RU2840044C1 - Canine alpha interleukin-4 receptor antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are a mammal antibody or an antigen-binding fragment thereof, which bind to a canine α-receptor of interleukin-4, as well as nucleic acids coding them, expression vectors and host cells for producing antibodies. Invention also relates to the use of antibodies for treating atopic dermatitis in dogs.

EFFECT: invention provides high binding affinity to canine α-receptor IL-4 and blocking the binding of canine IL-4 and/or IL-13 with canine α-receptor IL-4.

18 cl, 4 dwg, 5 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 63/015,220, filed April 24, 2020, 63/015,209, filed April 24, 2020, 62/951,793, filed December 20, 2019, the contents of each of which are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to antibodies against canine IL-4 alpha receptor that exhibit high binding affinity to canine IL-4 alpha receptor, which can block the binding of canine IL-4 and/or IL-13 to canine IL-4 alpha receptor. The present invention also relates to epitopes that are bound by antibodies to canine IL-4 alpha receptor. The present invention also relates to the use of antibodies of the present invention in the treatment of atopic dermatitis in dogs.

LEVEL OF TECHNOLOGY

The immune system consists of a network of resident and recirculating specialized cells that function together to protect the host from infectious diseases and cancer. The ability of the immune system to perform this function depends largely on the biological activity of a group of proteins secreted by white blood cells, collectively referred to as interleukins. Among the well-studied interleukins are four important molecules identified as interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-31 (IL-31), and interleukin-22 (IL-22). IL-4 and IL-13 are closely related proteins that can be secreted by many cell types, including CD4 ⁺ Th2 cells, natural killer T (NKT) cells, macrophages, mast cells, and basophils. IL-4 and IL-13 have many overlapping functions and are critical for the development of T cell-dependent humoral immune responses. IL-4 is known to bind with high affinity to two receptors, i.e. IL-4 receptors type I and type II. IL-4 receptor type I consists of the IL-4 receptor α chain and the common γ-C chain. IL-4 receptor type II consists of the IL-4 receptor α chain and the IL-13 receptor α1 chain. IL-13 binds to IL-4 receptor type II and to a unique receptor designated IL-13 receptor α2. Binding of IL-13 to IL-13 receptor α2 does not transduce a signal, and this receptor is also secreted in soluble form. Accordingly, IL-13 receptor α2 is often referred to as a decoy receptor. Although IL-4, IL-13, IL-22, and IL-31 are important cytokines for the development of immune responses that are required to defend against extracellular pathogens (e.g., tissue- or luminal-resident parasites), these cytokines are also involved in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

Atopic dermatitis (AD) is a recurrent, pruritic, and chronic inflammatory skin disease characterized by immune dysregulation and epidermal barrier abnormalities in humans. The pathologic and immunologic features of atopic dermatitis have been the subject of extensive research [for review, see Rahman et al. Inflammation & Allergy-drug target 10:486–496 (2011) and Harskamp et al., Seminar in Cutaneous Medicine and Surgery 32:132–139 (2013)]. Atopic dermatitis is also common in companion animals, particularly dogs, with a prevalence of approximately 10–15% in the canine population. Pathogenesis of atopic dermatitis in dogs and cats [see reviewed by Nuttall et al., Veterinary Records 172(8):201–207 (2013)] demonstrates significant similarities to the pathogenesis of human atopic dermatitis, including skin infiltration by various immune cells and a CD4 ⁺ Th2 polarized cytokine milieu including a predominance of IL-4, IL-13, and IL-31. In addition, IL-22 has been implicated in increased epithelial cell proliferation leading to epidermal hyperplasia, which is characteristic of atopic dermatitis.

For example, antibodies to canine IL-31 have been shown to have a significant effect on pruritus associated with atopic dermatitis in dogs [US 8790651 B2; US 10,093,731 B2]. Additionally, an antibody to the human IL-31 receptor alpha (IL-31RA) was tested and found to have a significant effect on pruritus associated with atopic dermatitis in humans [Ruzicka, et al., New England Journal of Medicine, 376(9), 826-835 (2017)]. Accordingly, blocking IL-31 binding to the IL-31RA receptor results in relief of pruritus associated with atopic dermatitis.

Monoclonal antibodies to human IL-4 receptor alpha (IL-4 Rα) have been developed, and some of these antibodies have been extensively tested for therapeutic activity in the treatment of atopic dermatitis in humans [see, e.g., US2015/0017176 A1]. More recently, caninized antibodies to canine IL-4 Rα have also been described that block the binding of canine IL-4 to canine IL-4 Rα [US 2018/0346580A1, incorporated herein by reference in its entirety]. Since the IL-4 receptor type II consists of the IL-4 receptor α-chain and the IL-13 receptor α1-chain, antibodies to canine IL-4 Rα have been developed that can block the binding of both canine IL-4 and canine IL-13 to canine IL-4 receptor type II, thereby blocking the inflammation associated with atopic dermatitis [US2018/0346580A1].

Interleukin-22 (IL-22), also known as IL-10 T-cell-like inducible factor (IL-TIF), belongs to the IL-10 cytokine family. IL-22 is produced by normal T cells upon anti-CD3 stimulation in humans. Murine IL-22 expression is also induced in various organs by lipopolysaccharide injection, suggesting that IL-22 may be involved in inflammatory responses. IL-22 specifically binds to and signals through a receptor complex consisting of a heterodimeric complex of IL-10R2 (also known as IL-10R beta) and the interleukin-22 receptor (IL-22R) [see Lee et al., Pharmacology Research & Perspectives, pp. 1–13 (2018:e00434)]. The interleukin-22 receptor is also known as interleukin-22R, alpha 1; IL-22RA1; IL-22R1; zcytor11; and CRF2-9 [Xu et al., Proc. Nat. Acad. Sci. 98(17):9511–9516 (2001); Gelebart and Lai, Atlas of Genetics and Cytogenetics 14(12):1106–1110 (2010)]. IL-22 induces epithelial cell proliferation during wound healing, and its deficiency can lead to uncontrolled proliferation and enhanced tumor progression [Huber et al., Nature 491:259–263 (2012)]. IL-22 has been shown to activate STAT-1 and STAT-3 in several hepatoma cell lines and enhance the production of acute phase proteins. Anti-IL-22 and IL-22R antibodies act as antiproliferative agents by blocking the interaction of IL-22 with IL-22R and thereby the associated signaling pathway, which leads to proliferation of epithelial cells.

However, despite recent success in the treatment of atopic dermatitis, none of the currently used treatments provide rapid onset of antipruritic action accompanied by significant effects on skin inflammation with improvement of skin barrier function. Therefore, there is a need to develop more effective treatments that can eliminate one or more symptoms of atopic dermatitis.

The citation of any reference in this specification shall not be construed as an admission that such reference may be used as "prior art" with respect to the present application.

ESSENCE OF THE INVENTION

The present invention provides novel caninized antibodies to canine IL-4R-alpha (IL-4Rα), which are isolated in specific embodiments and which have superior properties compared to antibodies known in the art, such as binding more tightly than prior art canine IL-4 receptor alpha antibodies. In particular embodiments, the present invention relates to mammalian antibodies or antigen-binding fragments thereof that specifically bind to a canine interleukin-4 alpha receptor comprising a heavy chain that comprises a set of three heavy chain complementarity determining regions (CDRs), heavy chain CDR 1 (HCDR1), heavy chain CDR 2 (HCDR2), and heavy chain CDR 3 (HCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:24, HCDR2 comprises the amino acid sequence of SEQ ID NO:26, and HCDR3 comprises the amino acid sequence of SEQ ID NO:28. In other embodiments, the present invention relates to mammalian antibodies or antigen-binding fragments thereof that specifically bind to a canine interleukin-4 alpha receptor comprising a heavy chain that comprises a set of three heavy chain CDRs, HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:24, HCDR2 comprises the amino acid sequence of SEQ ID NO:26, and HCDR3 comprises the amino acid sequence of SEQ ID NO:49.

In related embodiments, mammalian antibodies or antigen-binding fragments thereof that specifically bind to canine interleukin-4 receptor α (IL-4Rα) further comprise a light chain that comprises a set of three light chain CDRs: light chain CDR 1 (LCDR1), light chain CDR 2 (LCDR2), and light chain CDR 3 (LCDR3), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:30, LCDR2 comprises the amino acid sequence of SEQ ID NO:32, and LCDR3 comprises the amino acid sequence of SEQ ID NO:34. In preferred embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine IL-4Rα and blocks the binding of canine IL-4Rα to canine interleukin-4 (cIL-4). In related embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine IL-4Rα and blocks the binding of canine IL-4Rα to canine interleukin-13 (cIL-13). In other embodiments, the mammalian antibody or antigen-binding fragment thereof binds to canine IL-4Rα and blocks the binding of canine IL-4Rα to cIL-4 and to cIL-13.

In particular embodiments, the mammalian antibody to canine IL4R-alpha is a murine antibody. In related embodiments, the mammalian antibody to canine IL4R-alpha is a caninized murine antibody. In particular embodiments, the caninized antibody comprises a heavy chain that comprises an IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO:6. In other embodiments, the caninized antibody comprises a heavy chain that comprises an IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO:7. In other embodiments, the caninized antibody comprises a heavy chain that comprises IgG-D cFc, but the naturally occurring IgG-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO: 8. In other embodiments, the caninized antibody comprises a heavy chain that comprises IgG D cFc, but the naturally occurring IgG-D hinge region is replaced with a hinge region comprising the amino acid sequence of SEQ ID NO: 9.

In particular embodiments, the caninized antibody comprises a heavy chain comprising a modified canine IgG-B (IgG-Bm) comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:36. In other embodiments, the caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:37. In other embodiments, the caninized antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:38. In particular embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO:35. In alternative embodiments, the caninized antibody further comprises a light chain that comprises the amino acid sequence of SEQ ID NO:43.

In specific embodiments, the caninized antibodies or antigen-binding fragments thereof bind to SEQ ID NO:46. In more specific embodiments, the caninized antibodies or antigen-binding fragments thereof bind to one, two, three, four, five, six, or all seven of the following amino acid residues of canine IL-4Rα: _S111 , _H112 , _T113 , _T119 , _Y122 , _T124 , and _H127 of SEQ ID NO:5. In related embodiments, the caninized antibodies or antigen-binding fragments thereof bind to SEQ ID NO:47. In more specific embodiments, the caninized antibodies or antigen-binding fragments thereof bind to one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following amino acid residues of canine IL-4Rα: _Y150 , _T153 , Y154, _T158 , _R160 , _S164 , _T165 , _S168 , _S171 , _Y172 , _{S173 of} SEQ ID NO:5. In even more specific embodiments, the caninized antibodies or antigen-binding fragments thereof bind to both SEQ ID NO:46 and SEQ ID NO:47. In even more specific embodiments of this type, the caninized antibodies or antigen-binding fragments thereof bind to one, two, three, four, five, six, or all seven of the following amino acid residues of canine IL-4Rα: _S111 , _H112 , _T113 , _T119 , _Y122 , _T124 , and _H127 of SEQ ID NO:5 and/or to one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following amino acid residues of canine IL-4Rα: _Y150 , _T153 , _Y154 , _T158 , _R160 , _S164 , _T165 , _{S168 , S171} , Y172, _S173 of SEQ ID NO:5.

The present invention also relates to nucleic acids, including isolated nucleic acids, that encode CDRs, heavy chains of caninized antibodies or antigen-binding fragments thereof, and/or light chains of caninized antibodies or antigen-binding fragments thereof. In addition, the present invention relates to expression vectors that contain such nucleic acids and host cells that contain such expression vectors.

The present invention further relates to pharmaceutical compositions that comprise caninized antibodies and antigen-binding fragments thereof of the present invention together with a pharmaceutically acceptable carrier and/or diluent. The present invention further relates to methods for treating atopic dermatitis, comprising administering one of the above-mentioned compositions to a canine suffering from atopic dermatitis. In particular embodiments, the present invention relates to methods for blocking inflammation associated with atopic dermatitis, comprising administering to a canine in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention.

These and other aspects of the present invention will be better understood with reference to the following brief description of the drawings and detailed description.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a graph showing the inhibition of IL-4-mediated STAT-6 phosphorylation by anti-IL-4Ralpha (IL-4Rα) antibodies. Two different caninized monoclonal antibodies to canine IL-4Rα, designated c4H3 [see WO2016/156588] and c152H11-H3L3, were assessed 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. A control of IL-4 in the absence of anti-IL-4Ralpha (IL-4Rα) antibodies is shown in the upper right portion of the graph.

Figure 2 is a graph showing the inhibition of IL-13-mediated STAT-6 phosphorylation by anti-IL-4Ralpha antibodies. Two different caninized monoclonal antibodies to canine IL-4Ralpha, designated c4H3 [see WO2016/156588] and c152H11-H3L3, were assessed 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 anti-IL-4Ralpha antibodies (IL-4Ralpha) is shown in the upper right portion of the graph.

