Antibodies to canine interleukin-4 receptor alpha

Abstract

To provide a safe and effective treatment option for atopic dermatitis in companion animals.SOLUTION: The present invention discloses antibodies and blocking antibodies to canine IL-4 receptor alpha that have specific sequences and a high binding affinity for canine IL-4 receptor α. The present invention also discloses the use of the antibodies of the present invention in the treatment of atopic dermatitis in dogs. The present invention further discloses unique epitopes that bind to the antibodies to canine IL-4 receptor alpha.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 142,108, filed April 2, 2015. No. 62 / 269,486 filed December 18, 2015 and March 18, 2016 No. 62 / 310,250 filed on 25 Dec. 2002, the entire contents of all of which are incorporated herein by reference. (which will be incorporated herein by reference) claims priority under 35 U.S.C. § 119(e). That is why.

[0002] FIELD OF THE INVENTION The present invention relates to a method for producing a canine IL-4 receptor alpha antibody having high binding affinity and specific binding to the canine IL-4 receptor alpha. an antibody against canine IL-4 receptor alpha (canine IL-4 receptor alpha) having the sequence The present invention further relates to canine IL-4-binding proteins (including some capable of blocking the binding of canine IL-4 to canine IL-4). The present invention also relates to a unique epitope that binds to an antibody against receptor alpha 4. The present invention relates to the use of the antibodies and epitopes of the present invention in the treatment of atopic dermatitis. [Background technology]

[0003] The immune system is a complex network of resident and recurrent immune cells that function cooperatively to protect the host from infection and cancer. The ability of the immune system to perform this function is due to the presence of white blood cells, which contain a network of circulating specialized cells. It is highly dependent on the biological activity of a group of proteins collectively known as interleukins, which are secreted by the immune system. Interleukins that have been studied in detail include interleukin 4 (IL-4 Two key molecules have been identified as IL-13 (IL-14) and IL-15 (IL-16). IL-4 and IL-13 are involved in the expression of CD4 + Th2 cells, natural killer T cells It is expressed by multiple cell types, including NKT cells, macrophages, mast cells, and basophils. IL-4 and IL-13 are two closely related proteins that can be secreted by many They exhibit overlapping functions and are crucial for the generation of T cell-dependent humoral immune responses. Despite their similarities in structure, cellular origin and biological function, these sites Each of these interleukins mediates a specialized function, which is The receptors and downstream signaling pathways that mediate both their common and unique biological activities This has stimulated much research aimed at identifying nulling pathways.

[0004] IL-4 has high affinity to two receptors, namely, type I and type II IL-4 receptors. It is now known that type I IL-4 receptor binds to the IL-4 receptor α chain in a cellular context. , several other interleukins, including IL-2, IL-7, IL-9, and IL-15 The type II IL-4 receptor consists of the common γC chain, which is also part of the IL-4 receptor. IL-13 consists of the type II IL-4 receptor α chain and the IL-13 receptor α1 chain. IL-1 binds to the IL-13 receptor and binds to a specific receptor called IL-1 receptor α2. Binding of IL-13 to the α2 receptor does not signal, and this receptor is also expressed in soluble form. Therefore, IL-13 receptor α2 is often called a decoy receptor. .

[0005] Genes encoding IL-4 proteins from various species have been cloned and are available in bacteria and For example, mature human IL-4 has an estimated molecular weight of 15 Kd. The human IL-4 gene encoding the IL-4 gene was found to be a secreted polypeptide of 129 amino acids with a molecular weight of 1. The cDNA has been shown to be SA.83(16):5894-5898(1986)]. The corresponding cDNA has also been identified, which has 40% identity to human IL-413. It has been shown to encode a polypeptide of two amino acids [van der Kaai J et al., Immunogenetics 49:142-143 (1999)]. Human IL The gene encoding -13 has been cloned and expressed in various host systems. Minty et al., Nature 362:248-50 (1993)]. Mature IL-13 is Human IL-13 is a secreted polypeptide with an apparent molecular weight of 12.4 Kd. A cDNA encoding canine IL-13 has also been identified. [Yang et al., J. Interferon and Cytokine Research rch 20:779-785(2000)]. The predicted mature canine IL-13 polypeptide is It consists of 111 amino acids and shares 61.8% identity with human IL-13.

[0006] The genes encoding the human and mouse IL-4 receptor α chains have been cloned and various The cDNA encoding the human IL-4 receptor α chain is expressed in a host system. According to Gallizzi et al. [International Immunology 2(7): 669-675 (1990)], which encodes the mouse IL-4 receptor α chain. The cDNA used was prepared as described by Mosley et al. [Cell, 59(2):335-348(1989)]. The cDNA for the human IL-4 receptor α chain is a sequence of 24 amino acid residues. The mouse protein encodes 825 amino acid residues including the signal sequence. Although 15 amino acid residues shorter than α-glucan, both proteins are closely related and share overall sequence identity. Sex is 50% at the amino acid level.

[0007] Genes encoding the α-chain of the equine, canine and feline IL-4 receptor have also been disclosed [US See Patent Publication No. 7,208,579 B2. Also, the canine IL-4 receptor α isoform A putative cDNA corresponding to one of the forms was found in the Genbank database. Therefore, the present invention provides a method for determining the IL-4 receptor α chain cDNA. and unambiguously determining the encoded polypeptide sequence.

[0008] IL-4 and IL-13 are essential for the defense against extracellular pathogens (e.g., tissue- or lumen-dwelling parasites) is a critical cytokine for the development of the Th2 immune response required for protection against However, both cytokines are involved in various allergic diseases (asthma and It has been implicated in the pathogenesis of various diseases, including atopic dermatitis. Asthma is a common This is a serious respiratory disease that can cause pneumonia, hypersensitivity of the bronchial airways to external stimuli, and respiratory The pathophysiology of allergic asthma is characterized by structural modifications of the wall tissue. lla et al [Journal of Asthma and Allergy 7:123 -130 (2014)]. Asthma is caused by high amounts of IL-4 and IL-1. CD4 Produces 3 and Modulates Immune-Inflammatory Responses in Allergic Airways + Th2 cells Recent advances in understanding the asthma response have highlighted the role of IL-4 in the pathogenesis of the disease. This paper highlights the important role played by both IL-13 and IL-2. Cytokines also play a role in the switching of immunoglobulin isotypes from IgM to IgE in B cells. This allergen-specific IgE stimulates mast cell degranulation and inflammation in the airways. Both IL-4 and IL-13 contribute to the release of mediators in bronchial smooth muscle. It enhances the contraction of the airways and stimulates the recruitment of eosinophils, which activate the corresponding receptors on eosinophils. It can also induce degranulation in response to cross-linking of allergen-bound IgE to IL-1. -13 also stimulates mucus secretion and promotes airway remodeling, which includes goblet cell hyperplasia, collagen This occurs by stimulating the deposition of inflammatory cytokines and proliferation of airway smooth muscle cells. L-4 and IL-13 promote the development of asthma attacks, including enhanced bronchoconstriction and airway hyperactivity. It is now clear that cerebrospinal fluid (CEF) is closely involved in the pathological changes that lead to cerebrospinal fluid (CEF) damage.

[0009] Atopic dermatitis (AD) is characterized by immune system dysregulation and epidermal barrier abnormalities. AD is a recurrent, pruritic, inflammatory skin disease characterized by a variety of skin conditions. The pathological and immunological characteristics of AD have been extensively studied. [Rahman et al., Inflammation & Allergy -drug target 10:486-496(2011) and Harskamp Seminar in Cutaneous Medicine and Surg [Reviewed in [Dr.] J.D., 2013, 32:132-139]. AD is the most common form of AD in humans. It is also a common skin disease, affecting 2-10% of the adult population in the United States and approximately 25% of children worldwide. In humans, AD skin lesions are caused by Th2 cells, eosinophils, mast cells, and dendritic cells. In the acute phase of AD, these lesions are characterized by IL-4 AD shows predominant expression of Th2-type cytokines, including IL-13 and IL-14. It is also characterized by elevated levels of CD4 in the skin. + IL-4 and Th2 cells AD is classified as a Th2 disease, but the expression of Th 1, Other T cell subsets, such as Th22 and Th17, also contribute to the pathogenesis of the disease. Despite the increasing incidence of AD worldwide, symptoms may be relieved by topical medications. For patients not adequately suppressed by steroids, available treatment options are oral corticosteroids, oral Oral cyclosporine and narrow-band UVB phototherapy are the only treatments available. Furthermore, their use is associated with various safety implications. α Unique to Effective monoclonal antibodies have been developed, and some of these antibodies are useful in the treatment of atopic dermatitis. have been extensively tested for their therapeutic utility in humans for See 20150017176 A1.

