatory agent in animals, including mammals such as dogs, cats, and / or cats. In certain embodiments, the anti-inflammatory antibody is an antibody capable of inhibiting IL-4 or receptor I. L-4R α It binds to specific proteins in the IL-4 / IL-13 signaling pathway, such as Anti-inflammatory antibodies against their corresponding antigens (e.g., IL-4 or IL-4R) α ) is bound to , e.g., IL-4R for IL-4 α It blocks the binding of ATP to ATP, disrupting signal transduction in this pathway and and / or prevent, thereby disrupting or preventing the chronic inflammation associated with atopic dermatitis do.

[0048] As used herein, "homology" refers to the degree of similarity between two polynucleotide sequences or between two polynucleotide sequences or when their polypeptide sequences are optimally aligned refers to the sequence similarity between two polypeptide sequences. are occupied by the same base or amino acid residue, e.g., two DNA molecules If a position in each is occupied by an adenine, the molecule will The percent homology is the number of positions shared by the two sequences divided by the total number of positions compared. For example, when sequences are optimally aligned, if two If 6 of the 10 positions in a sequence are identical or homologous, then the two sequences are 60% homology. Generally, the comparison is made so that the two sequences have the highest percent homology. Sequence identity is determined when two sequences are optimally aligned. The degree to which the amino acids in two polypeptides are the same in equivalent positions.

[0049] As used herein, when the amino acid residues in both sequences are identical, one amino acid is An amino acid sequence is 100% "identical" to a second amino acid sequence. An amino acid sequence is considered to be a second amino acid sequence if 50% of the amino acid residues in the first amino acid sequence are identical. 50% "identity" to the sequence. Sequence comparison is performed to determine the identity of a given protein, e.g., A contiguous block of amino acid residues comprised by a portion of a protein or polypeptide In certain embodiments, the otherwise matching between two amino acid sequences is performed. Selected deletions or insertions that may alter the homology are taken into account. It includes residues and non-identical biochemically related amino acids. Biochemically related amino acids that may be interchangeable.

[0050] "Conservatively modified variants" or "conservative substitutions" are those that maintain the biological activity of a protein. Similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) Those skilled in the art will generally understand that substitution of amino acids in a non-essential region of a polypeptide is a substitution of amino acids in a protein. It is recognized that single amino acid substitutions within the region do not substantially alter biological activity [e.g. For example, Watson et al., Molecular Biology of the Gene,The Benjamin / Cummings Pub.Co.,p.22 4 (4th Ed.; 1987)]. Furthermore, structurally or functionally similar Substitutions of amino acids are less likely to destroy biological activity. Exemplary conservative substitutions are listed immediately below. See Table A below.

[0051] [Table 2]

[0052] Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. "Function-conservative variants" are those that alter desired properties such as antigen affinity and / or specificity. The term "antibody" refers to an antibody or fragment in which one or more amino acid residues have been altered without affecting the overall function of the antibody or fragment. Variants include amino acids with similar properties, such as conservative amino acid substitutions in Table A above. This includes, but is not limited to, amino acid substitutions.

[0053] An "isolated nucleic acid molecule" is any molecule of genomic, mRNA, cDNA, or synthetic origin, or The isolated polynucleotide is a DNA or RNA of any combination of these. The polynucleotide is not associated with all or part of the polynucleotide found in it in nature. or an isolated nucleic acid molecule is a polynucleotide to which it is not naturally linked. In the present disclosure, a nucleic acid comprising a particular nucleotide sequence It should be understood that "molecule" does not encompass intact chromosomes. An isolated nucleic acid molecule "comprising" a sequence may contain up to 10 or up to 2 sequences in addition to the specified sequence. Code for zero or more other proteins or parts or fragments thereof sequences or sequences operably linked to control expression of the coding regions of the described nucleic acid sequences. It may contain regulatory sequences and / or may contain vector sequences.

[0054] The present invention provides isolated caninized antibodies of the invention, for use in treating conditions such as atopy in dogs. The present invention provides a method for the use of antibodies in the treatment of allergic dermatitis in dogs, designated A, B, C, and D. There are four IgG heavy chains that are used in the treatment of leukemia. These heavy chains are IgG-A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC) and IgG-D (or Each of the two heavy chains represents four different subclasses of canine IgG, called IgG1, IgG2, and IgG3. It consists of one variable domain (VH) and three subdomains called CH-1, CH-2, and CH-3. The CH-1 domain is called the "hinge" or "hinge region." It is connected to the CH-2 domain via an amino acid sequence that

[0055] The nucleic acid and amino acid sequences of these four heavy chains are described by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)] The amino acid and nucleic acid sequences for these heavy chains were first identified by GenBank. For example, the amino acid sequence of the IgGA heavy chain is available from the Accession No. IgGB has accession number AAL35301.1, and IgGB has accession number AAL35 302.1, IgGC has accession number AAL35303.1, and IgG D has the accession number (AAL35304.1). The canine antibody is The DNA and amino acid sequences of these light chains are available from GenBank. For example, the kappa light chain amino acid sequence can be obtained from the Accession Number kappa Database. The λ light chain has accession number ABY57289.1, and the λ light chain has accession number ABY55 It has 569.1.

[0056] In the present invention, the amino acid sequences for each of the four canine IgG Fc fragments are: The identity of the CH1 and CH2 domains determined by Tang et al., supra Based on the boundaries established by the Canine IL-4R α As a caninized mouse anti-dog antibody that binds to Examples include, but are not limited to, canine IgG-A, IgG-B, IgG-C, and IgG-D heavy chains. and / or canine kappa or lambda light chains in a mouse anti-canine IL-4R α Book included with CDR The present invention therefore provides an antibody against canine IL-4R. α binds to its natural and its binding partners, canine IL-4 and / or canine IL-13. An isolated caninized mouse anti-dog antibody of the invention is provided that blocks binding of 4Rα.

[0057] Thus, the present invention further provides caninized murine antibodies and methods for treating conditions, e.g., in dogs. The present invention also provides methods of using the antibodies of the invention in the treatment of atopic dermatitis in humans.

[0058] The present invention further allows for matching with the corresponding light chain to create caninized antibodies. The present invention further provides a caninized mouse anti-dog antibody of the present invention. Antigens and antibodies (including isolated caninized mouse anti-dog antibodies), as well as methods for treating conditions, e.g., Methods of using the antibodies of the invention in the treatment of atopic dermatitis in humans are provided.