Figure 3 shows the binding of caninized anti-canine IL-4Rα antibodies containing lambda or kappa light chains as determined 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 in a similar manner to caninized anti-canine IL-4Rα antibodies containing the same CDRs but with a kappa light chain (c152H11-H3L3). 152mc is a positive control of the chimeric mouse-dog antibody, and Iso-Ctr, which serves as a negative control, is an unrelated caninized antibody.

Figure 4 shows the epitope of antibody c152H11-H3L3 comprising the amino acid sequences of SEQ ID NO:46 and SEQ ID NO:47 on canine IL-4Rα.

DETAILED DESCRIPTION OF THE INVENTION

In response to the need for more effective methods for treating atopic dermatitis, the present invention provides caninized antibodies, caninized antibody formulations, and methods that can have a significant effect on skin inflammation associated with atopic dermatitis.

ABBREVIATIONS

The following abbreviations are used in the detailed description and examples of the invention:

ADCC - Antibody-dependent cellular cytotoxicity

CDC - Complement-dependent cytotoxicity

CDR - Complementarity-determining region in immunoglobulin variable regions, defined using the Kabat numbering system

EC50 - Concentration resulting in 50% efficacy or binding

ELISA - Enzyme-linked immunosorbent assay

FR - Antibody framework region: the variable regions of the immunoglobulin, excluding the CDR regions.

IC50 - Concentration resulting in 50% inhibition

IgG - Immunoglobulin G

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

mAb - Monoclonal antibody (also Mab or MAb)

V region - A segment of the IgG chains whose sequence varies among antibodies. According to Kabat numbering, it extends to residue 109 in the light chain and 113 in the heavy chain.

VH - Variable region of the immunoglobulin heavy chain

VL - Variable region of the immunoglobulin light chain

VK - Variable region of the kappa light chain of immunoglobulin

DEFINITIONS

In order to make the invention more understandable, some technical and scientific terms are defined below. Unless otherwise specifically defined in this description, all other technical and scientific terms used have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

As used in the present specification, including the appended claims, references to singular forms such as "a," "an," and "the" include corresponding plural references unless the context clearly dictates otherwise.

"Administering" and "treating" with respect to an animal, such as a canine, cell, tissue, organ, or biological fluid, refers to contacting an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the animal, such as a canine, cell, tissue, organ, or biological fluid. Treating a cell includes contacting a reagent with the cell, as well as contacting a reagent with a fluid, wherein the fluid is in contact with the cell.

"Administering" and "treating" also mean in vitro and ex vivo treatment, such as of a cell with a reagent, a diagnostic agent, a binding compound, or another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a canine, feline, or human), and most preferably a canine.

"Treat" or "treating" means administering a therapeutic agent, such as a composition comprising any of the antibodies of the present invention, internally or externally, for example, to a canine or patient with one or more symptoms of, or suspected of having, a condition for which said agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate and/or reduce one or more symptoms of the disease/condition in the subject or population being treated, either by inducing regression or inhibiting the progression of such symptom(s) by any clinically measurable amount. The amount of therapeutic agent that is effective to reduce any particular symptom of the disease/condition (also referred to as a "therapeutically effective amount") may vary depending on factors such as the severity of the disease/condition, the age and weight of the patient (e.g., the canine), and the ability of the pharmaceutical composition to elicit the desired response in the subject. The alleviation or amelioration of a symptom of a disease/condition can be assessed using any clinical measurement commonly used by veterinarians or other qualified medical professionals to assess the severity or progression of that symptom. Although an embodiment of the present invention (e.g., a method of treatment or an article of manufacture) may not be effective in alleviating the symptom(s) of the target disease/condition in every subject, it should alleviate the symptom(s) of the target disease/condition in a statistically significant number of subjects as determined by any statistical test known in the art, such as the Student t-test, the chi-square test, the Mann-Whitney U-test, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

"Treatment" in relation to a human, veterinary (e.g., canine), or research subject includes therapeutic treatment as well as research and diagnostic applications. "Treatment" in relation to a human, veterinary (e.g., canine), or research subject, or a cell, tissue, or organ includes contacting antibodies of the present invention with, for example, a canine or other animal, cell, tissue, physiological compartment, or physiological fluid.

As used herein, the term "canine" includes all domestic dogs, Canis lupus familiaris or Canis familiaris , unless otherwise specified.

As used herein, the term "feline" refers to any member of the family Felidae. Members of this family include wild cats, zoo cats, and domestic cats, including pet, purebred, and/or mongrel companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

As used herein, the term "canine framework" refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the hypervariable region residues, defined herein as CDR residues. With respect to a caninized antibody, in most embodiments, the amino acid sequences of the native canine CDRs are replaced by the corresponding foreign CDRs (e.g., from a murine antibody) in both chains. Optionally, the heavy and/or light chains of the canine antibody may contain some foreign residues other than the CDR residues, for example, to maintain the conformation of the foreign CDRs within the canine antibody and/or to modify Fc function, as shown below and/or disclosed in U.S. Pat. No. 10,106,607 B2, incorporated herein by reference in its entirety.

The "crystallizable fragment region," abbreviated "Fc," corresponds to the CH3-CH2 portion of an antibody that interacts with cell surface receptors called Fc receptors. The canine crystallizable fragment (cFc) regions of each of the four canine IgGs were first described by Tang et al. [Vet. Immunol. Immunopathol. 80:259–270 (2001); see also Bergeron et al., Vet. Immunol. Immunopathol. 157:31–41 (2014) and U.S. 10,106,607 B2].

The canine Fc (cFc) "IgG-Bm" as used herein is a canine IgG-B Fc comprising two (2) amino acid residue substitutions, D31A and N63A, in the amino acid sequence of SEQ ID NO:10 of IgG-B (see below), which lacks the C-terminal lysine ("K"). In IgG-Bm, both residues, the aspartic acid (D) residue at position 31 of SEQ ID NO:10 and the asparagine (N) residue at position 63 of SEQ ID NO:10, are substituted with an alanine (A) residue. These two amino acid residue substitutions substantially reduce the antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of naturally occurring canine IgG-B [see U.S. Patent No. 1,100, or the American National Antibody Convention (ANC). 10,106,607 B2, the contents of which are incorporated herein by reference in their entirety]. IgG-Bm also provides additional amino acid substitutions similar to those that may be introduced in parallel in IgG-B and which may include amino acid substitutions that facilitate the formation of heterodimers in bispecific antibodies. The amino acid sequence of IgG-B, SEQ ID NO:45, is as follows:

The amino acid sequence of IgG-Bm is shown below, SEQ ID NO:10.

lggpsvfifppkpkdtlliartpevtcvvvaldpedpevqiswfvdgkqmqtaktqpreeqfagtyrvvsvlpighqdwlkgkqftckvnnkalpspiertiskarg qahqpsvyvlppsreelskntvsltclikdffppdidvewqsngqqepeskyrttppqldedgsyflysklsvdksrwqrgdtficavmhealhnhytqeslshspg

In the context of the present description, the term "substitution of an amino acid residue" with another amino acid residue in the amino acid sequence of an antibody, for example, is equivalent to the term "replacement of an amino acid residue" with another amino acid residue and means that a specific amino acid residue at a specific position of the amino acid sequence is replaced (or is substituted) with another amino acid residue. Such substitutions can be specifically intended, i.e. can be a targeted replacement of alanine with serine at a specific position of the amino acid sequence, for example, made by recombinant DNA technology. Alternatively, a specific amino acid residue or strand of amino acid residues of an antibody can be replaced by one or more amino acid residues by more natural selection processes, for example, based on the ability of an antibody produced by a cell to bind to a given region on an antigen, for example, which contains an epitope or a portion thereof, and/or by selecting an antibody containing a specific CDR with a preserved canonical structure that is the same as the CDR it replaces. Such substitutions/replacements may result in "variant" CDRs and/or variant antibodies.

As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. An antibody may be a monomer, a dimer, or a larger multimer. It is therefore used in its broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies, and camelized (camelid-derived) single-domain antibodies. "Parent antibodies" are antibodies produced by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as caninization of the antibody for use as a canine therapeutic antibody.

As used herein, antibodies of the present invention that "block" or are "blocking" or "block binding" of, for example, a canine receptor to its binding partner (ligand), are antibodies that block (partially or completely) the binding of the canine receptor to the canine ligand and vice versa, as determined by standard binding assays (e.g., BIACore®, ELISA, or flow cytometry).

Typically, the antibody or antigen-binding fragment thereof of the invention retains at least 10% of its canine antigen binding activity (compared to the parent antibody), expressed in molar units. Preferably, the antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the parent antibody's affinity for binding the canine antigen. It is also contemplated that the antibody or antigen-binding fragment of the invention may include conservative or non-conservative amino acid substitutions (called "conservative variants" or "functional variants" of the antibody) that do not substantially alter its biological activity.

"Isolated antibody" refers to the state of purification and in this context means that the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term "isolated" does not refer to the complete absence of such material or to the absence of water, buffers, or salts unless they are present in amounts that actually interfere with the experimental or therapeutic use of the disclosed binding compound.

As used herein, the term "chimeric antibody" is an antibody that has a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species. [U.S. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)]. Typically, the variable domains are derived from an antibody of an experimental animal (the "parent antibody"), such as a rodent, and the constant domain sequences are derived from antibodies of a subject animal, such as a human or canine, such that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human or canine, respectively, compared to the parent (e.g., rodent) antibody.

As used herein, the term "caninized antibody" refers to forms of antibodies that comprise both canine and non-canine (e.g., murine) antibody sequences. Typically, a caninized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., comprising 6 CDRs as shown below) and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining framework regions) are regions of a canine immunoglobulin sequence. As shown herein, a caninized antibody comprises three heavy chain CDRs and three light chain CDRs of a murine antibody to a canine antigen, together with a canine framework or a modified canine framework. The modified canine scaffold comprises one or more amino acid substitutions as described herein that further optimize the efficacy of the caninized antibody, such as by increasing its binding to a canine antigen and/or the ability to block binding of the canine antigen to a natural binding partner of the canine antigen.

The variable regions of each light/heavy chain pair form the binding site of the antibody. Thus, typically an intact antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are usually the same. Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are typically aligned with the framework regions, allowing for binding to a specific epitope. Typically, in the N-terminal to C-terminal direction, the variable domains of both the light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain typically follow the definitions described in Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 ^th ed.; NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32:1-75 (1978); Kabat, et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989).

As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region comprises the amino acid residues of the "complementarity determining region" or "CDR" (i.e., LCDR1, LCDR2, and LCDR3 in the variable domain of the light chain and HCDR1, HCDR2, and HCDR3 in the variable domain of the heavy chain). [See Kabat et al. Sequences of Proteins of Immunological Interest, ^5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), for identification of antibody CDR regions by sequence; see also Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), for identification of antibody CDR regions by structure]. As used herein, the term "framework" or "FR" residues refers to those residues of the variable domain that are not hypervariable region residues, defined herein as CDR residues.

There are four known subtypes of IgG heavy chains of canine IgG, which are designated IgG-A, IgG-B, IgG-C, and IgG-D. Two known subtypes of the light chain are called lambda and kappa. In particular embodiments, in addition to binding to and activating canine immune cells, a canine or caninized antibody to an antigen of the present invention optimally has two properties:

1) it lacks effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and

2) It is easily purified on a large scale using standard industrial methods such as protein A chromatography.

None of the naturally occurring canine IgG isotypes meets both criteria. For example, IgG-B can be purified using protein A, but has a high level of ADCC activity. On the other hand, IgG-A weakly binds to protein A and also exhibits ADCC activity. Moreover, neither IgG-C nor IgG-D can be purified on protein A columns, although IgG-D does not exhibit ADCC activity. (IgG-C has significant ADCC activity.) One way in which the present invention solves these problems is to provide modified canine IgG-B antibodies of the present invention specific for an antigen of the present invention that lack effector functions such as ADCC and can be readily purified by standard protein A chromatography.

As used herein, an "anti-inflammatory antibody" is an antibody that can act as an anti-inflammatory agent in an animal, including a mammal, such as a human, canine, and/or feline, particularly with respect to atopic dermatitis. In specific embodiments, the anti-inflammatory antibody binds to specific proteins of the IL-4/IL-13 signaling pathway, such as IL-4 or the IL-4Rα receptor. Binding of the anti-inflammatory antibody to its corresponding antigen (e.g., IL-4 or IL-4Rα) inhibits the binding of, for example, IL-4 to IL-4Rα and interferes with and/or prevents signaling in this pathway, thereby preventing or preventing the occurrence of chronic inflammation associated with atopic dermatitis.