[0010] AD is also a common disease in companion animals, especially dogs, and its prevalence in dogs is It is estimated that AD accounts for approximately 10-15% of the total dog population. Pathogenesis [Nuttall et al., Veterinary Records 172(8) :201-207(2013)] has significant correlation with AD in humans. Similarities include a predominance of IL-4 and IL-13 cytokines, -This includes a Th2-biased cytokine environment and skin infiltration of various immune cells. As in the case of dogs and cats, the current therapy for atopic dermatitis is shampooing. Palliative treatments such as lotions and moisturizers, or oral or systemic corticosteroids and As with AD in humans, symptomatic treatment relies on the use of oral cyclosporine. However, these therapies do not address the underlying mechanisms of the disease and have significant safety implications. Therefore, a safe and effective treatment for AD in companion animals is needed. There is an unmet medical need for options. Such treatments are preferably , should interfere with the underlying mechanisms of the disease.

[0011] Citation of any reference herein does not imply that such reference is an "Prior Art" to this application. This document should not be construed as an admission that the information contained herein is available for download. Summary of the Invention

[0012] Overview of the invention The present invention relates to canine IL-4R α Anti-dog interferon antibody with high binding affinity to Interleukin-4 receptor alpha (IL-4R α ) antibodies. In more particular embodiments , the anti-canine interleukin-4 receptor alpha (IL-4R α ) antibodies are type I or II Blocking the binding of canine IL-4 and canine IL-13 to the IL-4 receptor and subsequently inhibiting canine IL-13 It also has the ability to inhibit signaling from both IL-4 and IL-13. In the form of such anti-canine IL-4R α The antibody is mouse anti-canine IL-4R α With antibodies In more particular embodiments, the anti-canine IL-4R α The antibody is canine IL-4R α to It has high binding affinity to canine IL-4 receptors, and binds to type I and type II IL-4 receptors. and has the ability to block the binding of canine IL-13.

[0013] Furthermore, the present invention relates to the complementarity determining regions (CDRs) contained in these antibodies, and CDRs (e.g., mouse anti-canine IL-4R α antibody) in a canine frame Incorporated caninized anti-canine IL-4R α The present invention also relates to the preparation of antibodies against atopic dermatitis. conditions such as dermatitis and / or type I and / or type II IL-4 receptor Downstream effects of signaling from binding of canine IL-4 and / or canine IL-13 The present invention relates to the use of such antibodies in the treatment of various cancers and other adverse conditions.

[0014] Thus, the present invention utilizes 14 exemplified murine anti-canine IL-4R antibodies. α C from antibodies The fourteen exemplified mouse antibodies provide a unique set (combination) of DRs. Canine IL-4R α Antibodies have a unique set of CDRs: three light chain CDRs, CD1, CD2, and CD3. CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2) and CDR light chain 3 (CDRL3) ) and three heavy chain CDRs: CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDR H2) and CDR heavy chain 3 (CDRH3). As described in detail below However, there is considerable sequence homology and even some overlap within each group of CDRs (e.g., (See the set of CDRL1 below.) Thus, the present invention provides 14 exemplified Mouse anti-canine IL-4R α By simply providing the amino acid sequences of the six CDRs from an antibody, Furthermore, conservatively modified variants of these CDRs, as well as the same standards (canonical IL-4R) structure and / or α The epitope Canine IL-4R α At one or more (e.g., 1 to 4 or more) of the amino acid residues of Binding variants are also provided.

[0015] Therefore, the present invention provides SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 1 29, a light chain complementarity determining region comprising the amino acid sequence of SEQ ID NO: 130 or SEQ ID NO: 131 1 (VL CDR1) and / or SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: No. 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, A light chain complement comprising the amino acid sequence of SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134 Sex-determining region 2 (VL CDR2) and / or SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: No. 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 or A light chain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence of SEQ ID NO: 139 and / or or SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: No. 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO: 14 1. Heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 142 or SEQ ID NO: 143 (VH CDR1) and / or SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, Sequence number 144, sequence number 145, sequence number 146, sequence number 147 or sequence number 14 Heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of SEQ ID NO: 8 and / or SEQ ID NO: SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, Sequence number 98, sequence number 99, sequence number 100, sequence number 149, sequence number 150, sequence number heavy chain complementarity determination comprising the amino acid sequence of SEQ ID NO: 151, SEQ ID NO: 152 or SEQ ID NO: 153 IL-4R, including region 3 (VH CDR3) α Antibodies that specifically bind to or their antigens In certain embodiments, the antibody is a mammalian antibody. In more particular embodiments, the antibody is a caninized antibody.

[0016] Therefore, the caninized antibody or antigen-binding fragment thereof of the present invention can be identified as SEQ ID NO: 74 , SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142 or a heavy chain complementarity determining region 1 (VH CDR1) having the amino acid sequence of SEQ ID NO: 143 In another embodiment, the heavy chain complementarity determining region 2 (VH CDR 2) are SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: No. 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 144, SEQ ID NO: 14 5, the amino acid sequence of SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NO: 148. In another embodiment, the heavy chain complementarity determining region 3 (VH CDR3) is SEQ ID NO: 9 2, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: No. 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 1 51, SEQ ID NO: 152 or SEQ ID NO: 153. In embodiments, the caninized antibody or antigen-binding fragment has the sequence SEQ ID NO: 74, Sequence number 75, sequence number 76, sequence number 77, sequence number 78, sequence number 79, sequence number 80 , SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 or VH CDR1 comprising the amino acid sequence of SEQ ID NO: 143 and VH CDR2 comprising the amino acid sequence of SEQ ID NO: 83 and SEQ ID NO: 84 , SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 14 7 or SEQ ID NO: 148. In such embodiments, the caninized antibody or antigen-binding fragment has the sequence No. 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: VH CDR1 comprising the amino acid sequence of SEQ ID NO: 142 or SEQ ID NO: 143, and VH CDR1 comprising the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 9 8, SEQ ID NO: 99 or SEQ ID NO: 100, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 1 51, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 152 or SEQ ID NO: 153. In yet another such embodiment, the caninized antibody or antigen-binding The functional fragments are SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: No. 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 144, Amino acid sequence of SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NO: 148 and a VH CDR2 comprising SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100, No. 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152 or SEQ ID NO: 153 and a VH CDR3 comprising the amino acid sequence. In the above, the caninized antibody or antigen-binding fragment is selected from the group consisting of SEQ ID NO: 74, SEQ ID NO: 7 5, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 or SEQ ID NO: VH CDR1 comprising the amino acid sequence of SEQ ID NO: 143, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: SEQ ID NO: 91, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NO: VH CDR2 comprising the amino acid sequence of SEQ ID NO: 148, and SEQ ID NO: 92 and SEQ ID NO: 93 , SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152 or SEQ ID NO: 153.

[0017] In certain embodiments, the caninized antibody or antigen-binding fragment also has the sequence No. 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 129, SEQ ID NO: 130 or It contains a light chain complementarity determining region 1 (VL CDR1) containing the amino acid sequence of SEQ ID NO: 131. In a related embodiment, the light chain complementarity determining region 2 (VL CDR2) is SEQ ID NO: 56, the sequence No. 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134 In yet another embodiment, the light chain complementarity determining region 3 (VL) CDR3) are SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 6 9, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 or SEQ ID NO: 139. In certain embodiments of this type, the caninized antibody or antigen-binding fragment The sequences are SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: No. 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 129, SEQ ID NO: 13 0 or SEQ ID NO: 131, and a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 56, SEQ ID NO: SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, Sequence number 63, sequence number 64, sequence number 132, sequence number 133 or sequence number 134 The VL CDR2 contains both the amino acid sequence.