[0059] The present invention also provides methods for enhancing, reducing, or eliminating one or more effector functions. The antibody of the present invention comprises a canine fragment crystallizable region (cFc region) in which the cFc is genetically modified. In one embodiment of the present invention, the genetically modified cFc has one or more effectors. In another embodiment of the present invention, a genetically modified c The Fc enhances one or more effector functions. The engineered cFc region is a genetically modified canine IgGB Fc region. In the same manner, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In certain embodiments, the effector function is enhanced, reduced, or eliminated. In another embodiment, the effector function is antibody-dependent cellular cytotoxicity (ADCC). Enhanced, reduced, or eliminated complement-dependent cytotoxicity (CDC). In some embodiments, the cFc region enhances and reduces both ADCC and CDC, and has been genetically modified to remove it.

[0060] To generate mutant canine IgGs lacking effector function, multiple mutant canine IgGs were generated. These variants contained the following single amino acid sequence in the Fc portion of the heavy chain: or may contain one or more of the following substitutions in combination: P4A, D31A, N63A, G64P , T65A, A93G, and P95A. Mutant heavy chains (i.e., those with such amino acid substitutions) ) into an expression plasmid containing a gene encoding the light chain The plasmid was transfected into HEK293 cells. To evaluate these possibilities, Fc γ For binding to RI and C1q, Intact antibodies expressed and purified from HEK293 cells were evaluated [full text]. See U.S. Pat. No. 10,106,607, which is incorporated herein by reference. .]

[0061] The present invention also provides a method for the preparation of a human IgG-D hinge region comprising, in place of the native IgG-D hinge region, a hinge region from The present invention provides a modified canine IgG-D comprising: IgG-A: FNECRCTDTPPCPVPEP SEQ ID NO: 6 IgG-B:PKRENGRVPRPPDCPKCPAPEM SEQ ID NO:7; or IgG-C:AKECECKCNCNNCPCPGCGL SEQ ID NO:8.

[0062] Alternatively, the IgG-D hinge region may be modified by substituting serine residues with proline residues. can be genetically modified, i.e., PKESTCKCI P PCPVPES, sequence no. No. 9 (proline residue (P) is underlined and shown in bold, naturally occurring serine Such modifications result in canine IgG-D lacking Fab arm exchange. The modified canine IgG-D can be produced using standard methods of recombinant DNA technology [e.g., Ma niatis et al.,Molecular Cloning,A Labora These variants can be constructed using the [Tory Manual (1982)]. To construct the modified IgG-D, the amino acid sequence of canine IgG-D was The nucleic acid encoding the nucleic acid sequence can then be modified. For protein expression, the protein is cloned into an expression plasmid.

[0063] The six complementarity determining regions (CDRs) of the caninized murine anti-dog antibody described herein are: A canine antibody kappa light chain containing mouse light chains LCDR1, LCDR2, and LCDR3 and a mouse heavy and a canine antibody heavy chain IgG comprising chains HCDR1, HCDR2 and HCDR3. Cut.

[0064] nucleic acid The present invention further includes nucleic acids encoding the antibodies of the invention (see, e.g., the Examples below). ).

[0065] The algorithm is designed to maximize the match between each sequence over the entire length of each reference sequence. If the comparison is performed by the BLAST algorithm, the algorithm parameters are selected. and the amino acid sequence of the caninized antibody provided herein, excluding the CDRs that remain unchanged. at least about 70% identical, preferably at least about 80% identical, more preferably at least more preferably at least about 90% identical, most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, Immunoglobulin polypeptides containing amino acid sequences that are The present invention also encompasses nucleic acids encoding the respective reference sequences. The algorithm parameters are selected to give the largest match between the respective sequences. When a comparison is performed using the BLAST algorithm, At least about 70% similar, preferably at least about 80% similar, more preferably at least At least about 90% similar, most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 2 Immunoglobulin polypeptides containing amino acid sequences that are identical to those of the 7%, 98%, 99%, and 100% Nucleic acids encoding the following are further provided and encompassed by the present invention:

[0066] As used herein, percent identity of nucleotide and amino acid sequences is Using the alignment and identity initialization parameters, C, MacVec tor (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Gr. Determined using the PLC (1996) and Clustal W algorithms. The same or similar default parameters can be used to determine sequence similarity. Alternatively, you can use these commercially available programs to GCG (Genetics Computer Group, Pr gram Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program to Advanced Blast searches can be performed with set filter conditions.

[0067] The following references concern the BLAST algorithm, which is often 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(19 96);Altschul, SF, et al., Nucleic Acids R es.25:3389-3402(1997);Zhang,J.,et al.,Ge nome Res.7:649-656(1997);Wootton,JC,et al.,Comput.Chem.17:149-163(1993);Hancoc k,J.M.et al.,Comput.Appl.Biosci.10:67-70 (1994);ALIGNMENT SCORING SYSTEMS:Dayhoff ,M.O.,et al.,“A model of evolutionary ch ange in proteins.”in Atlas of Protein Se quence and Structure,vol.5,suppl.3.M.O.D ayhoff(ed.),pp.345-352,(1978);Natl.Biome d.Res.Found.,Washington,DC;Schwartz,R.M. ,et al.,“Matrices for detecting distant relationships.”in Atlas of Protein Seque nce and Structure,vol.5,suppl.3.”(1978), M.O.Dayhoff(ed.),pp.353-358(1978),Natl.B iomed.Res.Found.,Washington,DC;Altschul, S.F.,J.Mol.Biol.219:555-565(1991);States ,D.J.,et al.,Methods 3:66-70(1991);Henik off,S.,et al.,Proc.Natl.Acad.Sci.USA 89: 10915-10919(1992);Altschul,S.F.,et al.,J .Mol.Evol.36:290-300(1993);ALIGNMENT STA TISTICS: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.22:2022-2 039(1994); and Altschul, S.F. “Evaluating the e statistical significance of multiple d istinct local alignments.” in Theoretica l and Computational Methods in Genome Re search(S.Suhai,ed.),pp.1-14,Plenum,New Y ork (1997).

[0068] The antibodies of the present invention can be produced recombinantly by methods known in the art. Mammalian cells available as hosts for expressing the antibodies or fragments disclosed herein Strains are well known in the art and are available from American Type Culture Collection. These include many immortalized cell lines available from the American College of Cancer (ATCC). Among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, He La cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocytes Carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, HEK-293 cells, and Mammalian host cells include human, mouse, rat, dog, and many other cell lines. These include monkey, pig, goat, bovine, horse, and hamster cells. Which cell lines have high expression? By determining which cell lines have the highest levels of ATP, particularly preferred cell lines can be selected. Other cell lines that can be obtained include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and and fungal cells. When a recombinant expression vector encoding an antigen-binding fragment is introduced into a mammalian host cell, the antigen-binding fragment The body is responsible for the expression of the antibody in the host cells, or more preferably, for the host cells to be grown in. The host cells are cultured for a period of time sufficient to allow secretion of the antibody into the culture medium. It is produced by nurturing.