"Homology," as used herein, refers to the similarity between two polynucleotide sequences or two polypeptide sequences when optimally aligned. When a position in both sequences being compared is occupied by the same base or amino acid residue, such as a position in each of two DNA molecules occupied by an adenine, then the molecules are homologous at that position. Percent homology is the number of homologous positions common to the two sequences divided by the total number of positions being compared x 100. For example, if 6 out of 10 positions in two sequences are the same or homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Typically, comparisons are made with the two sequences aligned to obtain the maximum percent homology. Sequence identity refers to the degree to which the amino acids of the two polypeptides are the same at equivalent positions when the two sequences are optimally aligned.

In the context of the present description, one amino acid sequence is "identical" to a second amino acid sequence by 100%, when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is "identical" to a second amino acid sequence by 50%, when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed on a contiguous block of amino acid residues contained in a given protein, for example, a protein or a portion of a polypeptide to be compared. In particular embodiments, selected deletions or insertions that could otherwise change the correspondence between the two amino acid sequences are taken into account. Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids are amino acids that have similar properties and can be interchanged.

"Conservatively modified variants" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that such changes can often be made without altering the biological activity of the protein. Those skilled in the art recognize that, in general, substitutions of individual amino acids in non-essential regions of a polypeptide do not result in significant changes in biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 ( ^4th ed., 1987)]. Furthermore, substitution of structurally or functionally similar amino acids is less likely to result in impairment of biological activity. Examples of conservative substitutions are provided in Table A below.

TABLE A

Examples of conservative amino acid substitutions

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

The present invention also contemplates functionally conservative variants of the antibodies of the invention. "Functionally conservative variants," as used herein, refer to antibodies or fragments in which one or more amino acid residues are substituted without altering a desired property, such as affinity and/or specificity for an antigen. Such variants include, but are not limited to, substitution of an amino acid with an amino acid having similar properties, such as the conservative amino acid substitutions in Table A above.

"An isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that is not associated with all or part of a polynucleotide in which the isolated polynucleotide naturally occurs, or that is associated with a polynucleotide to which it is not naturally associated. For purposes of the present disclosure, it should be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules "comprising" particular nucleic acid sequences may include, in addition to the said sequences, sequences encoding up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the said nucleic acid sequences and/or may include vector sequences.

The present invention relates to isolated caninized antibodies of the present invention, methods of using these antibodies in the treatment of a condition, for example, in the treatment of atopic dermatitis in canines. In canines, four IgG heavy chains are found, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgG-A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC), and IgG-D (or IgGD). Each of the two heavy chains consists of one variable domain (VH) and three constant domains, designated CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2 domain by an amino acid sequence called a "hinge" or alternatively a "hinge region."

The nucleic acid and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80:259–270 (2001)]. The amino acid and nucleic acid sequences of these heavy chains are also available in the GenBank databases. For example, the amino acid sequence of the heavy chain of IgGA is available under the accession number AAL35301.1, IgGB is available under the accession number AAL35302.1, IgGC is available under the accession number AAL35303.1, and IgGD is available under the accession number (AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequence of these light chains can be obtained from the GenBank databases. For example, the amino acid sequence of the kappa light chain is available under the accession number ABY 57289.1, and the lambda light chain is available under the accession number ABY 55569.1.

In the present invention, the amino acid sequence for each of the four Fc regions of canine IgG is based on the identified boundary of the CH1 and CH2 domains as determined by Tang et al., supra. Caninized mouse anti-dog antibodies that bind to canine IL-4Rα include, but are not limited to, antibodies of the present invention that comprise canine heavy chains and/or canine light chains of kappa or lambda IgG-A, IgG-B, IgG-C, and IgG-D together with mouse CDRs to canine IL-4Rα. Accordingly, the present invention provides isolated caninized mouse anti-dog antibodies of the present invention that bind to canine IL-4Rα and block the binding of the canine IL-4Rα to canine IL-4 and/or canine IL-13, its natural binding partners.

Accordingly, the present invention further relates to caninized murine antibodies and methods of using the antibodies of the present invention in the treatment of a condition, such as in the treatment of atopic dermatitis in dogs.

The present invention also provides full-length canine heavy chains that can be paired with corresponding light chains to produce a caninized antibody. Accordingly, the present invention further provides caninized mouse antibodies to a canine antigen (including isolated caninized mouse antibodies to a canine antigen) of the present invention and methods of using the antibodies of the present invention in the treatment of a condition, such as in the treatment of atopic dermatitis in dogs.

The present invention also relates to antibodies of the present invention that comprise a canine crystallizable fragment region (cFc region), wherein the cFc is genetically modified to increase, decrease, or eliminate one or more effector functions. In one aspect of the present invention, the genetically modified cFc decreases or eliminates one or more effector functions. In another aspect of the invention, the genetically modified cFc enhances one or more effector functions. In some embodiments, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a specific embodiment, the effector function is antibody-dependent cytotoxicity (ADCC) that is enhanced, decreased, or eliminated. In another embodiment, the effector function is complement-dependent cytotoxicity (CDC) that is enhanced, decreased, or eliminated. In another embodiment, the cFc region is genetically modified to increase, decrease, or eliminate both ADCC and CDC.

To generate variants of canine IgG lacking effector functions, a series of mutant canine IgGB heavy chains were generated. These variants may include one or more of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. The heavy chain variants (i.e., containing these amino acid substitutions) were cloned into expression plasmids and transfected into HEK 293 cells along with a plasmid containing the gene encoding the light chain. Intact antibodies expressed and purified from HEK 293 cells were assayed for binding to FcγRI and C1q to assess their potency in mediating immune effector functions. [See U.S. Patent No. 1,388,100.] 10,106,607 B2, the contents of which are incorporated herein by reference in their entirety.]

The present invention also relates to modified canine IgG-D that, instead of the natural hinge region of IgG-D, comprises a hinge region of:

IgG A: FNECRCTDTPPCPVPEP SEQ ID NO:6

IgG B: PKRENGRVPPRPPPDCPKCPAPEM SEQ ID NO:7; or

IgG C: AKECECKCNCNNCPCPGCGL SEQ ID NO:8.

Alternatively, the hinge region of IgG-D can be genetically modified by replacing the serine residue with a proline residue, i.e., PKESTCKCI P PCPVPES, SEQ ID NO:9 (with the proline (P) residue underlined and in bold replacing the natural serine residue). Such modifications can produce canine IgG-D that does not undergo fab arm exchange. Modified canine IgG-D can be constructed using standard recombinant DNA techniques [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To generate these variants, nucleic acids encoding the canine IgG-D amino acid sequence can be modified to encode modified IgG-D. The modified nucleic acid sequences are then cloned into expression plasmids for protein expression.

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

Nucleic acids

The present invention also relates to nucleic acids encoding the antibodies of the present invention (see, for example, the examples below).

The present invention also relates to nucleic acids encoding immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of caninized antibodies, excluding the unaltered CDRs, as described herein, when compared using the BLAST algorithm, wherein the algorithm parameters are selected to obtain the maximum match between the corresponding sequences over the entire length of the corresponding reference sequences. The present invention also provides nucleic acids encoding immunoglobulin polypeptides that comprise amino acid sequences that are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences, when compared using the BLAST algorithm, wherein the algorithm parameters are selected to obtain the maximum match between the corresponding sequences over the entire length of the corresponding reference sequences.

The percentage of nucleotide and amino acid sequence identity used herein can be determined using the algorithm of C. MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and Clustal W. with default alignment parameters and default identification parameters. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, an advanced Blast search can be used under default filtering conditions, such as with the PILEUP GCG program (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) with default parameters.

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

The antibodies of the present invention can be produced recombinantly by methods known in the art. Mammalian cell lines available as hosts for expressing the antibodies or fragments of the present disclosure are well known in the art and include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and a variety of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, equine, and hamster cells. Cell lines that are particularly preferred are selected by identifying cell lines with high expression levels. Other cell lines that can be used include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When recombinant expression vectors encoding the heavy chain or an antigen-binding portion or fragment thereof, the light chain and/or an antigen-binding fragment thereof are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, sufficient to secrete the antibody into the culture medium in which the host cells are grown.

Antibodies can be isolated from the culture medium by standard protein purification techniques. In addition, expression of the antibodies of the invention (or other fragments thereof) in producer cell lines can be enhanced by several known methods. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. A complete or partial overview of the GS system can be found in European Patent Nos. 0216846, 0256055 and 0323997 and European Patent Application No. 89303964.4.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern characteristic of glycoproteins produced in that cell line or transgenic animal. Accordingly, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, included within the present invention are all antibodies encoded by the nucleic acid molecules described herein or comprising the amino acid sequences described herein, regardless of the glycosylation pattern the antibodies may have. Likewise, in particular embodiments, antibodies with a glycosylation pattern comprising only non-fucosylated N-glycans may be preferred, since these antibodies have been shown to be generally more potent than their fucosylated counterparts, both in vitro and in vivo [see, e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775].

CANINE IL-4 RECEPTOR, ALPHA RECEPTOR

The cDNA encoding the predicted full-length canine IL-4 receptor alpha chain (SEQ ID NO:1) was identified by searching the Genbank database (accession number XM_547077.4; see also US 7208579 B2). This predicted cDNA encodes 823 amino acids (SEQ ID NO:2), including a 25-amino acid leader sequence, and is identified by accession number XP_547077.3. The predicted mature canine IL-4 receptor alpha chain protein (SEQ ID NO:4) is 65% identical to the human IL-4 receptor alpha chain (accession number NP_000409.1) and 70% identical to the porcine IL-4 receptor alpha chain (accession number NP_999505.1). The predicted mature canine IL-4 receptor α-chain protein is encoded by the nucleotide sequence identified as SEQ ID NO:3. Comparison of the predicted mature IL-4 receptor α-chain with known human IL-4 receptor α-chain sequences allowed identification of the extracellular domain (ECD) of the mature canine IL-4 receptor α-chain protein, designated as SEQ ID NO:5. All of this has been previously described in [US2018/0346580; incorporated herein in its entirety].

Full-length DNA of canine IL-4 receptor α-chain with signal sequence [SEQ ID NO:1]

atgggcagactgtgcagcggcctgaccttccccgtgagctgcctggtgctggtgtgggtggccagcagcggcagcgtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccacccca ccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccgagaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacct gtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaacctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcac agcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctacatgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggccca gacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagcacctgcccctgggcgtgagcatcagctgcctggtgatcctggccatctgcctgagctgctacttcagcatcatcaagatc aagaagggctggtgggaccagatccccaaccccgcccacagccccctggtggccatcgtgatccaggacagccaggtgagcctgtggggcaagagaagcagaggccaggagcccgccaagtgcccccactggaagacctgcctgaccaagctgc tgccctgcctgctggagcacggcctgggcagagaggaggagagccccaagaccgccaagaacggccccctgcagggccccggcaagcccgcctggtgccccgtggaggtgagcaagaccatcctgtggcccgagagcatcagcgtggtgcagtg cgtggagctgagcgaggcccccgtggacaacgaggaggaggaggaggtggaggaggacaagagaagcctgtgccccagcctggagggcagcggcggcagcttccaggagggcagagagggcatcgtggccagactgaccgagagcctgttcctgg acctgctgggcggcgagaacggcggcttctgcccccagggcctggaggagagctgcctgcccccccccagcggcagcgtgggcgcccagatgccctgggcccagttccccagagccggccccagagccgcccccgagggccccgagcagcccag aagacccgagagcgccctgcaggccagccccacccagagcgccggcagcagcgccttccccgagcccccccccgtggtgaccgacaaccccgcctacagaagcttcggcagcttcctgggccagagcagcgaccccggcgacggcgacagcgac cccgagctggccgacagacccggcgaggccgaccccggcatccccagcgccccccagccccccgagccccccgccgccctgcagcccgagcccgagagctgggagcagatcctgagacagagcgtgctgcagcacagagccgcccccgcccccg gccccggccccggcagcggctacagagagttcacctgcgccgtgaagcagggcagcgcccccgacgccggcggccccggcttcggccccagcggcgaggccggctacaaggccttctgcagcctgctgcccggcggcgccacctgccccggcacc agcggcggcgaggccggcagcggcgagggcggctacaagcccttccagagcctgacccccggctgccccggcgcccccacccccgtgcccgtgcccctgttcaccttcggcctggacaccgagccccccggcagcccccaggacagcctgggcg ccggcagcagccccgagcacctgggcgtggagcccgccggcaaggaggaggacagcagaaagaccctgctggcccccgagcaggccaccgaccccctgagagacgacctggccagcagcatcgtgtacagcgccctgacctgccacctgtgcggc cacctgaagcagtggcacgaccaggaggagagaggcaaggcccacatcgtgcccagcccctgctgcggctgctgctgcggcgacagaagcagcctgctgctgagccccctgagagcccccaacgtgctgcccggcggcgtgctgctggaggcca gcctgagccccgccagcctggtgcccagcggcgtgagcaaggagggcaagagcagccccttcagccagcccgccagcagcagcgcccagagcagcagccagacccccaagaagctggccgtgctgagcaccgagcccacctgcatgagcgccagc

Full-length canine IL-4 receptor α protein with signal sequence in bold [SEQ ID NO:2]