[0018] In other such embodiments, the caninized antibody or antigen-binding fragment comprises: SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 5 2, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 129, SEQ ID NO: 130 or VL CDR1 comprising the amino acid sequence of SEQ ID NO: 131 and VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65 and SEQ ID NO: 66 , SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 13 and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 139. In one such embodiment, the caninized antibody or antigen-binding fragment comprises: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 6 1, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133 or VL CDR2 comprising the amino acid sequence of SEQ ID NO: 134 and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 65 and SEQ ID NO: 66 , SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 13 8 or SEQ ID NO: 139.

[0019] In still other such embodiments, the caninized antibody or antigen-binding fragment SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: No. 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 129, SEQ ID NO: 130 or VL CDR1 comprising the amino acid sequence of SEQ ID NO: 131, SEQ ID NO: 56, SEQ ID NO: 5 7, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: No. 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134 VL CDR2 containing the amino acid sequence, and SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: No. 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 or SEQ ID NO: 13 The VL CDR3 comprises a sequence of 9 amino acids.

[0020] In certain embodiments, the caninized anti-canine IL-4R α Antibodies contain complementarity-determining regions (CD R), in which the CDRs are as follows for CDR1, CDR2, and CDR3 of the heavy chain: They have the canonical structures H1-1, H2-3A, and H3-12, respectively, i.e. CDR1 of the heavy chain has canonical structure class 1, and CDR2 of the heavy chain has canonical structure class 3A. and the CDR3 of the heavy chain has canonical structure class 12. In more particular embodiments and the corresponding light chain CDRs are L1-L1 for CDR1, CDR2 and CDR3 of the light chain, respectively. In other embodiments, the caninized antibody has the canonical structure of L1, L2-1, and L3-1. Canine IL-4R α Antibodies contain complementarity determining regions (CDRs), where the CDRs are the C of the heavy chain. H1-1, H2-2A and H3-7 for DR1, CDR2 and CDR3, respectively In a more particular embodiment of this type, the CD of the corresponding light chain R represents L1-2A, L2-1 and L3-2A for CDR1, CDR2 and CDR3 of the light chain, respectively. In yet another embodiment, the caninized anti-canine IL-1 antibody has the canonical structure of L3-1 and L3-2. 4R α The antibody comprises complementarity determining regions (CDRs), in this case CDRs are CDR1 of the heavy chain, CDR2 of the Standard structures of H1-1, H2-2B and H3-15 for DR2 and CDR3, respectively In more particular embodiments of this type, the CDRs of the corresponding light chain have the structure L1-4, L2-1 and L3- for CDR1, CDR2 and CDR3, respectively. 1. In yet another embodiment, the caninized anti-canine IL-4R α antibody The complementarity determining regions (CDRs) of the heavy chain include CDR1, CDR2 and CDR3 of the heavy chain. The CDR1 and CDR2 have the canonical structures H1-1, H2-1, and H3-15, respectively. In even more particular embodiments of this type, the CDRs of the corresponding light chain are light chain CDR1, Canonical structures of L1-3, L2-1 and L3-1 for CDR2 and CDR3, respectively In yet another embodiment, the caninized anti-canine IL-4R α Antibodies are complementary determinants In this case, the CDRs correspond to CDR1, CDR2, and CDR3 of the heavy chain. These have the standard structures H1-1, H2-2B, and H3-6, respectively. In even more particular embodiments, the CDRs of the corresponding light chain comprise light chain CDR1, CDR2 and CDR3. The CDRs have the canonical structures of L1-2A, L2-1, and L3-1 for CDR3, respectively.

[0021] In yet other embodiments, the caninized anti-canine IL-4R α Antibodies contain complementarity-determining regions (C CDRs include CDR1, CDR2, and CDR3 of the heavy chain. These have the standard structures H1-1, H2-2B, and H3-4, respectively. In particular embodiments, the CDRs of the corresponding light chain comprise light chain CDR1, CDR2 and CDR 3 have the standard structures of L1-6, L2-1, and L3-1, respectively. In an embodiment, the caninized anti-canine IL-4R αAntibodies contain complementarity determining regions (CDRs) In this case, the CDRs are H1-H1 for CDR1, CDR2 and CDR3 of the heavy chain, respectively. 1, H2-1 and H3-13 standard structures. In this embodiment, the CDRs of the corresponding light chain are identical with respect to CDR1, CDR2, and CDR3 of the light chain. Each of the L1-1, L2-1, and L3-1 structures has a standard structure. The caninized anti-canine IL-4R α The antibody comprises a complementarity determining region (CDR), in this case The CDRs are H1-1 and H2-2 for CDR1, CDR2, and CDR3 of the heavy chain, respectively. A and H3-6. In a more detailed embodiment of this type, and the corresponding light chain CDRs are L1-L1 for CDR1, CDR2 and CDR3 of the light chain, respectively. It has the standard structure of 2A, L2-1 and L3-1.

[0022] In yet other embodiments, the caninized anti-canine IL-4R α Antibodies contain complementarity-determining regions (C CDRs include CDR1, CDR2, and CDR3 of the heavy chain. They have the standard structures H1-1, H2-3A, and H3-15 or H3-13, respectively. In more particular embodiments of this type, the CDRs of the corresponding light chain are CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR11, CDR12, CDR13, CDR1 The canonical structures of L1-6, L2-1 and L3-1 for DR2 and CDR3, respectively, are In yet another embodiment, the caninized anti-canine IL-4R antibody α Antibodies are composed of complementarity-determining regions In this case, the CDRs are related to CDR1, CDR2, and CDR3 of the heavy chain. These have the standard structures H1-1, H2-2A, and H3-10, respectively. In even more particular embodiments, the CDRs of the corresponding light chain comprise light chain CDR1, CDR2 and CDR3. The CDR1 and CDR2 have the canonical structures of L1-6, L2-1, and L3-1, respectively. In yet another embodiment, the caninized anti-canine IL-4R α Antibodies contain complementarity determining regions (CDRs) ), where the CDRs are CDR1, CDR2, and CDR3 of the heavy chain, respectively. It has standard structures H1-1, H2-3A and H3-9. In a preferred embodiment, the CDRs of the corresponding light chain are CDR1, CDR2, and CDR3 of the light chain. They have the standard structures L1-3, L2-1 and L3-3, respectively.

[0023] The present invention also provides a method for the detection of IL-4R antibodies comprising a canine IgG heavy chain and a canine kappa or lambda light chain. α The present invention provides an isolated caninized antibody or antigen-binding fragment thereof that specifically binds to In a particular embodiment of this type, the three light chain complementarity determining regions (CDRs) are: CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2) and CDR light chain 3 (CDR a canine kappa or lambda light chain containing the three heavy chain CDRs, and a CDR heavy chain containing the three heavy chain CDRs, CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2) and CDR heavy chain 3 (CDRH3) Canine IgG heavy chain is a mouse anti-canine IL-4R α The caninized antibodies and Specific embodiments of the canine IL-4R and antigen-binding fragments thereof include α Binds to, and / or canine IL-4R to canine IL-4 α Blocks the binding of