[0069] Antibodies can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibodies of the invention (or other moieties therefrom) from production cell lines can be achieved in several ways. For example, the glutamine synthetase gene can be amplified using known techniques. The GS system is a general approach to enhance expression under certain conditions. The GS series is disclosed in European Patent Nos. 0216846, 0256055 and 0323 997, as well as in whole or in part in connection with European Patent Application No. 89303964.4. It is being discussed in detail.

[0070] Generally, glycoproteins produced in specific cell lines or transgenic animals is characteristic of the glycoprotein produced in the cell line or transgenic animal. Therefore, the specific glycosylation pattern of the antibody may be Depends on the particular cell line or transgenic animal used to produce the antibody However, the nucleic acid molecules provided herein or the All antibodies containing the amino acid sequences provided are intended to be representative of the glycosylation patterns that the antibodies may possess. Similarly, in certain embodiments, non-fucosylated Antibodies with glycosylation patterns containing only N-glycans are typically purified in vitro showed stronger efficacy than their fucosylated counterparts both in vitro and in vivo. These antibodies may be advantageous because they have been shown to al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent See Nos. 6,946,292 and 7,214,775.

[0071] Canine IL-4 receptor α receptor Genbank database (accession number XM_547077.4; U.S. Patent 7,208,579) predicted full-length canine IL-4 receptor A cDNA encoding the α chain (SEQ ID NO: 1) was identified. This predicted cDNA was It encodes 823 amino acids (SEQ ID NO: 2) including the amino acid leader sequence. The mature predicted canine IL-4 receptor is identified as XP_547077.3. The human IL-4 receptor alpha chain protein (SEQ ID NO: 4) was synthesized from the human IL-4 receptor alpha chain (accession no. NP _000409.1) and the porcine IL-4 receptor α chain (accession no. It shares 70% identity with the adult predicted canine I (number NP_999505.1). The L-4 receptor alpha chain protein is identified by the nucleotide sequence identified as SEQ ID NO:3. The predicted mature IL-4 receptor α chain is encoded by the known sequence of the human IL-4 receptor α chain. By comparing the sequence of the mature canine IL-4 receptor α chain protein with that of the extracellular domain ( ECD) has been identified and designated SEQ ID NO: 5. All of this has been previously described in U.S. Pat. Publication No. 2018 / 0346580; the entire contents of which are incorporated herein. It has been done.

[0072] Canine IL-4 receptor α chain full-length DNA with signal sequence [SEQ ID NO: 1] atgggcagactgtgcagcggcctgaccttccccgtgagc tgcctggtgctggtgtgggtggccagcagcggcagcgtga aggtgctgcacgagcccagctgcttcagcgactacatcag caccagcgtgtgccagtggaagatggaccaccccaccaac tgcagcgccgagctgagactgagctaccagctggacttca tgggcagcgagaaccacacctgcgtgcccgagaacagaga ggacagcgtgtgcgtgtgcagcatgcccatcgacgacgcc gtggaggccgacgtgtaccagctggacctgtgggccggcc agcagctgctgtggagcggcagcttccagcccagcaagca cgtgaagcccagaacccccggcaacctgaccgtgcacccc aacatcagccacacctggctgctgatgtggaccaacccct accccaccgagaaccacctgcacagcgagctgacctacat ggtgaacgtgagcaacgacaacgaccccgaggacttcaag gtgtacaacgtgacctacatgggccccaccctgagactgg ccgccagcaccctgaagagcggcgccagctacagcgccag agtgagagcctgggcccagacctacaacagcacctggagc gactggagccccagcaccacctggctgaactactacgagc cctgggagcagcacctgcccctgggcgtgagcatcagctg cctggtgatcctggccatctgcctgagctgctacttcagc atcatcaagatcaagaaggggctggtgggaccagatcccca accccgcccacagccccctggtggccatcgtgatccagga cagccaggtgagcctgtggggcaagagaagcagaggccag gagcccgccaagtgccccactggaagacctgcctgacca agctgctgccctgcctgctggagcacggcctgggcagaga ggaggagagccccaagaccgccaagaacggccccctgcag ggccccggcaagcccgcctggtgccccgtggaggtgagca agaccatcctgtggcccgagagcatcagcgtggtgcagtg cgtggagctgagcgaggccccctggacaacgaggagga gaggaggtggaggaggacaagagaagcctgtgccccagcc tggagggcagcggcggcagcttccaggagggcagagagggg catcgtggccagactgaccgagagcctgttcctggacctg ctgggcggcgagaacggcggcttctgcccccagggcctgg aggagagctgcctgcccccccccagcggcagcgtgggcgc ccagatgccctgggcccagttccccagagccggccccaga gccgcccccgagggccccgagcagcccagaagacccgaga gcgccctgcaggccagccccacccagagcgccggcagcag cgccttccccgagcccccccccgtggtgaccgacaacccc gcctacagaagcttcggcagcttcctgggccagagcagcg accccggcgacggcgacagcgaccccgagctggccgacag acccggcgaggccgaccccggcatccccagcgccccccag ccccccgagccccccgccgccctgcagcccgagcccgaga gctgggagcagatcctgagacagagcgtgctgcagcacag agccgcccccgcccccggccccggccccggcagcggctac agagagttcacctgcgccgtgaagcagggcagcgcccccg acgccggcggccccggcttcggccccagcggcgaggccgg ctacaaggccttctgcagcctgctgcccggcggcgccacc tgccccggcaccagcggcggcgaggccggcagcggcgagg gcggctacaagcccttccagagcctgacccccggctgccc cggcgcccccacccccgtgcccgtgcccctgttcaccttc ggcctggacaccgagccccccggcagcccccaggacagcc tgggcgccggcagcagccccgagcacctgggcgtggagcc cgccggcaaggaggaggacagcagaaagaccctgctggcc cccgagcaggccaccgaccccctgagagacgacctggcca gcagcatcgtgtacagcgccctgacctgccacctgtgcgg ccacctgaagcagtggcacgaccaggaggagagaggcaag gcccacatcgtgcccagcccctgctgcggctgctgctgcg gcgacagaagcagcctgctgctgagccccctgagagcccc caacgtgctgcccggcggcgtgctgctggaggccagcctg agccccgccagcctggtgcccagcggcgtgagcaaggagg gcaagagcagccccttcagccagcccgccagcagcagcgc ccagagcagcagccagacccccaagaagctggccgtgctg agcaccgagcccacctgcatgagcgccagc