MGRLCSGLTFPVSCLVLVWVASSGS

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

VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPR TPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNY YEPWEQHLPLGVSISCLVILAICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEHGLGREEESP KTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCPSLEGSGGSFQEGREGIVARLTESLFLDLLGGENGGFCPQ GLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQPRRPESALQASPTQSAGSSAFPEPPPVVTDNPAYRSFGSFLGQSSDPGDGDSDPELADRPGEADPGIPSAPQPPEPPAALQPEPESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSLLPGGATCPGTSGGEAG SGEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHLCGHLK QWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLEASLSPASLVPSGVSKEGKSSPFSQPASSSAQSSSQTPKKLAVLSTEPTCMSAS

Full-length mature canine IL-4 receptor α DNA without signal sequence [SEQ ID NO:3]

gtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccaccccaccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgt gcccgagaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacctgtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaac ccccggcaacctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcacagcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgt acaacgtgacctacatgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggcccagacctacaacagcacctggagcgactggagccccagcaccacctggctgaactact acgagccctgggagcagcacctgcccctgggcgtgagcatcagctgcctggtgatcctggccatctgcctgagctgctacttcagcatcatcaagatcaagaagggctggtgggaccagatccccaaccccgcccacagccccctggtg gccatcgtgatccaggacagccaggtgagcctgtggggcaagagaagcagaggccaggagcccgccaagtgcccccactggaagacctgcctgaccaagctgctgccctgcctgctggagcacggcctgggcagagaggaggagagcccc aagaccgccaagaacggccccctgcagggccccggcaagcccgcctggtgccccgtggaggtgagcaagaccatcctgtggcccgagagcatcagcgtggtgcagtgcgtggagctgagcgaggcccccgtggacaacgaggaggaggag gaggtggagggacaagagaagcctgtgccccagcctggagggcagcggcggcagcttccaggagggcagagagggcatcgtggccagactgaccgagagcctgttcctggacctgctgggcggcgagaacggcggcttctgcccccag ggcctggagaggagagctgcctgcccccccccagcggcagcgtgggcgcccagatgccctgggcccagttccccagagccggccccagagccgcccccgagggccccgagcagcccagaagacccgagagcgccctgcaggccagccccac ccagagcgccggcagcagcgccttccccgagcccccccccgtggtgaccgacaaccccgcctacagaagcttcggcagcttcctgggccagagcagcgaccccggcgacggcgacagcgaccccgagctggccgacagacccggcgaggc cgaccccggcatccccagcgccccccagccccccgagccccccgccgccctgcagcccgagcccgagagctgggagcagatcctgagacagagcgtgctgcagcacagagccgcccccgcccccggccccggccccggcagcggctaca gagagttcacctgcgccgtgaagcagggcagcgcccccgacgccggcggccccggcttcggccccagcggcgaggccggctacaaggccttctgcagcctgctgcccggcggcgccacctgccccggcaccagcggcggcgaggccggca gcggcgagggcggctacaagcccttccagagcctgacccccggctgccccggcgcccccacccccgtgcccgtgcccctgttcaccttcggcctggacaccgagccccccggcagcccccaggacagcctgggcgccggcagcagcccc gagcacctgggcgtggagcccgccggcaaggaggaggacagcagaaagaccctgctggcccccgagcaggccaccgaccccctgagagacgacctggccagcagcatcgtgtacagcgccctgacctgccacctgtgcggccacctgaag cagtggcacgaccaggaggagagaggcaaggcccacatcgtgcccagcccctgctgcggctgctgctgcggcgacagaagcagcctgctgctgagccccctgagagcccccaacgtgctgcccggcggcgtgctgctggaggccagcctg agccccgccagcctggtgcccagcggcgtgagcaaggagggcaagagcagccccttcagccagcccgccagcagcagcgcccagagcagcagccagacccccaagaagctggccgtgctgagcaccgagcccacctgcatgagcgccagc

Extracellular domain of canine IL-4 receptor α-chain [SEQ ID NO:5]

vkvlhepscfsdyistsvcqwkmdhptncsaelrlsyqldfmgsenhtcvpenredsvcvcsmpiddaveadvyqldlwagqqllwsgsfqpskhvkprtpgnl tvhpnishtwllmwtnpyptenhlhseltymvnvsndndpedfkvynvtymgptlrlaastlksgasysarvrawaqtynstwsdwspsttwlnyyepweqhlp

Protein engineering of antibodies

By way of example and without limitation, the canine heavy chain constant region may be derived from IgG-B or a modified cFc such as IgG-Bm as used herein [see U.S. 10,106,607 B2, which is incorporated herein by reference in its entirety], and the canine light chain constant region may be derived from the kappa chain.

Antibodies may be engineered to include modifications of the canine framework and/or residues of the canine framework within the variable domains of the parent (i.e., murine) monoclonal antibody, for example, to improve the properties of the antibody. In certain circumstances, an individual CDR may be modified, as shown below.

Pharmaceutical compositions and administration

To prepare pharmaceutical or sterile compositions containing the antibodies of the present invention, the antibodies can be mixed with a pharmaceutically acceptable carrier or excipient. [See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

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

The toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with another agent can be determined using standard pharmaceutical procedures, in cell cultures or experimental animals, for example, to determine the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The ratio of toxic to therapeutic effects caused by a dose is the therapeutic index (LD ₅₀ /ED ₅₀ ). In particular aspects, antibodies that exhibit high therapeutic indices are desirable. Data obtained from these cell culture assays and animal studies can be used in determining a dosage range for dogs. Dosing of such compounds is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dose may vary within this range depending on the dosage form and route of administration used.

The route of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial. In particular embodiments, the antibodies of the present invention may be administered by an invasive route, such as by injection. In further embodiments, the antibodies of the present invention or pharmaceutical compositions thereof are administered intravenously, subcutaneously, intramuscularly, intraarterially or by inhalation or aerosol delivery. Administration by non-invasive routes (e.g., orally; e.g., in the form of pills, capsules or tablets) is also within the scope of the present invention.

The compositions can be administered using medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection using a hypodermic needle, including, for example, a prefilled syringe or an autoinjector. The pharmaceutical compositions disclosed herein can also be administered using a needle-free hypodermic injection device; for example, using the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.

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

Alternatively, the antibodies of the present invention may be administered locally rather than systemically, often as a depot or sustained release formulation.

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

Determination of the appropriate dose is performed by the veterinarian, using, for example, parameters or factors known or suspected in the art to influence treatment. Typically, dosing is started at a quantity slightly less than the optimal dose, then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measurements include symptom measurements.

The antibodies disclosed herein can be administered by continuous infusion or by doses administered, for example, daily, 1-7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semiannually, annually, etc. Doses can be administered, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinal, or by inhalation. The total weekly dose is usually at least 0.05 mcg/kg body weight, more generally at least 0.2 mcg/kg, 0.5 mcg/kg, 1 mcg/kg, 10 mcg/kg, 100 mcg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/mL, 10 mg/kg, 25 mg/kg, 50 mg/kg or more [see, e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al . Cancer Immunol. Immunother. 52:133-144 (2003)]. Doses may also be provided to achieve a predetermined target concentration of the antibodies of the invention in canine serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/mL or more. In other embodiments, the antibodies of the invention are administered subcutaneously or intravenously weekly, biweekly, q4w, monthly, bimonthly, or quarterly at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg/subject.

As used herein, the terms "inhibit," "treat," or "treatment" include delaying the development of symptoms associated with a disorder and/or reducing the severity of symptoms of such a disorder. The terms also include alleviating existing uncontrollable or unwanted symptoms, preventing the occurrence of additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms mean achieving a beneficial outcome in a vertebrate subject (e.g., a canine) suffering from a disorder, condition, and/or having a symptom or risk of developing such a disorder, disease, or occurrence of a symptom.

As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to the amount of antibodies of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue or organism of a subject, such as a canine, is effective in causing a measurable improvement in one or more symptoms of a disease or condition or in inhibiting the progression of such a disease or condition. A therapeutically effective dose also refers to that amount of antibodies that is sufficient to produce at least partial alleviation of symptoms, such as treatment, cure, prevention or amelioration of the relevant medical condition, or an increase in the rate of treatment, healing, prevention or amelioration of such conditions. When applied to a combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that result in a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of a therapeutic agent results in an improvement in a diagnostic measurement or parameter by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%. An effective amount may also result in an improvement in a subjective measure in cases where subjective measures are used to assess the severity of a condition.

EXAMPLES

EXAMPLE 1

ANTIBODIES TO IL-4 ALPHA RECEPTOR

General materials and methods

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

Balb/c mice were immunized multiple times (10 μg each time) for 17 days. The immunizing antigen was a fusion protein of the extracellular domain (ECD) of canine IL-4R alpha chain and human Fc. Following immunization, serum was collected from each mouse and tested for reactivity with HIS-tagged canine IL-4 receptor alpha chain ECD protein. Mouse spleen cells with the highest serum IL-4 receptor alpha chain ECD titer were fused with the P3X63Ag8.653 myeloma cell line. Approximately 2 weeks after fusion, the supernatant of the putative hybridoma cells was tested by ELISA for reactivity to the tagged IL-4 receptor alpha chain ECD protein. Hybridomas generating strong positive signals in ELISA were subcloned by limiting dilution and retested for reactivity to the HIS-tagged ECD protein of canine IL-4 receptor alpha chain. Antibodies to canine IL-4 receptor alpha included c152H11VL3-cCLk-s/c152H11VH3-cIgG-Bm and c146E2VL3-cCLk-s/c146E2VH3-cIgG-Bm.

The sets of six (6) CDRs (three separate light chain (LC) sequences and three heavy chain (HC) sequences) for these two antibody families are presented below in Table 1A (nucleic acid sequences) and Table 1B (amino acid sequences). Table 1B includes the modified mouse HCDR3 for 152H11 containing the amino acid sequence of SEQ ID NO:28, while the remainder are unmodified mouse CDRs. Table 1A presents the nucleic acids that encode the twelve CDRs listed in Table 1B. Table 1C presents both the amino acid sequence (SEQ ID NO:49) of the unmodified mouse HCDR3 for 152H11 and the nucleic acid sequence (SEQ ID NO:48) that encodes this unmodified mouse HCDR3. The amino acid sequence (SEQ ID NO:28) of the modified mouse HCDR3 of the 152H11 antibody family differs from that of the corresponding unmodified mouse HCDR3 by the substitution of a serine residue at the C-terminal cysteine residue in the amino acid sequence of SEQ ID NO:49. The unmodified mouse HCDR3 (SEQ ID NO:49) of the 152H11 antibody family is found in both c152H11VH1-cIgGBm (SEQ ID NO:36) and c152H11VH2-cIgGBm (SEQ ID NO:37), whereas c152H11VH3-cIgGBm (SEQ ID NO:38) contains the modified mouse HCDR3 (SEQ ID NO:28). The amino acid sequences of the full-length light chains and heavy chains of the caninized antibodies are presented immediately following Table 1C.

NUCLEIC ACID AND AMINO ACID SEQUENCES OF CDR ANTIBODIES TO IL-4R-alpha

TABLE 1A CDR SEQ ID NO: 146E2 HCDR1 agatactggatgcac 11 HCDR2 atgattcaccccgacagcggcaacatcaactacaacgagcggttcaagacc 13 HCDR3 cagctgcggaacgccatggattat 15 LCDR1 agagccagcgagagcgtggacagctacggcaacagcttcctgaac 17 LCDR2 agagccagcaacctggcctct 19 LCDR3 cagcagaactacgagaaccccagaacc 21 152H11 HCDR1 agctacggcatgagc 23 HCDR2 acaatcagcagaggcggcgactacacctactatcccgacagcgtgaagggc 25 HCDR3 ^# ggcaccctgaacaaccggggctttgcttct 27 LCDR1 aaggccagccagaacgtgggcaccaatgtggcc 29 LCDR2 agcgccagctaccggtactct 31 LCDR3 cagcagtacaacagctacccctacacc 33

# See footnote for Table 1B below.

TABLE 1B 146E2 SEQ ID NO: 152H11 SEQ ID NO: HCDR1 RYWMH 12 SYGMS 24 HCDR2 MIHPDSGNINYNERFKT 14 TISRGGDYTYYPDSVKG 26 HCDR3 QLRNAMDY 16 ^# GTLNNRGFAS 28 LCDR1 RASESVDSYGNSFLN 18 KASQNVGTNVA 30 LCDR2 RASNLAS 20 SASYRYS 32 LCDR3 QQNYENPRT 22 QQYNSYPYT 34

# The C-terminal cysteine residue of HCDR3 152H11 is replaced by a serine residue as discussed above.

TABLE 1C CDR 152H11 NA AA SEQ ID
NO: HCDR3 ggcaccctgaacaaccgcggctttgcgtgc √ 48 HCDR3 GTLNNRGFAC √ 49