[0024] In a specific embodiment, the present invention provides a method for detecting the three light chain complementarity determining regions (CDRs): C CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL2) and CDR light chain 3 (CDRL 3) and three heavy chain CDRs, CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH 2) and CDR heavy chain 3 (CDRH3), IL-4R α An isolated mammalian antibody or antigen-binding fragment thereof that specifically binds to In one embodiment, CDRL1 is SEQ ID NO: 47, SEQ ID NO: 4 7, conservatively modified variants of SEQ ID NO: 47 (i.e., conservatively modified variants) , the amino acid sequence of a variant of SEQ ID NO: 47 containing one canonical structural class; SEQ ID NO: 48, the sequence Variants of number 48, conservatively modified variants of SEQ ID NO: 48, sequences containing the canonical structural class of 2A Variants of SEQ ID NO: 48, SEQ ID NO: 49, Variants of SEQ ID NO: 49, Conservatively modified variants of SEQ ID NO: 49 Variants of SEQ ID NO: 49, including the four canonical structural classes, SEQ ID NO: 50, SEQ ID NO: 50 Conservatively modified variants of SEQ ID NO: 50, variants of SEQ ID NO: 50 containing the three canonical structural classes Variant, SEQ ID NO: 51, mutant of SEQ ID NO: 51, conservatively modified variant of SEQ ID NO: 51, target of 3 Variants of SEQ ID NO: 51, SEQ ID NO: 52, variants of SEQ ID NO: 52, sequences containing parastructural classes Conservatively modified variants of SEQ ID NO: 52, variants of SEQ ID NO: 52 containing the canonical structural class 2A, sequence No. 53, variants of SEQ ID NO: 53, conservatively modified variants of SEQ ID NO: 53, six canonical structural classes SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 54 variants, SEQ ID NO: 54 variants Conservatively modified variants, variants of SEQ ID NO: 54 containing one canonical structural class, SEQ ID NO: 55, Variants of SEQ ID NO: 55, conservatively modified variants of SEQ ID NO: 55, sequences containing the canonical structural class of 2A Variant of sequence number 55, sequence number 129, variant of sequence number 129, conservation of sequence number 129 modified variants, variants of SEQ ID NO: 129 containing six canonical structural classes, SEQ ID NO: 130, Variants of SEQ ID NO: 130, conservatively modified variants of SEQ ID NO: 130, including 6 canonical structural classes Variants of SEQ ID NO: 130, SEQ ID NO: 131, Variants of SEQ ID NO: 131, Conservatively modified variants, the amino acid sequences of variants of SEQ ID NO: 131 containing the three canonical structural classes include.

[0025] The corresponding CDRL2 is SEQ ID NO: 56, a variant of SEQ ID NO: 56, a conservatively modified version of SEQ ID NO: 56 Modified variants, variants of SEQ ID NO: 56 containing one canonical structural class, SEQ ID NO: 57, SEQ ID NO: 5 7 variants, conservatively modified variants of SEQ ID NO: 57, SEQ ID NO: 57 containing the canonical structural class of 1 variants of SEQ ID NO: 58, variants of SEQ ID NO: 58, conservatively modified variants of SEQ ID NO: 58, 1 SEQ ID NO: 58 variants, SEQ ID NO: 59 variants of SEQ ID NO: 59, Conservatively modified variants of SEQ ID NO: 59, variants of SEQ ID NO: 59 containing one canonical structural class, SEQ ID NO: 60, variants of SEQ ID NO: 60, conservatively modified variants of SEQ ID NO: 60, standard structure class 1 Variants of SEQ ID NO: 60 containing ras, SEQ ID NO: 61, variants of SEQ ID NO: 61, SEQ ID NO: 61 Conservatively modified variants of SEQ ID NO: 61, variants of SEQ ID NO: 61 containing one canonical structural class, SEQ ID NO: 62, Variants of SEQ ID NO: 62, conservatively modified variants of SEQ ID NO: 62, sequences containing one canonical structural class Variants of SEQ ID NO: 62, SEQ ID NO: 63, Variants of SEQ ID NO: 63, Conservative modifications of SEQ ID NO: 63 Variants, variants of SEQ ID NO: 63 containing one canonical structural class, SEQ ID NO: 64, SEQ ID NO: 64 Conservatively modified variants of SEQ ID NO: 64, variants of SEQ ID NO: 64 containing one canonical structural class Variants, SEQ ID NO: 132, variants of SEQ ID NO: 132, conservatively modified variants of SEQ ID NO: 132 , variants of SEQ ID NO: 132 containing the canonical structural class of 1, SEQ ID NO: 133, SEQ ID NO: 133 Conservatively modified variants of SEQ ID NO: 133, including the canonical structural class of SEQ ID NO: 13 3 variants, SEQ ID NO: 134, variants of SEQ ID NO: 134, conservatively modified variants of SEQ ID NO: 134 The amino acid sequences of variants or variants of SEQ ID NO: 134 that contain one of the canonical structural classes are included.

[0026] The corresponding CDRL3 is SEQ ID NO: 65, a variant of SEQ ID NO: 65, a conservatively modified version of SEQ ID NO: 65 Modified variants, variants of SEQ ID NO: 65 containing one canonical structural class, SEQ ID NO: 66, SEQ ID NO: 6 6 variants, conservatively modified variants of SEQ ID NO: 66, SEQ ID NO: 66 containing 1 canonical structural class variants of SEQ ID NO: 67, variants of SEQ ID NO: 67, conservatively modified variants of SEQ ID NO: 67, 1 SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 68 variants, Conservatively modified variants of SEQ ID NO: 68, variants of SEQ ID NO: 68 containing one canonical structural class, SEQ ID NO: 69, variants of SEQ ID NO: 69, conservatively modified variants of SEQ ID NO: 69, standard structure class 1 Variants of SEQ ID NO: 69 containing ras, SEQ ID NO: 70, variants of SEQ ID NO: 70, SEQ ID NO: 70 Conservatively modified variants of SEQ ID NO: 70, variants of SEQ ID NO: 71, comprising the canonical structural class of 1; Variants of SEQ ID NO: 71, conservatively modified variants of SEQ ID NO: 71, sequences containing one canonical structural class Variants of SEQ ID NO: 71, SEQ ID NO: 72, Variants of SEQ ID NO: 72, Conservative modifications of SEQ ID NO: 72 Variants, variants of SEQ ID NO: 72 containing one canonical structural class, SEQ ID NO: 73, SEQ ID NO: 73 Conservatively modified variants of SEQ ID NO: 73, variants of SEQ ID NO: 73 containing one canonical structural class Variants, SEQ ID NO: 135, variants of SEQ ID NO: 135, conservatively modified variants of SEQ ID NO: 135 , variants of SEQ ID NO: 135 containing the canonical structural class of 1, SEQ ID NO: 136, SEQ ID NO: 136 a variant of SEQ ID NO: 136, a conservatively modified variant of SEQ ID NO: 13, 6, SEQ ID NO: 137, SEQ ID NO: 137, Conservatively modified variants of SEQ ID NO: 137 Variants, variants of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 1 38 variants, conservatively modified variants of SEQ ID NO: 138, SEQ ID NO: 139, containing 3 canonical structural classes 138, SEQ ID NO: 139, SEQ ID NO: 139, Conservative modifications of SEQ ID NO: 139 The amino acid sequence of a modified variant or variant of SEQ ID NO: 139 containing one of the canonical structural classes .

[0027] The corresponding CDRH1 is SEQ ID NO: 74, a variant of SEQ ID NO: 74, a conservative modification of SEQ ID NO: 74 Modified variants, variants of SEQ ID NO: 74 containing one canonical structural class, SEQ ID NO: 75, SEQ ID NO: 7 5 variants of SEQ ID NO: 75, conservatively modified variants of SEQ ID NO: 75, including 1 canonical structural class variants of SEQ ID NO: 76, variants of SEQ ID NO: 76, conservatively modified variants of SEQ ID NO: 76 or 76, SEQ ID NO: 77, SEQ ID NO: 77 variants containing one canonical structural class a conservatively modified variant of SEQ ID NO: 77 or a variant of SEQ ID NO: 77 containing one of the canonical structural classes Variant, SEQ ID NO: 78, mutant of SEQ ID NO: 78, conservatively modified variant of SEQ ID NO: 78, target of 1 Variants of SEQ ID NO: 78, SEQ ID NO: 79, variants of SEQ ID NO: 79, sequences Conservatively modified variants of SEQ ID NO: 79, variants of SEQ ID NO: 79 containing one canonical structural class, No. 80, variants of SEQ ID NO: 80, conservatively modified variants of SEQ ID NO: 80, canonical structural class 1 SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 81 variants ... SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, S A variant of SEQ ID NO: 82, a conservatively modified variant of SEQ ID NO: 82, or a sequence comprising one canonical structural class Variant of sequence number 82, sequence number 140, variant of sequence number 140, conservation of sequence number 140 modified variants, variants of SEQ ID NO: 140 containing one canonical structural class, SEQ ID NO: 141, Variants of sequence number 141, conservatively modified variants of sequence number 141, containing a canonical structural class of 1 Variants of SEQ ID NO: 141, SEQ ID NO: 142, Variants of SEQ ID NO: 142, Conservatively modified variants, variants of SEQ ID NO: 142 containing one canonical structural class, SEQ ID NO: 143 , a variant of SEQ ID NO: 143, a conservatively modified variant of SEQ ID NO: 143 or one of the canonical structural classes The amino acid sequence of a variant of SEQ ID NO: 143 containing the amino acid sequence of SEQ ID NO: 143.