[0073] Canine IL-4 receptor alpha full-length protein with signal sequence in bold [SEQ ID NO: 2] MGRLCSGLTFPVSCLVLVWVASSGSVKVLHEPSCFSDYI STSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENR EDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSK HVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTY MVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSA RVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLPLGVSIS CLVILAICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQ DSQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEHGLGR EEESPKTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQ CVELSEAPVDNEEEEEVEEDKRSLCPSLEGSGGSFQEGRE GIVARLTESLFLDLLGGENGGFCPQGLEESCLPPPSGSVG AQMPWAQFPRAGPRAGPRAPEGPEQPRRPESALQASPTQSAGS SAFPEPPPVVTDNPAYRSFGSFLGQSSDPDGDSDPELAD RPGEADPGIPSAPQPPEPPAALQPEPESWEQILRQSVLQH RAAPAPPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEA GYKAFCSLLPGGATCPGTSGGEAGSGEGGYKPFQSLTPGC PGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVE PAGKEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHLC GHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRA PNVLPGGVLLEASLSPASLVPSGVSKEGKSSPFSQPASSS AQSSSQTPKKLAVLSTEPTCMSAS

[0074] Canine IL-4 receptor alpha mature full-length protein without signal sequence [SEQ ID NO: 4] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQL DFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLW AGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWT NPYPTENHLHSELTYMVNVSNNDNDPEDFKVYNVTYMGPTL RLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNY YEPWEQHLPLGVSISCLVILAICLSCYFSIIKIKKGWWDQ IPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTC LTKLLPCLLEHGLGREEESPKTAKNGPLQGPGKPAWCPVE VSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLC PSLEGSGGSFQEGREGIVARLTESLFLDLLGGENGGFCPQ GLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQPRR PESALQASPTQSAGSSAFFPEPPPVVTDNPAYRSFGSFLGQ SSDPDGDSDPELADRPGEADPGIPSAPQPPEPPAALQPE PESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGS APDAGGPGFGPSGEAGYKAFCSLLPGGATCPGTSGGEAGS GEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQ DSLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDPLRDD LASSIVYSALTCHLCGHLKQWHDQEERGKAHIVPSPCCGC CCGDRSSLLLSPLRAPNVLPGGVLLEASLSPASLVPSGVS KEGKSSPFSQPASSSAQSSSQTPKKLAVLSTEPTCMSAS

[0075] Canine IL-4 receptor α mature full-length DNA without signal sequence [SEQ ID NO: 3] gtgaaggtgctgcacgagcccagctgcttcagcgactac atcagcaccagcgtgtgccagtggaagatggaccaccca ccaactgcagcgccgagctgagactgagctaccagctgga cttcatgggcagcgagaaccacacctgcgtgcccgagaac agagaggacagcgtgtgcgtgtgcagcatgcccatcgacg acgccgtggaggccgacgtgtaccagctggacctgtgggc cggccagcagctgctgtggagcggcagcttccagcccagc aagcacgtgaagcccagaacccccggcaacctgaccgtgc accccaacatcagccacacctggctgctgatgtggaccaa cccctaccccaccgagaaccacctgcacagcgagctgacc tacatggtgaacgtgagcaacgacaacgaccccgaggact tcaaggtgtacaacgtgacctacatgggccccaccctgag actggccgccagcaccctgaagagcggcgccagctacagc gccagagtgagagcctgggcccagacctacaacagcacct ggagcgactggagccccagcaccacctggctgaactacta cgagccctgggagcagcacctgcccctgggcgtgagcatc agctgcctggtgatcctggccatctgcctgagctgctact tcagcatcatcaagatcaagaagggctggtgggaccagat ccccaaccccgcccacagccccctggtggccatcgtgatc caggacagccaggtgagcctgtggggcaagagaagcagag gccaggagcccgccaagtgcccccactggaagacctgcct gaccaagctgctgccctgcctgctggagcacggcctgggc agagagggagagccccaagaccgccaagaacggcccccc tgcagggccccggcaagcccgcctggtgccccgtggaggt gagcaagaccatcctgtggcccgagagcatcagcgtggtg cagtgcgtggagctgagcgaggccccccgtggacaacgagg aggaggaggaggtggaggaggacaagagaagcctgtgccc cagcctggagggcagcggcggcagcttccaggagggcaga gagggcatcgtggccagactgaccgagagcctgttcctgg acctgctgggcggcgagaacggcggcttctgcccccaggg cctggaggagagctgccctgcccccccccagcggcagcgtg ggcgcccagatgccctgggcccagttccccagagccggcc ccagagccgccccgagggccccgagcagcccagaagacc cgagagcgccctgcaggccagccccaccagagcgccggc agcagcgcttccccgagccccccccccgtggtgaccgaca accccgcctacagaagcttcggcagcttcctgggccagag cagcgaccccggcgacggcgacagcgaccccgagctggcc gacagacccggcgaggccgaccccggcatccccagcgccc cccagccccccgagccccccgccgccctgcagcccgagcc cgagagctgggagcagatcctgagacagagcgtgctgcag cacagagccgcccccgcccccggccccggccccggcagcg gctacagagagttcacctgcgccgtgaagcagggcagcgc ccccgacgccggcggccccggcttcggccccagcggcgag gccggctacaaggccttctgcagcctgctgcccggcggcg ccacctgccccggcaccagcggcggcgaggccggcagcgg cgagggcggctacaagcccttccagagcctgacccccggc tgccccggcgcccccacccccgtgcccgtgcccctgttca ccttcggcctggacaccgagccccccggcagcccccagga cagcctgggcgccggcagcagccccgagcacctgggcgtg gagcccgccggcaaggaggaggacagcagaaagaccctgc tggcccccgagcaggccaccgaccccctgagagacgacct ggccagcagcatcgtgtacagcgccctgacctgccacctg tgcggccacctgaagcagtggcacgaccaggaggagagag gcaaggcccacatcgtgcccagcccctgctgcggctgctg ctgcggcgacagaagcagcctgctgctgagccccctgaga gcccccaacgtgctgcccggcggcgtgctgctggaggcca gcctgagccccgccagcctggtgcccagcggcgtgagcaa ggagggcaagagcagccccttcagccagcccgccagcagc agcgcccagagcagcagccagacccccaagaagctggccg tgctgagcaccgagcccacctgcatgagcgccagc

[0076] Canine IL-4 receptor α chain extracellular domain [SEQ ID NO: 5] VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQL DFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLW AGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWT NPYPTENHLHSELTYMVNVSNNDNDPEDFKVYNVTYMGPTL RLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNY YEPWEQHLP

[0077] Antibody Protein Engineering By way of example, and not limitation, the canine heavy chain constant region may be any of the IgG-B or IgG-C heavy chain constant regions used herein. Modified cFc such as IgG-Bm [incorporated herein by reference in its entirety] See U.S. Pat. No. 10,106,607 for the disclosure of the present invention, and the canine light chain constant region can be derived from κ. possible.