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

EIVMTQSPASLSLSQEEKVTITC KASQNVGTNVA WYQQKPGQAPKLLIY SASYRYS GLPDRFSGSGGTDFSFTISSLEPEDVAEFFC QQYNSYPYT FGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

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

EVQLVESGGDLVKPGGSLRLSCAASGTFFS SYGMS WVRQAPGKGLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAMYYCAK GTLNNRGFAC WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

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

EVQLVESGGDLVKPGGSLRLSCAASGTFFS SYGMS WVRQAPDKRLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAMYYCAR GTLNNRGFAC WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

c152H11VH3-cIgG-Bm (heavy chain): [SEQ ID NO:38]

EVQLVESGGDLVKPGGSLRLSCAASGTFFS SYGMS WVRQAPDKRLQWVA TISRGGDYTYYPDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAMYYCAR GTLNNRGFAS WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

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

DIVLTQTPLSLSVSPGETASIYC RASESVDSYGNSFLN WYQQKPGQPPKLLIY RASNLAS EIPDRFSGSGSRTEFTLKISRVEADDAGVYYC QQNYENPRT FGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

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

EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WVRQAPGAGLDWMG MIHPDSGNINYNERFKT RVTLTADTSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDY WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

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

EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WMKQAPGAGLDWIG MIHPDSGNINYNERFKT KATLTADTSTSTAYMELSSLRAGDIAVYYCAR QLRNAMDY WGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQI SWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

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

EVQLVQSGAEVKKPGASVKVSCKASGYTFA RYWMH WMKQAPGAGLDWIG MIHPDSGNINYNERFKT KATLTVdkststaymelsslragdiavyycar qlrnamdy wgqgtlvtvssasttapsvfplapscgstsgstvalaclvsgyfpepvtvswnsgsltsgvhtfpsvlqssglyslssmvtvpssr wpsetftcnvahpasktkvdkpvpkrengrvprppdcpkcpapemlggpsvfifppkpkdtlliartpevtcvvvaldpedpevqi swfvdgkqmqtaktqpreeqfagtyrvvsvlpighqdwlkgkqftckvnnkalpspiertiskargqahqpsvyvlppsreelskntvsltclikdffppdidvewqsngqqepeskyrttppqldedgsyflysklsvdksrwqrgdtficavmhealhnhytqeslshspg

In addition, light chains of canine IL-4 receptor alpha antibodies with a lambda light chain were also engineered as shown below.

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

QSVLTQPASVSGSLGQRVTISC KASQNVGTNVA WYQQLPGTSPRTLIY SASYRYS GVPDRFSGSRSGSTATLTISGLQAEDEADYYC QQYNSYPYT FGGGTHLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS

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

QSVLTQPASVSGSLGQRVTISC RASESVDSYGNSFLN WYQQLPGKAPSLLIY RASNLAS GVPERFSGSKSGSSATLTITGLQAEDEADYYC QQNYENPRT FGGGTHLTVLGQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS

EXAMPLE 2

STAT-6 INHIBITION

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

Materials

1. Actively growing DH82 cells

2. DH82 cell growth medium (ATCC® 302003™, Eagle's Minimum Essential Medium containing heat-inactivated fetal bovine serum at a final concentration of 15% w/v)

3. AlphaLISA p-STAT6 Assay Kit (Tyr641): Perkin Elmer Catalog: ALSU PST6 A HV

4. Recombinant Canine IL-4: R&D Systems, Catalog: 752-CL/CF

5. Recombinant Canine IL-13: R&D Systems, Catalog: 5894-CL/CF

6. Perkin Elmer Vision

a. Caninized monoclonal antibodies to canine IL-4Rα

b. c152H11-H3L3

c. Caninized 4H3 antibodies from US 2018/0346580

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

Methods

1. Two tissue culture plates were seeded with DH82 cells at ^8x104 per well (200 µl at a density of ^4x105 cells/ml) and incubated at 37°C overnight.

2. The test antibodies were pre-diluted to 500 μg/mL and then serially diluted 3-fold in DH82 cell growth medium. The medium was removed from the cell culture plates and 50 μL/well of the serially diluted test sample was added to each plate.

3. Canine IL-4 was diluted to 5 ng/ml in DH82 cell growth medium and 50 μl was added to each well of one plate. Canine IL-13 was diluted to 10 ng/ml in DH82 cell growth medium and 50 μl was added to each well of the second plate. The plates were incubated for 15 min at 37°C.

4. The medium was removed from the plates and 100 µl per well of freshly prepared 1x Lysis Buffer from the AlphaLISA p-STAT-6 Assay Kit was added to the plate. The plate was shaken on a plate shaker at 350 rpm for 10 minutes at room temperature.

5. Acceptor mixture was prepared from the AlphaLISA p-STAT6 Assay Kit, and 15 µl per well was added to 30 µl of cell lysate in 96-well 1/2 Area plates. The plates were sealed, shaken for 2 min at 350 rpm, and then incubated for 2 h at room temperature.

6. The donor mixture was prepared from the AlphaLISA p-STAT6 assay kit under low laboratory light and 15 µl/well was added to each plate. The plates were sealed, covered with foil, shaken for 2 minutes at 350 rpm and then incubated for 2 hours at room temperature.

7. Plates were read using AlphaScreen settings on a Perkin Elmer EnVison.

Two different caninized monoclonal antibodies to canine IL-4Rα, designated c4H3 [WO2016/156588; US2018/0346580] and c152H11-H3L3, were evaluated for their ability to inhibit αSTAT-6 phosphorylation by blocking the binding of canine IL-4 or canine IL-13 to canine IL-4Rα. The data presented in Fig. 1 demonstrate that both antibodies provide dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-4. However, the binding of the c152H11-H3L3 antibody was surprisingly stronger than that of the prior art c4H3 antibody to canine IL-4 receptor alpha [WO2016/156588]. The IL-4 control in the absence of IL-4Ralpha antibody (IL-4Rα) is shown in the upper right hand side of the graph. The data presented in Figure 2 also demonstrate that both antibodies resulted in dose-dependent inhibition of STAT-6 phosphorylation in the presence of IL-13, but again the binding of the c152H11-H3L3 antibody was stronger than that of the prior art c4H3 antibody to canine IL-4 receptor alpha. The IL-13 control in the absence of IL-4Ralpha antibody (IL-4Rα) is shown in the upper right hand side of the graph. Figure 3 shows that substitution of the kappa light chain with the lambda light chain did not affect the binding of c152H11-H3L3 to IL-4Rα.

EXAMPLE 3

EPITOPE MAPPING

The interaction of antibodies with their cognate protein antigens is mediated through the binding of specific amino acids of the antibodies (paratopes) to specific amino acids (epitopes) of the target antigens. An epitope is an antigenic determinant that elicits a specific immunoglobulin response. An epitope consists of a group of amino acids on the surface of an antigen. A protein of interest may contain several epitopes that are recognized by different antibodies. Epitopes recognized by antibodies are classified as linear or conformational epitopes. Linear epitopes are formed by a region of continuous amino acid sequence in a protein, while conformational epitopes consist of amino acids that are not continuous (e.g., located far from each other) in the primary amino acid sequence, but come close to each other to form the spatial structure of the protein.

Epitope mapping refers to the process of identifying amino acid sequences (i.e. epitopes) that are recognized by antibodies on target antigens. Identification of epitopes recognized by monoclonal antibodies (mAbs) on target antigens has important applications. For example, it may aid in the development of new therapeutics, diagnostics, and vaccines. Epitope mapping may also facilitate the selection of optimized therapeutic mAbs and elucidate their mechanisms of action. Information on IL-4 receptor alpha epitopes may also shed light on unique epitopes and determine the protective or pathogenic effects of vaccines. Epitope identification may also lead to the development of subunit vaccines based on chemical or genetic linkage of the identified peptide epitope to a carrier protein or other immunostimulatory agents.

Epitope mapping can be performed using polyclonal or monoclonal antibodies, and several methods are used to identify epitopes depending on the suspected nature of the epitope (i.e., linear or conformational). Linear epitope mapping is more straightforward and relatively easy to perform. For this purpose, commercial linear epitope mapping services often use peptide scanning. In this case, an overlapping set of short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind the antibody of interest. The strategy is rapid, high-throughput, and relatively inexpensive to perform. Discontinuous epitope mapping, on the other hand, is technically more challenging and requires more specialized techniques such as X-ray co-crystallography of the monoclonal antibody with the target protein, hydrogen-deuterium (H/D) exchange, mass spectrometry coupled to enzymatic digestion, and several other methods known to those skilled in the art.

Epitope mapping of canine IL-4 receptor alpha using mass spectroscopy:

To identify the epitopes recognized by mAbs to canine IL-4 receptor alpha, a method based on chemical cross-linking, mass spectrometric detection, and covalent labeling was used [CovalX Instruments Incorporated, located at 999 Broadway, Suite 305, Saugus, MA 01906-4510 USA).

Application of this method to canine IL-4 receptor alpha chain epitope mapping in a previous study demonstrated that the mAb recognizes specific peptide epitopes that are present in the extracellular domain of canine IL-4 receptor alpha [US2018/0346580]. A similar analysis performed on the c152H11-H3L3 anti-canine IL-4R alpha antibody shown in Figure 4 resulted in the identification of the amino acid sequences SEQ ID NO:46 and SEQ ID NO:47 for epitope(s) that are(are) reasonably similar to those previously identified. In addition, as shown in Fig. 4, amino acid residues _S111 , _H112 , T113, _T119 , _{Y122 , T124, H127, Y150, T153, Y154, T158, R160, S164, T165, S168, S171, Y172, and S173 were identified as specific contact points [ see , e.g. ,} SEQ ID NO:5, for amino _acid residue numbering _] .

SEQUENCE LIST TABLE SEQ ID NO: NA AA 1 Canine IL-4 receptor α chain with signal sequence √ 2 Canine IL-4 receptor α chain with signal sequence √ 3 Canine IL-4 receptor α chain without signal sequence √ 4 Canine IL-4 receptor α chain without signal sequence √ 5 Extracellular domain of canine IL-4 receptor α √ 6 Hinge region of IgG-A √ 7 IgG-B hinge region √ 8 IgG-C hinge region √ 9 Modified hinge region of IgG-D √ 10 Canine IgG-Bm √ 11 146 E2 HCDR1 √ 12 146 E2 HCDR1 √ 13 146 E2HCDR2 √ 14 146 E2 HCDR2 √ 15 146 E2 HCDR3 √ 16 146 E2 HCDR3 √ 17 146 E2 LCDR1 √ 18 146 E2 LCDR1 √ 19 146 E2 LCDR2 √ 20 146 E2 LCDR2 √ 21 146 E2 LCDR3 √ 22 146 E2 LCDR3 √ 23 152 H11 HCDR1 √ 24 152 H11 HCDR1 √ 25 152 H11 HCDR2 √ 26 152 H11 HCDR2 √ 27 152 H11 HCDR3 ^# √ 28 152 H11 HCDR3 ^# √ 29 152 H11 LCDR1 √ 30 152 H11 LCDR1 √ 31 152 H11 LCDR2 √ 32 152 H11 LCDR2 √ 33 152 H11 LCDR3 √ 34 152 H11 LCDR3 √ 35 c152H11VL3-cCLk-s (light kappa chain) √ 36 c152H11VH1-cIgG-Bm (heavy chain) √ 37 c152H11VH2-cIgG-Bm (heavy chain) √ 38 c152H11VH3-cIgG-Bm (heavy chain) √ 39 c146E2VL3-cCLk-s (light kappa chain) √ 40 c146E2VH1-cIgG-Bm (heavy chain) √ 41 c146E2VH2-cIgG-Bm (heavy chain) √ 42 c146E2VH3-cIgG-Bm (heavy chain) √ 43 c152H11LV1-cCl (light lambda chain) √ 44 c146E2LV1-cCl (light lambda chain) √ 45 Canine IgG-B √ 46 ishtwllmwtnpyptenhlhs √ 47 ndndpedfkvynvtymgptlrlaastlksgasysarvrawa √ 48 152 H11 HCDR3 √ 49 152 H11 HCDR3 √

# The C-terminal cysteine residue of HCDR3 152H11 is replaced by a serine residue.

--->

LIST OF SEQUENCES

<110> INTERVET INTERNATIONAL B.V.