[0028] The corresponding CDRH2 is SEQ ID NO: 83, a variant of SEQ ID NO: 83, a conservative modification of SEQ ID NO: 83 SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, S Variants of SEQ ID NO: 84, conservatively modified variants of SEQ ID NO: 84, SEQ ID NO: 2A containing canonical structural class Variants of SEQ ID NO: 84, SEQ ID NO: 85, Variants of SEQ ID NO: 85, Conservatively modified variants of SEQ ID NO: 85 or a variant of SEQ ID NO: 85 containing the canonical structural class of 2B, SEQ ID NO: 86, SEQ ID NO: 86 Conservatively modified variants of SEQ ID NO: 86, SEQ ID NO: 87, variants of SEQ ID NO: 87, Conservatively modified variants of sequence number 87, variants of sequence number 87 containing one canonical structural class, sequence No. 88, variants of SEQ ID NO: 88, conservatively modified variants of SEQ ID NO: 88, canonical structure of 2B Variants of SEQ ID NO: 88 containing ras, SEQ ID NO: 89, variants of SEQ ID NO: 89, SEQ ID NO: 89 Conservatively modified variants of SEQ ID NO: 89, variants of SEQ ID NO: 90 containing the canonical structural class of 2B , variants of SEQ ID NO: 90, conservatively modified variants of SEQ ID NO: 90, comprising one canonical structural class Variant of SEQ ID NO: 90, SEQ ID NO: 91, Variant of SEQ ID NO: 91, Conservative modification of SEQ ID NO: 91 SEQ ID NO: 144, SEQ ID NO: 91, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 15 Conservatively modified variants of SEQ ID NO: 144, sequences containing the canonical structural class of 3A Mutant of sequence number 144, sequence number 145, mutant of sequence number 145, conservation of sequence number 145 SEQ ID NO: 146, variants of SEQ ID NO: 145 containing the canonical structural class of 2A , variants of SEQ ID NO: 146, conservatively modified variants of SEQ ID NO: 146, canonical structural class 3A SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 147, SEQ ID NO: 1 47 conservatively modified variants, variants of SEQ ID NO: 147 containing the canonical structural class of 3A, SEQ ID NO: SEQ ID NO: 148, a variant of SEQ ID NO: 148, a conservatively modified variant of SEQ ID NO: 148 or a variant of 3A The amino acid sequences of variants of SEQ ID NO: 148 include parastructural classes.

[0029] The corresponding CDRH3 is SEQ ID NO: 92, a variant of SEQ ID NO: 92, a conservative modification of SEQ ID NO: 92 SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 93 variants, conservatively modified variants of SEQ ID NO: 93, SEQ ID NO: 9 containing 7 canonical structural classes 3, SEQ ID NO: 94, variants of SEQ ID NO: 94, conservatively modified variants of SEQ ID NO: 94 or or variants of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 95 containing 15 canonical structural classes Variants, conservatively modified variants of SEQ ID NO: 95 or SEQ ID NO: 9 containing 11 canonical structural classes 5, SEQ ID NO: 96, variants of SEQ ID NO: 96, conservatively modified variants of SEQ ID NO: 96, Variants of SEQ ID NO: 96, SEQ ID NO: 97, mutations of SEQ ID NO: 97, including 15 canonical structural classes Conservatively modified variants of SEQ ID NO: 97; variants of SEQ ID NO: 97 containing the six canonical structural classes , SEQ ID NO: 98, variants of SEQ ID NO: 98, conservatively modified variants of SEQ ID NO: 98, the standard structure of 4 Variants of SEQ ID NO: 98, SEQ ID NO: 99, variants of SEQ ID NO: 99, SEQ ID NO: 99 conservatively modified variants, variants of SEQ ID NO: 99 including 13 canonical structural classes, SEQ ID NO: 100, a variant of SEQ ID NO: 100, a conservatively modified variant of SEQ ID NO: 100, or the standard structure of 6 Variants of SEQ ID NO: 100, SEQ ID NO: 149, variants of SEQ ID NO: 149, Conservatively modified variants of SEQ ID NO: 149, variants of SEQ ID NO: 149 containing 15 canonical structural classes , SEQ ID NO: 150, variants of SEQ ID NO: 150, conservatively modified variants of SEQ ID NO: 150, 10 SEQ ID NO: 150 variants, SEQ ID NO: 151, SEQ ID NO: 151 variants, Variants, conservatively modified variants of SEQ ID NO: 151, including 15 canonical structural classes Variants of SEQ ID NO: 152, Variants of SEQ ID NO: 152, Conservatively modified variants of SEQ ID NO: 152 152, SEQ ID NO: 153, SEQ ID NO: 15 3 variants, conservatively modified variants of SEQ ID NO: 153 or sequences comprising 13 canonical structural classes The amino acid sequence of variant no. 153 is included.

[0030] In certain embodiments, mammalian antibodies (including chimeric mammalian antibodies) and / or or an antigen-binding fragment thereof, R α ) and / or bind to canine IL-4 and / or canine IL-13. NuIL-4R α In a related embodiment, the mammalian antibody of the invention and or its antigen-binding fragment is an IL-4 type I receptor and / or an IL-4 Blocks the binding of canine IL-4 and / or canine IL-13 to type II receptors. In embodiments, the mammalian antibody (whether isolated or not) is a caninized antibody. do.

[0031] Thus, in some embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is SEQ ID NO: 47, a variant of SEQ ID NO: 47, a conservatively modified variant of SEQ ID NO: 47, or a target of 1 CDRL2 comprises the amino acid sequence of SEQ ID NO: 47 variants comprising the substructure class; 56, a variant of SEQ ID NO: 56, a conservatively modified variant of SEQ ID NO: 56 or one of the canonical structural classes CDRL3 comprises the amino acid sequence of SEQ ID NO: 65, a variant of SEQ ID NO: 56, including the sequence A variant of SEQ ID NO: 65, a conservatively modified variant of SEQ ID NO: 65, or a sequence comprising one canonical structural class CDRH1 contains the amino acid sequence of the variant of SEQ ID NO: 74; Variants, conservatively modified variants of SEQ ID NO: 74 or SEQ ID NO: 74 comprising one canonical structural class CDRH2 comprises the amino acid sequence of SEQ ID NO: 83, variants of SEQ ID NO: 83, Conservatively modified variants of sequence number 83 and variants of sequence number 83 containing the canonical structural class of 3A CDRH3 comprises the amino acid sequence of SEQ ID NO:92, a variant of SEQ ID NO:92, SEQ ID NO:9 2 conservatively modified variants or variants of SEQ ID NO: 92 containing 12 canonical structural classes Contains the acid sequence.

[0032] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 48, a variant of SEQ ID NO: 48, a conservatively modified variant of SEQ ID NO: 48 or the canonical structure of 2A class comprises the amino acid sequence of the variant of SEQ ID NO: 48; CDRL2 comprises SEQ ID NO: 57; Variants of SEQ ID NO: 57, conservatively modified variants of SEQ ID NO: 57 or containing one canonical structural class CDRL3 includes the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 6 6, a conservatively modified variant of SEQ ID NO: 66 or a SEQ ID NO: containing the canonical structural class of 1 66 variants of the amino acid sequence of SEQ ID NO: 75; , conservatively modified variants of SEQ ID NO: 75 or variants of SEQ ID NO: 75 comprising one canonical structural class CDRH2 comprises the amino acid sequence of SEQ ID NO: 84, variants of SEQ ID NO: 84, SEQ ID NO: Conservatively modified variants of SEQ ID NO: 84 and variants of SEQ ID NO: 84 containing the canonical structural class of 2A. CDRH3 contains the amino acid sequence of SEQ ID NO:93, variants of SEQ ID NO:93, and conserved sequences of SEQ ID NO:93. The amino acid sequences of the variants of SEQ ID NO: 93 containing the structurally modified variants or the seven canonical structural classes are include.