[0078] Antibodies may be modified from the parental (i.e., murine) monoclonal antibody, e.g., to improve the properties of the antibody. Modifications to the canine framework and / or canine framework residues within the variable domains of the antibody In certain circumstances, individual The CDRs of the can be modified.

[0079] Pharmaceutical Compositions and Administration To prepare pharmaceutical or sterile compositions containing the antibodies of the present invention, these antibodies can be prepared in a pharmaceutical The compound may be mixed with a commercially acceptable carrier or excipient. on's Pharmaceutical Sciences and USPharm acopeia:National Formulary,Mack Publishing ng Company, Easton, PA (1984)].

[0080] Formulations of therapeutic and diagnostic agents may be, for example, lyophilized powders, slurries, aqueous solutions or It is prepared by mixing with an acceptable carrier, excipient or stabilizer in the form of a suspension. [For example, Hardman, et al. (2001) Goodman and G ilman's The Pharmacological Basis of The rapeutics,McGraw-Hill,New York,NY;Gennar o(2000) Remington: The Science and Practical e of Pharmacy, Lippincott, Williams, and Wi. lkins, New York, NY; Avis, et al. (eds.) (1993 )Pharmaceutical Dosage Forms: Parental Medications,Marcel Dekker,NY;Lieberman,e t al.(eds.)(1990)Pharmaceutical Dosage F orms:Tablets,Marcel Dekker,NY;Lieberman, et al.(eds.)(1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxi city ​​and Safety,Marcel Dekker,Inc.,New Y In one embodiment, the antibodies of the invention are dissolved in sodium acetate solution, pH 5 to 10. It is diluted to the appropriate concentration with 6 mL of NaCl or sucrose for isotonicity. To improve the quality, additional preparations such as polysorbate 20 or polysorbate 80 may be used. Active substances may be added.

[0081] Toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with other agents Gender, for example, LD 50 (a dose lethal to 50% of the population) and ED 50 (Collective in cell cultures or experimental animals to determine the therapeutically effective dose in 50% of cases The dose ratio between toxic and therapeutic effects can be determined by standard pharmaceutical procedures. The therapeutic index (LD 50 / ED 50 In certain situations, it exhibits a high therapeutic index. The data obtained from these cell culture assays and animal studies are A range of dosages for use in the present invention can be used in formulating such a formulation. The dosage of the compound is preferably sufficient to achieve ED with little or no toxicity. 50 Circulation including The dosage may vary within this range depending on the dosage form and route of administration used. May fluctuate.

[0082] The mode of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, and parenteral; Intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation In certain embodiments, the antibodies of the invention are administered by: It can be administered by an invasive route such as injection. The antibodies of the present invention or pharmaceutical compositions thereof can be administered intravenously, subcutaneously, intramuscularly, intraarterially, or intravenously. Administered by aerosol delivery; non-invasive routes (e.g., oral; e.g., pill, Administration by capsule or tablet is also within the scope of the present invention.

[0083] The compositions can be administered using medical devices known in the art. The pharmaceutical compositions are administered by injection, for example, with a prefilled syringe or a hypodermic needle, including an auto-injector. The pharmaceutical compositions disclosed herein can also be administered by injection. No. 6,620,135; No. 6,096,002; No. 5,399,163; No. No. 5,383,851; No. 5,312,335; No. 5,064,413; No. 4 ,941,880; disclosed in No. 4,790,824 or No. 4,596,556 The drug may be administered using a needleless hypodermic injection device, such as the device described herein.

[0084] The pharmaceutical compositions disclosed herein may also be administered by injection. Examples of well-known implants and modular forms of administering drugs include those that deliver drugs at a controlled rate. U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing substances No. 4,499,499, which discloses a drug infusion pump for delivering drugs at precise infusion rates. No. 47,233, a U.S. patent disclosing a variable flow rate implantable infusion device for continuous drug delivery. No. 4,447,224, which discloses an osmotic drug delivery system having multiple chamber compartments Many other such implants, including U.S. Patent No. 4,439,196, Systems and modules are well known to those skilled in the art.

[0085] Alternatively, the antibodies of the invention can be administered locally rather than systemically, often in a depot or sustained release formulation. It may be administered in a sustained release formulation.

[0086] The dosing regimen may be adjusted based on serum or tissue turnover rates of the therapeutic antibody, the level of symptoms, and the therapeutic antibody dose. Several factors, including the immunogenicity of the body, as well as the accessibility of target cells in the biological matrix Preferably, the administration regimen will be one that simultaneously minimizes undesirable side effects. deliver sufficient therapeutic antibodies to result in improvement of the targeted disease / symptom state while minimizing Thus, the amount of biologic delivered will depend, in part, on the specific therapeutic antibody and treatment. The dose depends on the severity of the condition being treated. Guidance is available for selecting the appropriate dose of a therapeutic antibody. It is possible [e.g., Wawrzynczak Antibody Therapy,B ios Scientific Pub.Ltd,Oxfordshire,UK(19 96);Kresina(ed.) Monoclonal Antibodies, Cy tokines and Arthritis,Marcel Dekker,New York, NY (1991); Bach (ed.) Monoclonal Antibo dies and Peptide Therapy in Autoimmune D iseases, Marcel Dekker, New York, NY (1993); Baert,et al.New Engl.J.Med.348:601-608(2 003);Milgrom et al.New Engl.J.Med.341:19 66-1973(1999);Slamon et al.New Engl.J.Me d.344:783-792(2001); Beniaminovitz et al. New Engl.J.Med.342:613-619(2000);Ghosh e t al.New Engl.J.Med.348:24-32(2003);Lips ky et al.New Engl.J.Med.343:1594-1602(20 00).

[0087] Determining the appropriate dosage is well known in the art and may affect treatment, for example. This is done by a veterinarian using the parameters or factors that are currently or suspected. It is recommended to start with a somewhat less than optimal dose and then increase the desired level relative to any negative side effects. or by small increments until optimal effect is achieved. Important diagnostic measures include: Includes diagnostic scales for:

[0088] The antibodies provided herein can be administered by continuous infusion or, for example, daily, once a week, or once a week. Depending on the dose, it may be given weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, or annually. Doses can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, or intravenously. It may be delivered intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0. 0.5 μg / kg body weight, more commonly at least 0.2 μg / kg, 0.5 μg / kg, 1μg / kg, 10μg / kg, 100μg / kg, 0.25mg / kg, 1.0mg / kg, 2.0mg / kg, 5.0mg / ml, 10mg / kg, 25mg / kg, 50m g / kg or more [e.g., Yang, et al. New Engl .J.Med.349:427-434(2003);Herold,et al.Ne w Engl.J.Med.346:1692-1698(2002);Liu,et al.J.Neurol.Neurosurg.Psych.67:451-456(1 999);Portielji,et al.Cancer Immunol.Immu nother.52:133-144(2003)]. 0.1, 0.3, 1, 3, 10, The concentration of the compounds of the present invention in dog serum, such as 30, 100, 300 μg / ml or more Dosages to achieve a predetermined target concentration of the antibody may also be provided. The antibodies are available in 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject subcutaneously weekly, every other week, every 4 weeks, monthly, every other month, or every 3 months It is administered intravenously.