MORSI, Mohamad

ZHANG, Yuanzheng

<120> ANTIBODIES TO CANINE INTERLEUKIN-4 ALPHA RECEPTOR

<130> 24981

<150> 63/015,220

<151> 2020-04-24

<150> 63/015,209

<151> 2020-04-24

<150> 62/951,793

<151> 2019-12-20

<150> 62/951,778

<151> 2019-12-20

<160> 49

<170> PatentIn version 3.5

<210> 1

<211> 2469

<212> DNA

<213> Canis familiaris

<400> 1

atgggcagac tgtgcagcgg cctgaccttc cccgtgagct gcctggtgct ggtgtgggtg 60

gccagcagcg gcagcgtgaa ggtgctgcac gagcccagct gcttcagcga ctacatcagc 120

accagcgtgt gccagtggaa gatggaccac cccaccaact gcagcgccga gctgagactg 180

agctaccagc tggacttcat gggcagcgag aaccacacct gcgtgcccga gaacagagag 240

gacagcgtgt gcgtgtgcag catgcccatc gacgacgccg tggaggccga cgtgtaccag 300

ctggacctgt gggccggcca gcagctgctg tggagcggca gcttccagcc cagcaagcac 360

gtgaagccca gaacccccgg caacctgacc gtgcacccca acatcagcca cacctggctg 420

ctgatgtgga ccaaccccta ccccaccgag aaccacctgc acagcgagct gacctacatg 480

gtgaacgtga gcaacgacaa cgaccccgag gacttcaagg tgtacaacgt gacctacatg 540

ggccccaccc tgagactggc cgccagcacc ctgaagagcg gcgccagcta cagcgccaga 600

gtgagagcct gggcccagac ctacaacagc acctggagcg actggagccc cagcaccacc 660

tggctgaact actacgagcc ctgggagcag cacctgcccc tgggcgtgag catcagctgc 720

ctggtgatcc tggccatctg cctgagctgc tacttcagca tcatcaagat caagaagggc 780

tggtgggacc agatccccaa ccccgcccac agccccctgg tggccatcgt gatccaggac 840

agccaggtga gcctgtgggg caagagaagc agaggccagg agcccgccaa gtgcccccac 900

tggaagacct gcctgaccaa gctgctgccc tgcctgctgg agcacggcct gggcagagag 960

gagggagagcc ccaagaccgc caagaacggc cccctgcagg gccccggcaa gcccgcctgg 1020

tgccccgtgg aggtgagcaa gaccatcctg tggcccgaga gcatcagcgt ggtgcagtgc 1080

gtggagctga gcgaggcccc cgtggacaac gaggaggagg aggaggtgga gggaggacaag 1140

agaagcctgt gccccagcct ggagggcagc ggcggcagct tccaggaggg cagagaggc 1200

atcgtggcca gactgaccga gagcctgttc ctggacctgc tgggcggcga gaacggcggc 1260

ttctgccccc agggcctgga ggagagctgc ctgccccccc ccagcggcag cgtgggcgcc 1320

cagatgccct gggcccagtt ccccagagcc ggccccagag ccgcccccga gggccccgag 1380

cagcccagaa gacccgagag cgccctgcag gccagcccca cccagagcgc cggcagcagc 1440

gccttccccg agcccccccc cgtggtgacc gacaaccccg cctacagaag cttcggcagc 1500

ttcctgggcc agagcagcga ccccggcgac ggcgacagcg accccgagct ggccgacaga 1560

cccggcgagg ccgaccccgg catccccagc gccccccagc cccccgagcc ccccgccgcc 1620

ctgcagcccg agcccgagag ctgggagcag atcctgagac agagcgtgct gcagcacaga 1680

gccgcccccg cccccggccc cggccccggc agcggctaca gagagttcac ctgcgccgtg 1740

aagcaggca gcgcccccga cgccggcggc cccggcttcg gccccagcgg cgaggccggc 1800

tacaaggcct tctgcagcct gctgcccggc ggcgccacct gccccggcac cagcggcggc 1860

gaggccggca gcggcgaggg cggctacaag cccttccaga gcctgacccc cggctgcccc 1920

ggcgccccca cccccgtgcc cgtgcccctg ttcaccttcg gcctggacac cgagcccccc 1980

ggcagccccc aggacagcct gggcgccggc agcagccccg agcacctggg cgtggagccc 2040

gccggcaagg aggaggacag cagaaagacc ctgctggccc ccgagcaggc caccgacccc 2100

ctgagagacg acctggccag cagcatcgtg tacagcgccc tgacctgcca cctgtgcggc 2160

cacctgaagc agtggcacga cccaggagg agaggcaagg cccacatcgt gcccagcccc 2220

tgctgcggct gctgctgcgg cgacagaagc agcctgctgc tgagccccct gagagccccc 2280

aacgtgctgc ccggcggcgt gctgctggag gccagcctga gccccgccag cctggtgccc 2340

agcggcgtga gcaagggaggg caagagcagc cccttcagcc agcccgccag cagcagcgcc 2400

cagagcagca gccagacccc caagaagctg gccgtgctga gcaccgagcc cacctgcatg 2460

agcgccagc 2469

<210> 2

<211> 823

<212> Protein

<213> Canis familiaris

<400> 2

Met Gly Arg Leu Cys Ser Gly Leu Thr Phe Pro Val Ser Cys Leu Val

1 5 10 15

Leu Val Trp Val Ala Ser Ser Gly Ser Val Lys Val Leu His Glu Pro

20 25 30

Ser Cys Phe Ser Asp Tyr Ile Ser Thr Ser Val Cys Gln Trp Lys Met

35 40 45

Asp His Pro Thr Asn Cys Ser Ala Glu Leu Arg Leu Ser Tyr Gln Leu

50 55 60

Asp Phe Met Gly Ser Glu Asn His Thr Cys Val Pro Glu Asn Arg Glu

65 70 75 80

Asp Ser Val Cys Val Cys Ser Met Pro Ile Asp Asp Ala Val Glu Ala

85 90 95

Asp Val Tyr Gln Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Ser

100 105 110

Gly Ser Phe Gln Pro Ser Lys His Val Lys Pro Arg Thr Pro Gly Asn

115 120 125

Leu Thr Val His Pro Asn Ile Ser His Thr Trp Leu Leu Met Trp Thr

130 135 140

Asn Pro Tyr Pro Thr Glu Asn His Leu His Ser Glu Leu Thr Tyr Met

145 150 155 160

Val Asn Val Ser Asn Asp Asn Asp Pro Glu Asp Phe Lys Val Tyr Asn

165 170 175

Val Thr Tyr Met Gly Pro Thr Leu Arg Leu Ala Ala Ser Thr Leu Lys

180 185 190

Ser Gly Ala Ser Tyr Ser Ala Arg Val Arg Ala Trp Ala Gln Thr Tyr

195 200 205

Asn Ser Thr Trp Ser Asp Trp Ser Pro Ser Thr Thr Trp Leu Asn Tyr

210 215 220

Tyr Glu Pro Trp Glu Gln His Leu Pro Leu Gly Val Ser Ile Ser Cys

225 230 235 240

Leu Val Ile Leu Ala Ile Cys Leu Ser Cys Tyr Phe Ser Ile Ile Lys

245 250 255

Ile Lys Lys Gly Trp Trp Asp Gln Ile Pro Asn Pro Ala His Ser Pro

260 265 270

Leu Val Ala Ile Val Ile Gln Asp Ser Gln Val Ser Leu Trp Gly Lys

275 280 285

Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Thr Cys

290 295 300

Leu Thr Lys Leu Leu Pro Cys Leu Leu Glu His Gly Leu Gly Arg Glu

305 310 315 320

Glu Glu Ser Pro Lys Thr Ala Lys Asn Gly Pro Leu Gln Gly Pro Gly

325 330 335

Lys Pro Ala Trp Cys Pro Val Glu Val Ser Lys Thr Ile Leu Trp Pro

340 345 350

Glu Ser Ile Ser Val Val Gln Cys Val Glu Leu Ser Glu Ala Pro Val

355 360 365

Asp Asn Glu Glu Glu Glu Glu Val Glu Glu Asp Lys Arg Ser Leu Cys

370 375 380

Pro Ser Leu Glu Gly Ser Gly Gly Ser Phe Gln Glu Gly Arg Glu Gly

385 390 395 400

Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly Gly

405 410 415

Glu Asn Gly Gly Phe Cys Pro Gln Gly Leu Glu Glu Ser Cys Leu Pro

420 425 430

Pro Pro Ser Gly Ser Val Gly Ala Gln Met Pro Trp Ala Gln Phe Pro

435 440 445

Arg Ala Gly Pro Arg Ala Ala Pro Glu Gly Pro Glu Gln Pro Arg Arg

450 455 460

Pro Glu Ser Ala Leu Gln Ala Ser Pro Thr Gln Ser Ala Gly Ser Ser

465 470 475 480

Ala Phe Pro Glu Pro Pro Pro Val Val Thr Asp Asn Pro Ala Tyr Arg

485 490 495

Ser Phe Gly Ser Phe Leu Gly Gln Ser Ser Asp Pro Gly Asp Gly Asp

500 505 510

Ser Asp Pro Glu Leu Ala Asp Arg Pro Gly Glu Ala Asp Pro Gly Ile

515 520 525

Pro Ser Ala Pro Gln Pro Pro Glu Pro Pro Ala Ala Leu Gln Pro Glu

530 535 540

Pro Glu Ser Trp Glu Gln Ile Leu Arg Gln Ser Val Leu Gln His Arg

545 550 555 560

Ala Ala Pro Ala Pro Gly Pro Gly Pro Gly Ser Gly Tyr Arg Glu Phe

565 570 575

Thr Cys Ala Val Lys Gln Gly Ser Ala Pro Asp Ala Gly Gly Pro Gly

580 585 590

Phe Gly Pro Ser Gly Glu Ala Gly Tyr Lys Ala Phe Cys Ser Leu Leu

595 600 605

Pro Gly Gly Ala Thr Cys Pro Gly Thr Ser Gly Gly Glu Ala Gly Ser

610 615 620

Gly Glu Gly Gly Tyr Lys Pro Phe Gln Ser Leu Thr Pro Gly Cys Pro

625 630 635 640

Gly Ala Pro Thr Pro Val Pro Val Pro Leu Phe Thr Phe Gly Leu Asp

645 650 655

Thr Glu Pro Pro Gly Ser Pro Gln Asp Ser Leu Gly Ala Gly Ser Ser

660 665 670

Pro Glu His Leu Gly Val Glu Pro Ala Gly Lys Glu Glu Asp Ser Arg

675 680 685

Lys Thr Leu Leu Ala Pro Glu Gln Ala Thr Asp Pro Leu Arg Asp Asp

690 695 700

Leu Ala Ser Ser Ile Val Tyr Ser Ala Leu Thr Cys His Leu Cys Gly

705 710 715 720

His Leu Lys Gln Trp His Asp Gln Glu Glu Arg Gly Lys Ala His Ile

725 730 735

Val Pro Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp Arg Ser Ser Leu

740 745 750

Leu Leu Ser Pro Leu Arg Ala Pro Asn Val Leu Pro Gly Gly Val Leu

755 760 765

Leu Glu Ala Ser Leu Ser Pro Ala Ser Leu Val Pro Ser Gly Val Ser

770 775 780

Lys Glu Gly Lys Ser Ser Pro Phe Ser Gln Pro Ala Ser Ser Ser Ala

785 790 795 800

Gln Ser Ser Ser Gln Thr Pro Lys Lys Leu Ala Val Leu Ser Thr Glu

805 810 815

Pro Thr Cys Met Ser Ala Ser

820

<210> 3

<211> 2394

<212> DNA

<213> Canis familiaris

<400> 3

gtgaaggtgc tgcacgagcc cagctgcttc agcgactaca tcagcaccag cgtgtgccag 60

tggaagatgg accaccccac caactgcagc gccgagctga gactgagcta ccagctggac 120

ttcatgggca gcgagaacca cacctgcgtg cccgagaaca gagaggacag cgtgtgcgtg 180

tgcagcatgc ccatcgacga cgccgtggag gccgacgtgt accagctgga cctgtgggcc 240

ggccagcagc tgctgtggag cggcagcttc cagcccagca agcacgtgaa gcccagaacc 300

cccggcaacc tgaccgtgca ccccaacatc agccacacct ggctgctgat gtggaccaac 360

ccctacccca ccgagaacca cctgcacagc gagctgacct acatggtgaa cgtgagcaac 420

gacaacgacc ccgaggactt caaggtgtac aacgtgacct acatgggccc caccctgaga 480

ctggccgcca gcaccctgaa gagcggcgcc agctacagcg ccagagtgag agcctgggcc 540

cagacctaca acagcacctg gagcgactgg agccccagca ccacctggct gaactactac 600

gagccctggg agcagcacct gcccctgggc gtgagcatca gctgcctggt gatcctggcc 660

atctgcctga gctgctactt cagcatcatc aagatcaaga agggctggtg ggaccagatc 720

cccaaccccg cccacagccc cctggtggcc atcgtgatcc aggacagcca ggtgagcctg 780

tggggcaaga gaagcagagg ccaggagccc gccaagtgcc cccactggaa gacctgcctg 840

accaagctgc tgccctgcct gctggagcac ggcctgggca gagaggagga gagccccaag 900

accgccaaga acggccccct gcagggcccc ggcaagcccg cctggtgccc cgtggaggtg 960

agcaagacca tcctgtggcc cgagagcatc agcgtggtgc agtgcgtgga gctgagcgag 1020

gcccccgtgg acaacgagga ggaggaggag gtggaggagg acaagagaag cctgtgcccc 1080

agcctggagg gcagcggcgg cagcttccag gagggcagag agggcatcgt ggccagactg 1140

accgagagcc tgttcctgga cctgctgggc ggcgagaacg gcggcttctg cccccagggc 1200

ctggaggaga gctgcctgcc cccccccagc ggcagcgtgg gcgcccagat gccctgggcc 1260

cagttcccca gagccggccc cagagccgcc cccgaggggcc ccgagcagcc cagaagaccc 1320

gagagcgccc tgcaggccag ccccacccag agcgccggca gcagcgcctt ccccgagccc 1380

ccccccgtgg tgaccgacaa ccccgcctac agaagcttcg gcagcttcct gggccagagc 1440

agcgaccccg gcgacggcga cagcgacccc gagctggccg acagacccgg cgaggccgac 1500

cccggcatcc ccagcgcccc ccagcccccc gagccccccg ccgccctgca gcccgagccc 1560

gagagctggg agcagatcct gagacagagc gtgctgcagc acagagccgc ccccgccccc 1620

ggccccggcc ccggcagcgg ctacagagag ttcacctgcg ccgtgaagca gggcagcgcc 1680

cccgacgccg gcggccccgg cttcggcccc agcggcgagg ccggctacaa ggccttctgc 1740

agcctgctgc ccggcggcgc cacctgcccc ggcaccagcg gcggcgaggc cggcagcggc 1800

gagggcggct acaagccctt ccagagcctg acccccggct gccccggcgc ccccaccccc 1860

gtgcccgtgc ccctgttcac cttcggcctg gacaccgagc cccccggcag cccccaggac 1920

agcctgggcg ccggcagcag ccccgagcac ctgggcgtgg agcccgccgg caaggaggag 1980

gacagcagaa agaccctgct ggcccccgag caggccaccg accccctgag agacgacctg 2040

gccagcagca tcgtgtacag cgccctgacc tgccacctgt gcggccacct gaagcagtgg 2100

cacgaccagg aggagagagg caaggcccac atcgtgccca gcccctgctg cggctgctgc 2160

tgcggcgaca gaagcagcct gctgctgagc cccctgagag cccccaacgt gctgcccggc 2220

ggcgtgctgc tggaggccag cctgagcccc gccagcctgg tgcccagcgg cgtgagcaag 2280

gagggcaaga gcagcccctt cagccagccc gccagcagca gcgcccagag cagcagccag 2340

acccccaaga agctggccgt gctgagcacc gagcccacct gcatgagcgc cagc 2394

<210> 4

<211> 798

<212> Protein

<213> Canis familiaris

<400> 4

Val Lys Val Leu His Glu Pro Ser Cys Phe Ser Asp Tyr Ile Ser Thr

1 5 10 15

Ser Val Cys Gln Trp Lys Met Asp His Pro Thr Asn Cys Ser Ala Glu

20 25 30

Leu Arg Leu Ser Tyr Gln Leu Asp Phe Met Gly Ser Glu Asn His Thr

35 40 45

Cys Val Pro Glu Asn Arg Glu Asp Ser Val Cys Val Cys Ser Met Pro

50 55 60

Ile Asp Asp Ala Val Glu Ala Asp Val Tyr Gln Leu Asp Leu Trp Ala

65 70 75 80

Gly Gln Gln Leu Leu Trp Ser Gly Ser Phe Gln Pro Ser Lys His Val

85 90 95

Lys Pro Arg Thr Pro Gly Asn Leu Thr Val His Pro Asn Ile Ser His

100 105 110

Thr Trp Leu Leu Met Trp Thr Asn Pro Tyr Pro Thr Glu Asn His Leu

115 120 125

His Ser Glu Leu Thr Tyr Met Val Asn Val Ser Asn Asp Asn Asp Pro

130 135 140

Glu Asp Phe Lys Val Tyr Asn Val Thr Tyr Met Gly Pro Thr Leu Arg

145 150 155 160

Leu Ala Ala Ser Thr Leu Lys Ser Gly Ala Ser Tyr Ser Ala Arg Val

165 170 175

Arg Ala Trp Ala Gln Thr Tyr Asn Ser Thr Trp Ser Asp Trp Ser Pro

180 185 190

Ser Thr Thr Trp Leu Asn Tyr Tyr Glu Pro Trp Glu Gln His Leu Pro

195 200 205

Leu Gly Val Ser Ile Ser Cys Leu Val Ile Leu Ala Ile Cys Leu Ser

210 215 220

Cys Tyr Phe Ser Ile Ile Lys Ile Lys Lys Gly Trp Trp Asp Gln Ile

225 230 235 240

Pro Asn Pro Ala His Ser Pro Leu Val Ala Ile Val Ile Gln Asp Ser

245 250 255

Gln Val Ser Leu Trp Gly Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys

260 265 270

Cys Pro His Trp Lys Thr Cys Leu Thr Lys Leu Leu Pro Cys Leu Leu

275 280 285

Glu His Gly Leu Gly Arg Glu Glu Glu Ser Pro Lys Thr Ala Lys Asn

290 295 300

Gly Pro Leu Gln Gly Pro Gly Lys Pro Ala Trp Cys Pro Val Glu Val

305 310 315 320

Ser Lys Thr Ile Leu Trp Pro Glu Ser Ile Ser Val Val Gln Cys Val

325 330 335

Glu Leu Ser Glu Ala Pro Val Asp Asn Glu Glu Glu Glu Glu Val Glu

340 345 350

Glu Asp Lys Arg Ser Leu Cys Pro Ser Leu Glu Gly Ser Gly Gly Ser

355 360 365