[0033] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: SEQ ID NO: 49, a variant of SEQ ID NO: 49, a conservatively modified variant of SEQ ID NO: 49 or the canonical structure of SEQ ID NO: 4 CDRL1 comprises the amino acid sequence of SEQ ID NO: 58, a variant of SEQ ID NO: 49 containing the ras; Variants of SEQ ID NO: 58, including conservatively modified variants of SEQ ID NO: 58 or 4 canonical structural classes CDRL3 comprises the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 67 a variant of SEQ ID NO: 67, a conservatively modified variant of SEQ ID NO: 6, or a variant of SEQ ID NO: 6, comprising one canonical structural class 7 variants; CDRH1 comprises the amino acid sequence of SEQ ID NO: 76, variants of SEQ ID NO: 76; Conservatively modified variants of SEQ ID NO: 76 or variants of SEQ ID NO: 76 comprising one canonical structural class CDRH2 comprises the amino acid sequence of SEQ ID NO: 85, a variant of SEQ ID NO: 85, SEQ ID NO: 8 5 conservatively modified variants and variants of SEQ ID NO: 85 containing the canonical structural class of 2B. CDRH3 contains the amino acid sequence SEQ ID NO:94, variants of SEQ ID NO:94, and conserved amino acids of SEQ ID NO:94. The amino acid sequences of the modified variants or variants of SEQ ID NO: 94 containing the 15 canonical structural classes are include.

[0034] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: SEQ ID NO: 51, a variant of SEQ ID NO: 51, a conservatively modified variant of SEQ ID NO: 51 or the canonical structure of SEQ ID NO: 3 CDRL1 comprises the amino acid sequence of a variant of SEQ ID NO: 51 containing the ras; CDRL2 comprises the amino acid sequence of SEQ ID NO: 60; Variants of SEQ ID NO: 60, conservatively modified variants of SEQ ID NO: 60, or canonical structural classes of 1 CDRL3 comprises the amino acid sequence of a variant of SEQ ID NO: 69; a variant of SEQ ID NO: 69, a conservatively modified variant of SEQ ID NO: 69, or a variant of SEQ ID NO: 69, comprising one canonical structural class 9 variants; CDRH1 is SEQ ID NO: 78, variants of SEQ ID NO: 78, Conservatively modified variants of SEQ ID NO: 78 or variants of SEQ ID NO: 78 comprising one canonical structural class CDRH2 comprises the amino acid sequence of SEQ ID NO: 87, a variant of SEQ ID NO: 87, SEQ ID NO: 8 Amino acid sequence of variants of SEQ ID NO: 87 containing 7 conservatively modified variants and 1 canonical structural class CDRH3 comprises the sequence SEQ ID NO:96, a variant of SEQ ID NO:96, a conservative variant of SEQ ID NO:96 Modified variants or variants of SEQ ID NO: 96 containing the amino acid sequence of 15 canonical structural classes. nothing.

[0035] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 52, a variant of SEQ ID NO: 52, a conservatively modified variant of SEQ ID NO: 52 or the canonical structure of 2A class includes the amino acid sequence of the variant of SEQ ID NO: 52; CDRL2 includes SEQ ID NO: 61; Variants of SEQ ID NO: 61, conservatively modified variants of SEQ ID NO: 61 or containing one canonical structural class CDRL3 includes the amino acid sequence of SEQ ID NO: 70, SEQ ID NO: 7 0, a conservatively modified variant of SEQ ID NO: 70 or a SEQ ID NO: containing the canonical structural class of 1 70 variants; CDRH1 is SEQ ID NO: 79, variants of SEQ ID NO: 79 , a conservatively modified variant of SEQ ID NO: 79 or a mutation of SEQ ID NO: 79 comprising one canonical structural class CDRH2 comprises the amino acid sequence of SEQ ID NO: 88, variants of SEQ ID NO: 88, SEQ ID NO: Conservatively modified variants of SEQ ID NO: 88 and variants of SEQ ID NO: 88 containing the canonical structural class of 2B. CDRH3 contains the amino acid sequence of SEQ ID NO:97, variants of SEQ ID NO:97, and conserved sequences of SEQ ID NO:97. Amino acid sequences of variants of SEQ ID NO: 97 containing structurally modified variants or variants of the 16 canonical structural classes Includes.

[0036] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: SEQ ID NO: 53, a variant of SEQ ID NO: 53, a conservatively modified variant of SEQ ID NO: 53 or the canonical structure of SEQ ID NO: 6 CDRL1 comprises the amino acid sequence of SEQ ID NO: 62, a variant of SEQ ID NO: 53 containing the ras; Variants of SEQ ID NO: 62, conservatively modified variants of SEQ ID NO: 62, or canonical structural classes of 1 CDRL3 comprises the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 71 a variant of SEQ ID NO: 71, a conservatively modified variant of SEQ ID NO: 7, or a variant of SEQ ID NO: 7, comprising one canonical structural class CDRH1 comprises the amino acid sequence of SEQ ID NO: 80, a variant of SEQ ID NO: 80, Conservatively modified variants of SEQ ID NO: 80 or variants of SEQ ID NO: 80 comprising one of the canonical structural classes CDRH2 comprises the amino acid sequence of SEQ ID NO: 89, a variant of SEQ ID NO: 89, SEQ ID NO: 8 9 conservatively modified variants and variants of SEQ ID NO: 89 containing the canonical structural class of 2B. CDRH3 contains the amino acid sequence SEQ ID NO:98, variants of SEQ ID NO:98, and conserved amino acids of SEQ ID NO:98. The amino acid sequence of SEQ ID NO: 98 includes a modified variant or variant of the four canonical structural classes. nothing.

[0037] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: SEQ ID NO: 54, a variant of SEQ ID NO: 54, a conservatively modified variant of SEQ ID NO: 54 or a standard structural variant of SEQ ID NO: 54 CDRL1 comprises the amino acid sequence of SEQ ID NO: 63, a variant of SEQ ID NO: 54 containing the ras; Variants of SEQ ID NO: 63, including conservatively modified variants of SEQ ID NO: 63 or canonical structural classes of 1 CDRL3 comprises the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 72 a variant of SEQ ID NO: 72, a conservatively modified variant of SEQ ID NO: 7, or a variant of SEQ ID NO: 7, comprising one canonical structural class CDRH1 comprises the amino acid sequence of SEQ ID NO: 81, a variant of SEQ ID NO: 81, Conservatively modified variants of SEQ ID NO: 81 or variants of SEQ ID NO: 81 containing one canonical structural class CDRH2 comprises the amino acid sequence of SEQ ID NO: 90, a variant of SEQ ID NO: 90, SEQ ID NO: 9 Amino acid sequence of variants of SEQ ID NO: 90 containing 0 conservatively modified variants and 1 canonical structural class CDRH3 comprises the sequence SEQ ID NO:99, a variant of SEQ ID NO:99, a conservative variant of SEQ ID NO:99 Modified variants or variants of SEQ ID NO: 99 containing the amino acid sequence of 13 canonical structural classes. In certain embodiments of this type, the antibody (or antigen-binding fragment thereof) Canine interleukin-4 receptor α (IL-4R α ), the antibody binds to SEQ ID NO: No. 157 or SEQ ID NO: 158, or SEQ ID NO: 157 and SEQ ID NO: At least one amino acid residue, preferably 2 to 5 amino acids, in both 158 and 159 The amino acid residue is preferably 3 to 8 or more amino acid residues.