[0089] As used herein, "inhibit" or "treat" or "treatment" refers to the treatment of a disorder. delaying the onset of symptoms associated with and / or reducing the severity of symptoms of such disorders. These terms further include improving existing uncontrolled or undesired symptoms, Preventing further symptoms and ameliorating or preventing the underlying causes of such symptoms. Thus, these terms include disorders, conditions and / or symptoms. or a vertebrate subject (e.g., a mammalian animal) that has the potential to develop such a disorder, disease, or symptom. It indicates that beneficial results have been achieved in animals (e.g., dogs).

[0090] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to The term "antibody" refers to a therapeutic agent, alone or in combination with an additional therapeutic agent, that is administered to a cell, tissue, or subject, e.g., an injectable steroid, or a steroid drug. When administered to a subject, the compound or compound(s) may induce one or more symptoms of a disease or condition or cause the development of such a disease or condition. It refers to an amount of an antibody of the invention that is effective to cause a measurable improvement in the progression of symptoms. An effective dose may also result in at least partial improvement of symptoms, e.g., treatment of associated medical conditions. , cure, prevention or amelioration, or the rapid treatment, cure, prevention or amelioration of such symptoms When applied to a combination, it refers to an amount of antibody sufficient to produce an increase in the degree of therapeutic efficacy. The doses, whether administered in combination, sequentially, or simultaneously, are sufficient to produce a therapeutic effect. An effective amount of a therapeutic agent refers to the combined amount of active ingredients that results in a diagnostic measure or parameter At least 10% of the data; usually at least 20%; preferably at least about 30%; more preferably More preferably, it results in an improvement of at least 40%, and most preferably at least 50%. An effective amount also includes a subjectively measured amount when a subjective scale is used to assess the severity of symptoms. This can result in improvements in objective measures. [Example]

[0091] [Example 1] Anti-IL-4 receptor α antibody General Materials and Methods Recombinant proteins are prepared by synthesizing the amino acid sequence of a selected protein from a commercial manufacturer (ATU M, Newark, California) and commercially manufactured The vendor selected the appropriate nucleotide sequence that encodes this amino acid sequence. Code sequences can also be obtained from publicly available DNA databases such as GenBank®. Commercial manufacturers then chemically synthesize the nucleic acids and then use the synthesized nucleic acids. The nucleic acid is then transfected into an expression plasmid to produce the corresponding recombinant protein. The vector was cloned into HEK-2 (pD2610-v10; available from AUTM). Plasmids were placed into either 93 cells or CHO cells to express the recombinant protein. Expression was allowed to proceed and the expressed recombinant protein was then isolated by conventional methods.

[0092] Balb / c mice were immunized multiple times (10 μg each time) over a period of 17 days. The original product was a canine IL-4R α chain extracellular domain (ECD)-human Fc fusion protein. After immunization, serum was collected from each mouse and purified with the canine IL-4 receptor α-chain ECD HIS-tagged antibody. The highest serum anti-IL-4 receptor α chain ECD titer was 1.25. Spleen cells from mice bearing the antibody were fused to the myeloma P3X63Ag8.653 cell line. Approximately 2 weeks after the treatment, IL-4 receptor α chain ECD HIS-tagged protein was detected by ELISA. Supernatants from putative hybridoma cells were tested for reactivity to proteins. Hybridomas that gave strong positive signals in A were subcloned by limiting dilution. The reactivity of the canine IL-4 receptor α chain ECD HIS-tagged protein was investigated. The anti-canine IL-4 receptor α antibody was the antibody c152H11VL3-cCL ks / c152H11VH3-cIgG-Bm and antibody c146E2VL3-cCL ks / c146E2VH3-cIgG-Bm.

[0093] The six CDRs for these two antibody families (three individual light chains (LCs) and The set of three heavy chain (HC) sequences is shown in Table 1A (nucleic acid sequences) and Table 1B (antibody sequences). Table 1B shows the amino acid sequence of 152H11, which contains the amino acid sequence of SEQ ID NO:28. It contains a modified mouse HCDR3 with the remainder being unmodified mouse CDRs. Table 1A provides nucleic acids encoding the 12 CDRs listed in Table 1B. Table 1C provides nucleic acids encoding the 12 CDRs listed in Table 1B. Amino acid sequence of unmodified mouse HCDR3 for 52H11 (SEQ ID NO: 49) and the nucleic acid sequence encoding this unmodified mouse HCDR3 (SEQ ID NO: 48) The amino acids of the modified mouse HCDR3 of the 152H11 antibody family are The sequence (SEQ ID NO: 28) is a sequence in which the C-terminal cysteine ​​residue of the amino acid sequence of SEQ ID NO: 49 is substituted with seryl. The amino acid residues are replaced with amino acid residues of the corresponding unmodified mouse HCDR3. The unmodified murine HCDR of the 152H11 antibody family differs from the amino acid sequence. 3 (SEQ ID NO: 49) is a clone of c152H11VH1-cIgGBm (SEQ ID NO: 36) and c 152H11VH2-cIgGBm (SEQ ID NO: 37), but c152H 11VH3-cIgGBm (SEQ ID NO: 38) has an altered mouse HCDR3 (SEQ ID NO: 2 The amino acid sequences of the full-length light and heavy chains for the caninized antibodies are shown in Table 1C. later provided.