Phe Gln Glu Gly Arg Glu Gly Ile Val Ala Arg Leu Thr Glu Ser Leu

370 375 380

Phe Leu Asp Leu Leu Gly Gly Glu Asn Gly Gly Phe Cys Pro Gln Gly

385 390 395 400

Leu Glu Glu Ser Cys Leu Pro Pro Pro Ser Gly Ser Val Gly Ala Gln

405 410 415

Met Pro Trp Ala Gln Phe Pro Arg Ala Gly Pro Arg Ala Ala Pro Glu

420 425 430

Gly Pro Glu Gln Pro Arg Arg Pro Glu Ser Ala Leu Gln Ala Ser Pro

435 440 445

Thr Gln Ser Ala Gly Ser Ser Ala Phe Pro Glu Pro Pro Pro Val Val

450 455 460

Thr Asp Asn Pro Ala Tyr Arg Ser Phe Gly Ser Phe Leu Gly Gln Ser

465 470 475 480

Ser Asp Pro Gly Asp Gly Asp Ser Asp Pro Glu Leu Ala Asp Arg Pro

485 490 495

Gly Glu Ala Asp Pro Gly Ile Pro Ser Ala Pro Gln Pro Pro Glu Pro

500 505 510

Pro Ala Ala Leu Gln Pro Glu Pro Glu Ser Trp Glu Gln Ile Leu Arg

515 520 525

Gln Ser Val Leu Gln His Arg Ala Ala Pro Ala Pro Gly Pro Gly Pro

530 535 540

Gly Ser Gly Tyr Arg Glu Phe Thr Cys Ala Val Lys Gln Gly Ser Ala

545 550 555 560

Pro Asp Ala Gly Gly Pro Gly Phe Gly Pro Ser Gly Glu Ala Gly Tyr

565 570 575

Lys Ala Phe Cys Ser Leu Leu Pro Gly Gly Ala Thr Cys Pro Gly Thr

580 585 590

Ser Gly Gly Glu Ala Gly Ser Gly Glu Gly Gly Tyr Lys Pro Phe Gln

595 600 605

Ser Leu Thr Pro Gly Cys Pro Gly Ala Pro Thr Pro Val Pro Val Pro

610 615 620

Leu Phe Thr Phe Gly Leu Asp Thr Glu Pro Pro Gly Ser Pro Gln Asp

625 630 635 640

Ser Leu Gly Ala Gly Ser Ser Pro Glu His Leu Gly Val Glu Pro Ala

645 650 655

Gly Lys Glu Glu Asp Ser Arg Lys Thr Leu Leu Ala Pro Glu Gln Ala

660 665 670

Thr Asp Pro Leu Arg Asp Asp Leu Ala Ser Ser Ile Val Tyr Ser Ala

675 680 685

Leu Thr Cys His Leu Cys Gly His Leu Lys Gln Trp His Asp Gln Glu

690 695 700

Glu Arg Gly Lys Ala His Ile Val Pro Ser Pro Cys Cys Gly Cys Cys

705 710 715 720

Cys Gly Asp Arg Ser Ser Leu Leu Leu Ser Pro Leu Arg Ala Pro Asn

725 730 735

Val Leu Pro Gly Gly Val Leu Leu Glu Ala Ser Leu Ser Pro Ala Ser

740 745 750

Leu Val Pro Ser Gly Val Ser Lys Glu Gly Lys Ser Ser Pro Phe Ser

755 760 765

Gln Pro Ala Ser Ser Ser Ala Gln Ser Ser Ser Gln Thr Pro Lys Lys

770 775 780

Leu Ala Val Leu Ser Thr Glu Pro Thr Cys Met Ser Ala Ser

785 790 795

<210> 5

<211> 208

<212> Protein

<213> Canis familiaris

<400> 5

Val Lys Val Leu His Glu Pro Ser Cys Phe Ser Asp Tyr Ile Ser Thr

1 5 10 15

Ser Val Cys Gln Trp Lys Met Asp His Pro Thr Asn Cys Ser Ala Glu

20 25 30

Leu Arg Leu Ser Tyr Gln Leu Asp Phe Met Gly Ser Glu Asn His Thr

35 40 45

Cys Val Pro Glu Asn Arg Glu Asp Ser Val Cys Val Cys Ser Met Pro

50 55 60

Ile Asp Asp Ala Val Glu Ala Asp Val Tyr Gln Leu Asp Leu Trp Ala

65 70 75 80

Gly Gln Gln Leu Leu Trp Ser Gly Ser Phe Gln Pro Ser Lys His Val

85 90 95

Lys Pro Arg Thr Pro Gly Asn Leu Thr Val His Pro Asn Ile Ser His

100 105 110

Thr Trp Leu Leu Met Trp Thr Asn Pro Tyr Pro Thr Glu Asn His Leu

115 120 125

His Ser Glu Leu Thr Tyr Met Val Asn Val Ser Asn Asp Asn Asp Pro

130 135 140

Glu Asp Phe Lys Val Tyr Asn Val Thr Tyr Met Gly Pro Thr Leu Arg

145 150 155 160

Leu Ala Ala Ser Thr Leu Lys Ser Gly Ala Ser Tyr Ser Ala Arg Val

165 170 175

Arg Ala Trp Ala Gln Thr Tyr Asn Ser Thr Trp Ser Asp Trp Ser Pro

180 185 190

Ser Thr Thr Trp Leu Asn Tyr Tyr Glu Pro Trp Glu Gln His Leu Pro

195 200 205

<210> 6

<211> 17

<212> Protein

<213> Canis familiaris

<400> 6

Phe Asn Glu Cys Arg Cys Thr Asp Thr Pro Pro Cys Pro Val Pro Glu

1 5 10 15

Pro

<210> 7

<211> 22

<212> Protein

<213> Canis familiaris

<400> 7

Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys

1 5 10 15

Cys Pro Ala Pro Glu Met

20

<210> 8

<211> 20

<212> Protein

<213> Canis familiaris

<400> 8

Ala Lys Glu Cys Glu Cys Lys Cys Asn Cys Asn Asn Cys Pro Cys Pro

1 5 10 15

Gly Cys Gly Leu

20

<210> 9

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> modified dog

<400> 9

Pro Lys Glu Ser Thr Cys Lys Cys Ile Pro Pro Cys Pro Val Pro Glu

1 5 10 15

Ser

<210> 10

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> modified dog

<400> 10

Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr

1 5 10 15

Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu

20 25 30

Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys

35 40 45

Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly

50 55 60

Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu

65 70 75 80

Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser

85 90 95

Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro

100 105 110

Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr

115 120 125

Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp

130 135 140

Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg

145 150 155 160

Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser

165 170 175

Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile

180 185 190

Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser

195 200 205

Leu Ser His Ser Pro Gly

210

<210> 11

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 11

agatactgga tgcac 15

<210> 12

<211> 5

<212> Protein

<213> Mus musculus

<400> 12

Arg Tyr Trp Met His

1 5

<210> 13

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 13

atgattcacc ccgacagcgg caacatcaac tacaacgagc ggttcaagac c 51

<210> 14

<211> 17

<212> Protein

<213> Mus musculus

<400> 14

Met Ile His Pro Asp Ser Gly Asn Ile Asn Tyr Asn Glu Arg Phe Lys

1 5 10 15

Th

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 15

cagctgcgga acgccatgga ttat 24

<210> 16

<211> 8

<212> Protein

<213> Mus musculus

<400> 16

Gln Leu Arg Asn Ala Met Asp Tyr

1 5

<210> 17

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 17

agagccagcg agagcgtgga cagctacggc aacagcttcc tgaac 45

<210> 18

<211> 15

<212> Protein

<213> Mus musculus

<400> 18

Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Leu Asn

1 5 10 15

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 19

agagccagca acctggcctc t 21

<210> 20

<211> 7

<212> Protein

<213> Mus musculus

<400> 20

Arg Ala Ser Asn Leu Ala Ser

1 5

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 21

cagcagaact acgagaaccc cagaacc 27

<210> 22

<211> 9

<212> Protein

<213> Mus musculus

<400> 22

Gln Gln Asn Tyr Glu Asn Pro Arg Thr

1 5

<210> 23

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 23

agctacggca tgagc 15

<210> 24

<211> 5

<212> Protein

<213> Mus musculus

<400> 24

Ser Tyr Gly Met Ser

1 5

<210> 25

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 25

acaatcagca gaggcggcga ctacacctac tatcccgaca gcgtgaaggg c 51

<210> 26

<211> 17

<212> Protein

<213> Mus musculus

<400> 26

Thr Ile Ser Arg Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 27

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 27

ggcaccctga acaaccgggg ctttgcttct 30

<210> 28

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> modified mouse

<400> 28

Gly Thr Leu Asn Asn Arg Gly Phe Ala Ser

1 5 10

<210> 29

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 29

aaggccagcc agaacgtggg caccaatgtg gcc 33

<210> 30

<211> 11

<212> Protein

<213> Mus musculus

<400> 30

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210> 31

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 31

agcgccagct accggtactc t 21

<210> 32

<211> 7

<212> Protein

<213> Mus musculus

<400> 32

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210> 33

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on amino acid

CDR sequences

<400> 33

cagcagtaca acagctaccc ctacacc 27

<210> 34

<211> 9

<212> Protein

<213> Mus musculus

<400> 34

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

<210> 35

<211> 217

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 35

Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Gln Glu

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Leu Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Val Ala Glu Phe Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Asn Asp Ala Gln

100 105 110

Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln Leu His Thr Gly

115 120 125

Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp Thr Gly Ile Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His Glu Leu Tyr Ser

180 185 190

Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu Ile Lys Ser Phe

195 200 205

Gln Arg Ser Glu Cys Gln Arg Val Asp

210 215

<210> 36

<211> 453

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Lys Gly Thr Leu Asn Asn Arg Gly Phe Ala Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu

130 135 140

Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser

180 185 190

Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg

210 215 220

Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu Asp

260 265 270

Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln

275 280 285

Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys

305 310 315 320

Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro

325 330 335

Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser

340 345 350

Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val

355 360 365

Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val

370 375 380

Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr

385 390 395 400

Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys

405 410 415

Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys

420 425 430

Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu

435 440 445

Ser His Ser Pro Gly

450

<210> 37

<211> 453

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Asp Lys Arg Leu Gln Trp Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Leu Asn Asn Arg Gly Phe Ala Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu

130 135 140

Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser

180 185 190

Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg

210 215 220

Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu Asp

260 265 270

Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln

275 280 285

Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys

305 310 315 320

Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro

325 330 335

Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser

340 345 350

Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val

355 360 365

Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val

370 375 380

Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr

385 390 395 400

Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys

405 410 415

Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys

420 425 430

Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu

435 440 445

Ser His Ser Pro Gly

450

<210> 38

<211> 453

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Asp Lys Arg Leu Gln Trp Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Leu Asn Asn Arg Gly Phe Ala Ser Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu

130 135 140

Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser

180 185 190

Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala

195 200 205

Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg

210 215 220

Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu Asp

260 265 270

Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln

275 280 285

Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys

305 310 315 320

Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro

325 330 335

Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser

340 345 350

Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val

355 360 365

Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val

370 375 380

Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr

385 390 395 400

Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys

405 410 415

Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys

420 425 430

Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu

435 440 445

Ser His Ser Pro Gly

450

<210> 39

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 39

Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Thr Ala Ser Ile Tyr Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Glu Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Glu Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Asp Asp Ala Gly Val Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Glu Asn Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Asn Asp Ala Gln Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln

115 120 125

Leu His Thr Gly Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr

130 135 140

Pro Lys Asp Ile Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp

145 150 155 160

Thr Gly Ile Gln Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His

180 185 190

Glu Leu Tyr Ser Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu

195 200 205

Ile Lys Ser Phe Gln Arg Ser Glu Cys Gln Arg Val Asp

210 215 220

<210> 40

<211> 451

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 40

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Arg Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Asp Trp Met

35 40 45

Gly Met Ile His Pro Asp Ser Gly Asn Ile Asn Tyr Asn Glu Arg Phe

50 55 60

Lys Thr Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ala Gly Asp Ile Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Arg Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys

130 135 140

Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg

180 185 190

Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys

195 200 205

Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro

210 215 220

Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile

245 250 255

Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu Asp Pro Glu

260 265 270

Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln

275 280 285

Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys

305 310 315 320

Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu

325 330 335

Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr

340 345 350

Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu

355 360 365

Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp

370 375 380

Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro

385 390 395 400

Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser

405 410 415

Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser His

435 440 445

Ser Pro Gly

450

<210> 41

<211> 451

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 41

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Arg Tyr

20 25 30

Trp Met His Trp Met Lys Gln Ala Pro Gly Ala Gly Leu Asp Trp Ile

35 40 45

Gly Met Ile His Pro Asp Ser Gly Asn Ile Asn Tyr Asn Glu Arg Phe

50 55 60

Lys Thr Lys Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ala Gly Asp Ile Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Arg Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys

130 135 140

Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg

180 185 190

Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys

195 200 205

Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro

210 215 220

Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile

245 250 255

Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu Asp Pro Glu

260 265 270

Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln

275 280 285

Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys

305 310 315 320

Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu

325 330 335

Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr

340 345 350

Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu

355 360 365

Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp

370 375 380

Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro

385 390 395 400

Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser

405 410 415

Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser His

435 440 445

Ser Pro Gly

450

<210> 42

<211> 451

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 42

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Arg Tyr

20 25 30

Trp Met His Trp Met Lys Gln Ala Pro Gly Ala Gly Leu Asp Trp Ile

35 40 45

Gly Met Ile His Pro Asp Ser Gly Asn Ile Asn Tyr Asn Glu Arg Phe

50 55 60

Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ala Gly Asp Ile Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Arg Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys

130 135 140

Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg

180 185 190

Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys

195 200 205

Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro

210 215 220

Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile

245 250 255

Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Leu Asp Pro Glu

260 265 270

Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln

275 280 285

Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Ala Gly Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys

305 310 315 320

Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu

325 330 335

Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr

340 345 350

Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu

355 360 365

Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp

370 375 380

Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro

385 390 395 400

Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser

405 410 415

Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser His

435 440 445

Ser Pro Gly

450

<210> 43

<211> 212

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 43

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val

20 25 30

Ala Trp Tyr Gln Gln Leu Pro Gly Thr Ser Pro Arg Thr Leu Ile Tyr

35 40 45

Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Arg Ser Gly Ser Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr His Leu Thr Val Leu Gly Gln Pro Lys Ala Ser

100 105 110

Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gly Ala Asn

115 120 125

Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Ser Gly Val

130 135 140

Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Thr Gln Gly Val Glu

145 150 155 160

Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

165 170 175

Tyr Leu Ser Leu Thr Pro Asp Lys Trp Lys Ser His Ser Ser Phe Ser

180 185 190

Cys Leu Val Thr His Glu Gly Ser Thr Val Glu Lys Lys Val Ala Pro

195 200 205

Ala Glu Cys Ser

210

<210> 44

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> caninized mouse

<400> 44

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly

20 25 30

Asn Ser Phe Leu Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Ser

35 40 45

Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Glu Arg

50 55 60

Phe Ser Gly Ser Lys Ser Gly Ser Ser Ala Thr Leu Thr Ile Thr Gly

65 70 75 80

Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Gln Asn Tyr Glu

85 90 95

Asn Pro Arg Thr Phe Gly Gly Gly Thr His Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gly Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Ser Gly Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Thr

145 150 155 160

Gln Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Asp Lys Trp Lys Ser His

180 185 190

Ser Ser Phe Ser Cys Leu Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Lys Val Ala Pro Ala Glu Cys Ser

210 215

<210> 45

<211> 215

<212> Protein

<213> Canis familiaris

<400> 45

Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr

1 5 10 15

Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu

20 25 30

Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys

35 40 45

Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly

50 55 60

Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu

65 70 75 80

Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser

85 90 95

Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro

100 105 110

Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr

115 120 125

Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp

130 135 140

Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg

145 150 155 160

Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe Leu Tyr Ser

165 170 175

Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile

180 185 190

Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

195 200 205

Leu Ser His Ser Pro Gly Lys

210 215

<210> 46

<211> 21

<212> Protein

<213> Canis familiaris

<400> 46

Ile Ser His Thr Trp Leu Leu Met Trp Thr Asn Pro Tyr Pro Thr Glu

1 5 10 15

Asn His Leu His Ser

20

<210> 47

<211> 41

<212> Protein

<213> Canis familiaris

<400> 47

Asn Asp Asn Asp Pro Glu Asp Phe Lys Val Tyr Asn Val Thr Tyr Met

1 5 10 15

Gly Pro Thr Leu Arg Leu Ala Ala Ser Thr Leu Lys Ser Gly Ala Ser

20 25 30

Tyr Ser Ala Arg Val Arg Ala Trp Ala

35 40

<210> 48

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> computer generated based on the amino acid sequence of the CDR

<400> 48

ggcaccctga acaaccgcgg ctttgcgtgc 30

<210> 49

<211> 10

<212> Protein

<213> Mus musculus

<400> 49

Gly Thr Leu Asn Asn Arg Gly Phe Ala Cys

1 5 10

<---

Claims (24)

1. An isolated mammalian antibody, or antigen-binding fragment thereof, that binds to canine interleukin-4 receptor α (IL-4Rα), comprising a set of six complementarity determining regions (CDRs), three of which are heavy chain CDRs: heavy chain CDR 1 (HCDR1), heavy chain CDR 2 (HCDR2), and heavy chain CDR 3 (HCDR3), and three of which are light chain CDRs: light chain CDR 1 (LCDR1), light chain CDR 2 (LCDR2), and light chain CDR 3 (LCDR3); wherein (i) HCDR1 comprises the amino acid sequence of SEQ ID NO: 24; (ii) HCDR2 comprises the amino acid sequence of SEQ ID NO: 26; (iii) HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 28 and SEQ ID NO: 49; (iv) LCDR1 comprises the amino acid sequence of SEQ ID NO: 30; (v) LCDR2 comprises the amino acid sequence of SEQ ID NO: 32; and (vi) LCDR3 comprises the amino acid sequence of SEQ ID NO: 34. 2. An isolated mammalian antibody, or antigen-binding fragment thereof, according to claim 1, wherein the antibody, and antigen-binding fragment thereof, binds to canine IL-4Rα and blocks the binding of canine IL-4Rα to canine interleukin-4. 3. An isolated mammalian antibody, or antigen-binding fragment thereof, according to claim 1 or 2, which is a caninized antibody or caninized antigen-binding fragment thereof. 4. An isolated mammalian antibody, or antigen-binding fragment thereof, according to claim 3, which comprises a hinge region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. 5. An isolated mammalian antibody, or antigen-binding fragment thereof, according to claim 3, which comprises a heavy chain comprising a modified canine IgG-B (IgG-Bm) comprising the amino acid sequence of SEQ ID NO: 10. 6. An isolated mammalian antibody, or antigen-binding fragment thereof, according to claim 5, comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38. 7. An isolated mammalian antibody, or antigen-binding fragment thereof, according to claim 6, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 35. 8. An isolated mammalian antibody, or antigen-binding fragment thereof, according to claim 6, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 43. 9. An isolated nucleic acid encoding the heavy chain of an isolated mammalian antibody or antigen-binding fragment thereof according to claims 3, 4, 5, 6, 7 or 8. 10. An isolated nucleic acid encoding the light chain of an isolated antibody or antigen-binding fragment thereof according to claim 7 or 8. 11. An expression vector containing the isolated nucleic acid according to claim 9. 12. An expression vector containing the isolated nucleic acid according to claim 10. 13. An expression vector comprising the isolated nucleic acid of claim 9 and the isolated nucleic acid of claim 10. 14. A host cell containing the expression vector of claim 11 for producing the heavy chain of the isolated mammalian antibody or an antigen-binding fragment thereof. 15. A host cell containing the expression vector of claim 12 for producing the light chain of the isolated mammalian antibody or antigen-binding fragment thereof. 16. A host cell containing the expression vector of claim 13 for producing an isolated mammalian antibody or antigen-binding fragment thereof. 17. A pharmaceutical composition for reducing inflammation associated with atopic dermatitis, comprising an isolated mammalian antibody or antigen-binding fragment thereof according to claims 3, 4, 5, 6, 7 or 8 and a pharmaceutically acceptable carrier or diluent. 18. A method for reducing inflammation associated with atopic dermatitis, comprising administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 17.