[0038] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 55, a variant of SEQ ID NO: 55, a conservatively modified variant of SEQ ID NO: 55 or the canonical structure of 2A class comprises the amino acid sequence of the variant of SEQ ID NO: 55; CDRL2 comprises SEQ ID NO: 64; Variants of SEQ ID NO: 64, conservatively modified variants of SEQ ID NO: 64 or containing one canonical structural class CDRL3 includes the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 7 3 variants, conservatively modified variants of SEQ ID NO: 73 or SEQ ID NO: 1 containing the canonical structural class 73 variants of the amino acid sequence; CDRH1 is SEQ ID NO: 82, variants of SEQ ID NO: 82 , a conservatively modified variant of SEQ ID NO: 82 or a variant of SEQ ID NO: 82 comprising one canonical structural class CDRH2 comprises the amino acid sequence of SEQ ID NO:91, variants of SEQ ID NO:91, SEQ ID NO: 91 conservatively modified variants and variants of SEQ ID NO: 91 containing the canonical structural class 2A. CDRH3 contains the amino acid sequence SEQ ID NO: 100, variants of SEQ ID NO: 100, SEQ ID NO: 10 0 conservatively modified variants or variants of SEQ ID NO: 100 containing 6 canonical structural classes Contains the acid sequence.

[0039] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 129, a variant of SEQ ID NO: 129, a conservatively modified variant of SEQ ID NO: 129 or a standard of 6 The structural class comprises the amino acid sequence of a variant of SEQ ID NO: 129; CDRL2 comprises SEQ ID NO: 132, a variant of SEQ ID NO: 132, a conservatively modified variant of SEQ ID NO: 132 or the standard structure of 1 CDRL3 comprises the amino acid sequence of SEQ ID NO: 132, a variant of SEQ ID NO: 133; 35, a variant of SEQ ID NO: 135, a conservatively modified variant of SEQ ID NO: 135, or the canonical structure of 1 The class includes the amino acid sequence of variants of SEQ ID NO: 135; CDRH1 includes the amino acid sequence of variants of SEQ ID NO: 14 0, a variant of SEQ ID NO: 140, a conservatively modified variant of SEQ ID NO: 140 or the canonical structure of 1 CDRH2 comprises the amino acid sequence of a variant of SEQ ID NO: 144; , variants of SEQ ID NO: 144, conservatively modified variants of SEQ ID NO: 144 and the canonical structure of 3A CDRH3 comprises the amino acid sequence of a variant of SEQ ID NO: 149; , a variant of SEQ ID NO: 149, a conservatively modified variant of SEQ ID NO: 149 or 15 standard structural groups Specific embodiments of this type include the amino acid sequence of a variant of SEQ ID NO: 149 that contains the ras. In the method, the antibody (or antigen-binding fragment thereof) is Condition α(IL-4R α ), the antibody binds to the sequence of SEQ ID NO: 127 or SEQ ID NO: 128 At least one amino acid sequence in the amino acid sequence or in both SEQ ID NO: 127 and SEQ ID NO: 128 at least one amino acid residue, preferably 2 to 5 amino acid residues, and / or more preferably It binds to 3 to 8 or more amino acid residues.

[0040] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 130, a variant of SEQ ID NO: 130, a conservatively modified variant of SEQ ID NO: 130 or a standard of 6 The structural class comprises the amino acid sequence of variants of SEQ ID NO: 130; CDRL2 comprises SEQ ID NO: 133, a variant of SEQ ID NO: 133, a conservatively modified variant of SEQ ID NO: 133 or the standard structure of 1 CDRL3 comprises the amino acid sequence of SEQ ID NO: 133, a variant of SEQ ID NO: 134; 36, a variant of SEQ ID NO: 136, a conservatively modified variant of SEQ ID NO: 136, or the canonical structure of 1 The class includes the amino acid sequence of variants of SEQ ID NO: 136; CDRH1 includes the amino acid sequence of variants of SEQ ID NO: 14 1, a variant of SEQ ID NO: 141, a conservatively modified variant of SEQ ID NO: 141 or the canonical structure of 1 CDRH2 comprises the amino acid sequence of a variant of SEQ ID NO: 145; , variants of SEQ ID NO: 145, conservatively modified variants of SEQ ID NO: 145 and the canonical structure of 2A. CDRH3 comprises the amino acid sequence of a variant of SEQ ID NO: 145 containing RAS; , a variant of SEQ ID NO: 150, a conservatively modified variant of SEQ ID NO: 150 or 10 standard structural groups Specific embodiments of this type include the amino acid sequence of a variant of SEQ ID NO: 150 containing ras. In the method, the antibody (or antigen-binding fragment thereof) is Condition α(IL-4R α ), the antibody binds to the sequence of SEQ ID NO: 158 or SEQ ID NO: 162 At least one amino acid sequence in the amino acid sequence or in both SEQ ID NO: 158 and SEQ ID NO: 162 at least one amino acid residue, preferably 2 to 5 amino acid residues, and / or more preferably It binds to 3 to 8 or more amino acid residues.

[0041] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 129, a variant of SEQ ID NO: 129, a conservatively modified variant of SEQ ID NO: 129 or a standard of 6 The structural class comprises the amino acid sequence of a variant of SEQ ID NO: 129; CDRL2 comprises SEQ ID NO: 134, a variant of SEQ ID NO: 134, a conservatively modified variant of SEQ ID NO: 134 or the standard structure of 1 CDRL3 comprises the amino acid sequence of SEQ ID NO: 134, a variant of SEQ ID NO: 135; 37, a variant of SEQ ID NO: 137, a conservatively modified variant of SEQ ID NO: 137, or the canonical structure of 1 The class includes the amino acid sequence of variants of SEQ ID NO: 137; CDRH1 includes the amino acid sequence of variants of SEQ ID NO: 14 0, a variant of SEQ ID NO: 140, a conservatively modified variant of SEQ ID NO: 140 or the canonical structure of 1 CDRH2 comprises the amino acid sequence of a variant of SEQ ID NO: 146; , variants of SEQ ID NO: 146, conservatively modified variants of SEQ ID NO: 146 and the canonical structure of 3A CDRH3 comprises the amino acid sequence of the variant of SEQ ID NO: 146 containing RAS; , a variant of SEQ ID NO: 151, a conservatively modified variant of SEQ ID NO: 151 or a canonical structure of 15 Specific embodiments of this type include the amino acid sequence of a variant of SEQ ID NO: 151 that contains the ras. In the method, the antibody (or antigen-binding fragment thereof) is Condition α(IL-4R α ), the antibody binds to the sequence of SEQ ID NO: 125 or SEQ ID NO: 126 At least one amino acid sequence in the amino acid sequence or in both SEQ ID NO: 125 and SEQ ID NO: 126 at least one amino acid residue, preferably 2 to 5 amino acid residues, and / or more preferably It binds to 3 to 8 or more amino acid residues.

[0042] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 131, a variant of SEQ ID NO: 131, a conservatively modified variant of SEQ ID NO: 131 or a standard of 3 CDRL2 comprises the amino acid sequence of a variant of SEQ ID NO: 131, including the structural class 60, a variant of SEQ ID NO: 60, a conservatively modified variant of SEQ ID NO: 60 or one of the canonical structural classes CDRL3 comprises the amino acid sequence of SEQ ID NO: 138, a variant of SEQ ID NO: 60 containing the sequence Variants of sequence number 138, conservatively modified variants of sequence number 1385, or canonical structural class 3 CDRH1 comprises the amino acid sequence of SEQ ID NO: 138, a variant of SEQ ID NO: 142, A variant of SEQ ID NO: 142, a conservatively modified variant of SEQ ID NO: 142, or a canonical structural class of 1 CDRH2 comprises the amino acid sequence of SEQ ID NO: 147, the variant of SEQ ID NO: 142 comprising the amino acid sequence of SEQ ID NO: 147; The variants of SEQ ID NO: 147, conservatively modified variants of SEQ ID NO: 147 and the canonical structural class of 3A are shown. CDRH3 comprises the amino acid sequence of SEQ ID NO: 152, the variant of SEQ ID NO: 147 comprising the sequence SEQ ID NO: 152, a conservatively modified variant of SEQ ID NO: 152, or any of the 9 canonical structural classes. In particular embodiments of this type, the amino acid sequence of a variant of SEQ ID NO: 152 is The present invention relates to a method for treating canine interleukin-4 receptor alpha (IL-4R)-associated ... IL-4R α ), the antibody binds to SEQ ID NO: 154 or SEQ ID NO: 155 or the sequence At least one amino acid residue at position 156 or any combination thereof, preferably More preferably, 2 to 5 amino acid residues, and / or more preferably 3 to 8 or more amino acid residues. Attaches to the group.