[0094] [Table 3]

[0095] [Table 4]

[0096] [Table 5]

[0097] c152H11VL3-cCLk-s (κ light chain): [SEQ ID NO: 35] EIVMTQSPASLSLSQEEKVTITC KASQNVGTNVA WYQQK PGQAPKLLIY SASYRYS GLPDRFSGSGSGTDFSFTISSLE PEDVAEFFC QQYNSYPYT FGQGTKLEIKRNDAQPAVYLFQ PSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGI QESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKS LPSTLIKSFQRSECQRVD

[0098] c152H11VH1-cIgGBm (heavy chain): [SEQ ID NO: 36] EVQLVESGGDLVKPGGSLRLSCAASGFTFS SYGMS WVRQ APGKGLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTL YLQMNSLRAEDTAMYYCAK GTLNNRGFAC WGQGTLVTVSS ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS WNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSET FTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQ ISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWL KGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPS REELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYR TTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL HNHYTQESLSHSPG

[0099] c152H11VH2-cIgGBm (heavy chain): [SEQ ID NO: 37] EVQLVESGGDLVKPGGSLRLSCAASGFTFS SYGMS WVRQ APDKRLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTL YLQMNSLRAEDTAMYYCAR GTLNNRGFAC WGQGTLVTVSS ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS WNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSET FTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQ ISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWL KGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPS REELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYR TTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL HNHYTQESLSHSPG

[0100] c152H11VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 38] EVQLVESGGDLVKPGGSLRLSCAASGFTFS SYGMS WVRQ APDKRLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTL YLQMNSLRAEDTAMYYCAR GTLNNRGFAS WGQGTLVTVSS ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS WNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSET FTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEM LGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQ ISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWL KGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPS REELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYR TTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL HNHYTQESLSHSPG

[0101] c146E2VL3-cCLk-s (κ light chain): [SEQ ID NO: 39] DIVLTQTPLSLSVSPGETASIYC RASESVDSYGNSFLN W YQQKPGQPPKLLIY RASNLAS EIPDRFSGSGSRTEFTLKI SRVEADDAGVYYC QQNYENPRT FGQGTKLEIKRNDAQPAV YLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQ DTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEI THKSLPSTLIKSFQRSECQRVD

[0102] c146E2VH1-cIgGBm (heavy chain): [SEQ ID NO: 40] EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WVRQ APGAGLDWMG MIHPDSGNINYNERFKT RVTLTADTSTSTA YMELSSLRAGDIAVYYCAR QLRNAMDY WGQGTLVTVSSAS TTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWN SGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFT CNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLG GPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKG KQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSRE ELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTT PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG

[0103] c146E2VH2-cIgGBm (heavy chain): [SEQ ID NO: 41] EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WMKQ APGAGLDWIG MIHPDSGNINYNERFKT KATLTADTSTSTA YMELSSLRAGDIAVYYCAR QLRNAMDY WGQGTLVTVSSAS TTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWN SGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFT CNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLG GPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKG KQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSRE ELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTT PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG

[0104] c146E2VH3-cIgG-Bm (heavy chain): [SEQ ID NO: 42] EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WMKQ APGAGLDWIG MIHPDSGNINYNERFKT KATLTVDKSTSTA YMELSSLRAGDIAVYYCAR QLRNAMDY WGQGTLVTVSSAS TTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWN SGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFT CNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLG GPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQIS WFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKG KQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSRE ELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTT PPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHN HYTQESLSHSPG

[0105] Furthermore, the light chain of the canine IL-4 receptor α antibody is also composed of a λ light chain, as provided below. was built.

[0106] c152H11LV1-cCl (lambda light chain) [SEQ ID NO: 43] QSVLTQPASVSGSLGQRVTISC KASQNVGTNVA WYQQLP GTSPRTLIY SASYRYS GVPDRFSGSRSGSTATLTISGLQA EDEADYYC QQYNSYPYT FGGGTHLTVLGQPKASPSVTLFP PSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGV ETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEG STVEKKVAPAECS

[0107] c146E2LV1-cCl (lambda light chain) [SEQ ID NO: 44] QSVLTQPASVSGSLGQRVTISC RASESVDSYGNSFLN WY QQLPGKAPSLLIY RASNLAS GVPERFSGSKSGSSATLTIT GLQAEDEADYYC QQNYENPRT FGGGTHLTVLGQPKASPSV TLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPV TQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLV THEGSTVEKKVAPAECS

[0108] [Example 2] STAT-6 inhibition for its ability to inhibit STAT-6 phosphorylation in DH82 cells as follows: Antibodies against canine IL-4 receptor alpha were tested: material 1. Actively growing DH82 cells 2. DH82 Cell Growth Media (ATCC® 3020 03™, supplied with heat-inactivated fetal bovine serum to a final concentration of 15% w / v Eagle's Minimum Essential Medium) 3.AlphaLISA p-STAT6(Tyr641) Assay Kit:Pe rkin 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 / C F 6. Perkin Elmer Envision a. Caninized anti-canine IL-4R α Monoclonal antibodies b.c152H11-H3L3 c. 4H3 caninized antibody from U.S. Patent Application Publication No. 2018 / 0346580

[0109] for its ability to inhibit STAT-6 phosphorylation in DH82 cells as follows: Antibodies against canine IL-4 receptor alpha were tested: 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. Pre-diluting the test antibody to 500 μg / mL and then incubating it in DH82 Cell Grow The medium was removed from the cell culture plate and 50 μL / Serially diluted test samples from the wells were transferred to each plate. 3. Canine IL-4 at 5ng / mL in DH82 Cell Growth Media Diluted and added 50 μL to each well of one plate. DH82 Cell Growth Dilute canine IL-13 to 10 ng / mL with IL-13 Media and add 50 μL to the second plate. The plate was incubated at 37°C for 15 minutes. 4. Remove the medium from the plate and add 100 μL / well of AlphaLISA pS Add freshly prepared 1x lysis buffer from the TAT-6 Assay Kit to the plate. The plate was agitated at 350 rpm on a plate shaker at room temperature for 10 minutes. 5. AlphaLISA p-STAT6 Assay Kit Acceptor Prepare the mix and add 30 μL of cell lysis solution to 96-well 1 / 2 area plates. 15 μL / well of the solution was added. The plate was sealed and agitated at 350 rpm for 2 minutes. , and then incubated at room temperature for 2 hours. 6. Run the AlphaLISA p-STAT6 Assay under dimmed laboratory lighting. Donor Mix was prepared from the kit and added to each plate at 15 μL / well. The plate was sealed, covered with foil, and stirred at 350 rpm for 2 minutes, then left to stand at room temperature for 2 hours. I was invited. 7. Using AlphaScreen settings on Perkin Elmer EnVison The plate was read using

[0110] Canine IL-4R α Blocks the binding of either canine IL-4 or canine IL-13 to c4H3 [International Publication No. 2002 / 0030906] was investigated for its ability to inhibit αSTAT-6 phosphorylation by 016 / 156588; U.S. Patent Application Publication No. 2018 / 0346580] and c1 Two different caninized monoclonal anti-canine IL-4R antibodies, designated 52H11-H3L3 α The data shown in Figure 1 demonstrate that both antibodies inhibited STAT-6 activation in the presence of IL-4. Surprisingly, c152H11-H3L3 produced a dose-dependent inhibition of phosphorylation. It has been demonstrated that the antibody binds more strongly to the canine IL-4 receptor α than the conventional anti-canine IL-4 receptor α antibody c4H3. International Publication No. 2016 / 156588]. IL-4Rα (IL-4R α ) in the absence of antibody The IL-4 control is shown in the upper right portion of the graph. In the presence of IL-13, c152H1 resulted in a dose-dependent inhibition of STAT-6 phosphorylation 1-H3L3 also binds more tightly than the prior art anti-canine IL-4 receptor α antibody c4H3. We also demonstrate that IL-4Rα (IL-4R α ) IL-13 in the absence of antibodies The reference is shown in the upper right portion of the graph. Figure 3 shows that replacing the kappa light chain with a lambda light chain significantly reduces the number of c152 H11-H3L3 IL-4R α This indicates that the binding to β-lactamase was not affected.

[0111] [Example 3] Epitope mapping The interaction of an antibody with its cognate protein antigen is determined by the specific amino acids (paratope) of the antibody. The binding of a specific amino acid (epitope) to a target antigen is mediated by the An epitope is an antigenic determinant that elicits a specific response by immunoglobulins. The protein of interest consists of a group of amino acids on the surface of a protein that are recognized by different antibodies. The epitope recognized by the antibody may be a linear epitope. Epitopes are classified as linear or structural epitopes. Linear epitopes are those found in proteins. A structural epitope is formed by a series of consecutive amino acids. Although they are discontinuous (e.g., far apart) in the amino acid sequence, they are It is composed of amino acids that come together when consumed.

[0112] Epitope mapping is the process of identifying the amino acid sequence ( It refers to the process of identifying the target antigen (i.e., epitope) by a monoclonal antibody (mAb). The identification of the epitope recognized by b) has important applications, e.g., on a target antigen. Identification of epitopes recognized by monoclonal antibodies (mAbs) has led to the development of new therapeutic agents. Epitope mapping can also aid in the development of diagnostics and vaccines. Assist in the selection of optimized therapeutic mAbs and elucidate the mechanism of action of optimized therapeutic mAbs The epitope information on the IL-4 receptor alpha can also be used to identify unique epitopes. Epitope identification can be used to determine the protective or pathogenic effects of vaccines. The method involves chemical or immunological coupling of the identified peptide epitopes to carrier proteins or other immunostimulatory agents. or may lead to the development of subunit vaccines based on genetic coupling. .

[0113] Epitope mapping can be performed using polyclonal or monoclonal antibodies. can be determined in several ways depending on the suspected nature of the epitope (i.e., linear vs. structural). Methods have been used for epitope identification. Mapping of linear epitopes is more direct. For this purpose, linear epitope maps are used. Commercial services for peptide scanning often use peptide scanning. In this case, a set of overlapping short peptide sequences of the target protein are chemically synthesized and These antibodies are then tested for their ability to bind to the target antibody. This strategy is rapid, high-throughput, and On the other hand, mapping of discontinuous epitopes is technically difficult. X-ray co-binding of monoclonal antibodies with their target proteins is generally more difficult. Crystallography, hydrogen-deuterium (H / D) exchange, mass spectrometry coupled with enzymatic digestion, and methods known in the art This requires more specialized techniques than some other methods known to those skilled in the art.

[0114] Mapping the Canine IL-4 Receptor Alpha Epitope Using Mass Spectrometry: Used to identify the epitope recognized by anti-canine IL-4 receptor α mAb A method based on chemical cross-linking, mass spectrometric detection and covalent labeling [999 Broad Way, Suite 305, Saugus, MA 01906-4510USA CovalX Instruments Incorporated).

[0115] Previous studies have applied this technique to epitope mapping of the canine IL-4 receptor α chain. The mAb binds to a specific peptide epitope present in the extracellular domain of canine IL-4 receptor α. It has been shown that the nucleotide sequence can recognize the target protein [US Patent Application Publication No. 2018 / 0346580]. 4, performed on the c152H11-H3L3 antibody against canine IL-4Rα. Similar analyses using the same method identified epitopes with reasonable similarity to previously identified epitopes. The amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 47 were identified for the group(s). As shown in FIG. 4, the amino acid residue S 111 , H 112 , T 113 , T 119 , Y 122 , T 124 , H 127 Y 150 , T 153 , Y 154, T 158 , R 160 , S 164 , T 165 , S 168 , S 171 , Y 172 and S 173 However, as a specific contact point [See, e.g., SEQ ID NO: 5 for amino acid residue numbering].

[0116] [Table 6] TIFF2025166118000008.tif33160

Claims

1. Canine interleukin-4 receptor alpha (IL-4R) containing a series of six complementarity-determining regions (CDRs) 1. An isolated mammalian antibody or antigen-binding fragment thereof that binds to IL-4Rα, comprising: Three of them are heavy chain CDRs: CDR heavy 1 (HCDR1), CDR heavy 2 (HCDR2) and CDR heavy 3 (HCDR3), and three of them are light chain CDRs: CDR light 1 (LCDR1), CDR light 2 (LCDR2), and CDR light 3 (LCDR3) There, where (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24; (ii) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 26; (iii) the HCDR3 is selected from the group consisting of SEQ ID NO: 28 and SEQ ID NO:

49. comprising an amino acid sequence (iv) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 30; (v) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and (vi) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 34; An isolated mammalian antibody or antigen-binding fragment thereof.

2. The antibodies and antigen-binding fragments thereof are directed against canine IL-4R α binds to canine IL-4R α Noi 2. The isolated mammalian antibody of claim 1, which blocks binding to interleukin-4. or an antigen-binding fragment thereof.

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

4. 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 hinge region comprising the amino acid sequence 。

5. A modified canine IgG-B (IgG-Bm) comprising the amino acid sequence of SEQ ID NO:

10. The caninized antibody or antigen-binding fragment thereof of claim 3, comprising a heavy chain.

6. An amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:

38.

6. The caninized antibody or antigen-binding fragment thereof of claim 5, comprising a heavy chain comprising a sequence.

7. The caninized antibody of claim 6 or its derivatives, comprising a light chain comprising the amino acid sequence of SEQ ID NO:

35. An antigen-binding fragment of.

8. The caninized antibody of claim 6, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 43; or An antigen-binding fragment thereof.

9. The caninized antibody or antigen-binding fragment thereof according to claim 3, 4, 5, 6, 7 or 8. An isolated nucleic acid encoding a heavy chain.

10. The caninized antibody or antigen-binding fragment thereof according to claim 3, 4, 5, 6, 7 or 8. An isolated nucleic acid encoding a light chain.

11. An expression vector comprising the isolated nucleic acid of claim 9 or 10.

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

13. A caninized antibody according to claim 3, 4, 5, 6, 7 or 8, and a pharmaceutically acceptable carrier. or a diluent.

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

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