[0043] In yet other embodiments of mammalian antibodies (including caninized antibodies), CDRL1 is selected from the group consisting of SEQ ID NO: No. 129, a variant of SEQ ID NO: 129, a conservatively modified variant of SEQ ID NO: 129 or a standard of 6 The structural class comprises the amino acid sequence of a variant of SEQ ID NO: 129; CDRL2 comprises SEQ ID NO: 132, a variant of SEQ ID NO: 132, a conservatively modified variant of SEQ ID NO: 132 or the standard structure of 1 CDRL3 comprises the amino acid sequence of SEQ ID NO: 132, a variant of SEQ ID NO: 133; 39, a variant of SEQ ID NO: 139, a conservatively modified variant of SEQ ID NO: 139, or the canonical structure of 1 The class includes the amino acid sequence of variants of SEQ ID NO: 139; CDRH1 includes the amino acid sequence of variants of SEQ ID NO: 14 3. A variant of SEQ ID NO: 143, a conservatively modified variant of SEQ ID NO: 143, or a standard structure clone of 1. CDRH2 comprises the amino acid sequence of a variant of SEQ ID NO: 148; , variants of SEQ ID NO: 148, conservatively modified variants of SEQ ID NO: 148 and the canonical structure of 3A CDRH3 comprises the amino acid sequence of a variant of SEQ ID NO: 148 containing RAS; , a variant of SEQ ID NO: 153, a conservatively modified variant of SEQ ID NO: 153 or a canonical structure of 13 Specific embodiments of this type include the amino acid sequence of a variant of SEQ ID NO: 153 that contains the ras. In the method, the antibody (or antigen-binding fragment thereof) is Condition α(IL-4R α ), the antibody binds to SEQ ID NO: 159 or SEQ ID NO: 160 or at least one amino acid residue in SEQ ID NO: 161, or any combination thereof Preferably, the amino acid sequence is 2 to 5 amino acid residues, and / or more preferably 3 to 8 or more amino acid residues. It binds to amino acid residues.

[0044] The present invention relates to canine interleukin-4 receptor alpha (IL-4Rα ) specifically binds to In particular embodiments of this type, the present invention includes antibodies and antigen-binding fragments thereof. The antibody and its antigen-binding fragment bind to canine IL-4R α binds to canine IL- Canine IL-4R to IL-4 and / or IL-13 α As mentioned above, The isolated mammalian antibody or antigen-binding fragment thereof may be a caninized antibody or a caninized antigen. In other embodiments, the antibody may be an isolated mammalian antibody or a human binding fragment. The antigen-binding fragment is a murine antibody or a murine antigen-binding fragment thereof. It's possible.

[0045] The caninized antibodies or caninized antigen-binding fragments thereof of the present invention may include a hinge region. In a particular embodiment of this type, the hinge region has the amino acid sequence of SEQ ID NO: 101. In another embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 102. In yet another embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 103. In yet another embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO: 104. Contains columns.

[0046] In certain embodiments, the caninized antibody or antigen-binding fragment thereof has SEQ ID NO: In particular embodiments of this type, the heavy chain comprises the amino acid sequence of SEQ ID NO: 164. The heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 163. the caninized antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO: 166 In a particular embodiment of this type, the heavy chain comprises SEQ ID NO: 165. In yet other embodiments, the caninized antibody or or an antigen-binding fragment thereof, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:168. In particular embodiments of this type, the heavy chain comprises the nucleotide sequence of SEQ ID NO: 167 In certain embodiments of that type, the caninized antibody (or or its antigen-binding fragment) binds to canine interleukin-4 receptor α (IL-4R α ) When binding to SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: 156 or or at least one amino acid residue, preferably 2 to 5 amino acids, in any combination thereof. It binds to an amino acid residue, and / or more preferably to 3 to 8 or more amino acid residues.

[0047] In related embodiments, the caninized antibody or antigen-binding fragment thereof has SEQ ID NO: In particular embodiments of this type, the light chain comprises the amino acid sequence of SEQ ID NO:170. The light chain is encoded by the nucleotide sequence of SEQ ID NO: 169. the caninized antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO: 172 In a particular embodiment of this type, the light chain comprises SEQ ID NO: 171. In yet other embodiments, the caninized antibody or or an antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO:174. In particular embodiments of this type, the light chain has the nucleotide sequence of SEQ ID NO: 173 In certain embodiments of that type, the caninized antibody (or or its antigen-binding fragment) binds to canine interleukin-4 receptor α (IL-4R α ) When binding to SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: 156 or or at least one amino acid residue, preferably 2 to 5 amino acids, in any combination thereof. It binds to an amino acid residue, and / or more preferably to 3 to 8 or more amino acid residues.

[0048] The present invention further provides antibodies comprising such heavy and light chain combinations. In one embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 164 and the light chain comprises the amino acid sequence of SEQ ID NO: 170. In more particular embodiments of this type, the heavy chain comprises the amino acid sequence of SEQ ID NO: The light chain is encoded by the nucleotide sequence of SEQ ID NO: 169. In other embodiments, the heavy chain is encoded by the amino acid sequence of SEQ ID NO: 166. and the light chain comprises the amino acid sequence of SEQ ID NO: 172. In one embodiment, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 165, and the light chain is is encoded by the nucleotide sequence of SEQ ID NO: 171. The heavy chain comprises the amino acid sequence of SEQ ID NO: 168 and the light chain comprises the amino acid sequence of SEQ ID NO: 174. In more particular embodiments of this type, the heavy chain comprises the nucleotide sequence of SEQ ID NO: 167. The light chain is encoded by the nucleotide sequence of SEQ ID NO: 173. It will be downloaded.

[0049] In a related embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 164 and the light chain comprises the amino acid sequence of SEQ ID NO: 165. In other embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 164. and the light chain comprises the amino acid sequence of SEQ ID NO: 174. In one embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 166 and the light chain comprises the amino acid sequence of SEQ ID NO: 170. In yet another embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 166. and the light chain comprises the amino acid sequence of SEQ ID NO: 174. For example, the heavy chain comprises the amino acid sequence of SEQ ID NO: 168 and the light chain comprises the amino acid sequence of SEQ ID NO: 170. In other embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 168. , the light chain comprises the amino acid sequence of SEQ ID NO: 172.

[0050] In certain embodiments of such type, the caninized antibody (or its antigen-binding fragment) is a canine interleukin-4 receptor α (IL-4R α ), the antibody The antibody is SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: 156 or any combination thereof At least one amino acid residue, preferably 2 to 5 amino acid residues in the combination, and and / or more preferably, 3 to 8 or more amino acid residues.

[0051] Therefore, the present invention further provides a method for producing canine interleukin-4 receptor alpha (IL-4R α ) specific to An isolated mammalian antibody or antigen-binding fragment thereof (e.g., caninized antibody or or an antigen-binding fragment thereof), wherein the antibody is a canine IL-4R α when binding to SEQ ID NO: 125 or SEQ ID NO: 126 or SEQ ID NO: 127 or SEQ ID NO: No. 128 or SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: 156 or SEQ ID NO: No. 157 or SEQ ID NO: 158 or SEQ ID NO: 159 or SEQ ID NO: 160 or SEQ ID NO: At least one of SEQ ID NO: 161 or SEQ ID NO: 162 or any combination thereof amino acid residues, preferably 2 to 5 amino acid residues, and / or more preferably 3 to 5 amino acid residues In certain embodiments, the antibody or its antigen binds to eight or more amino acid residues. The binding fragment is canine IL-4R α binds to canine interleukin-4 -4R α Blocks the binding of

[0052] The present invention further provides a method for producing a 1×10 -12 A dissociation constant (Kd) lower than M (e.g., 1 × 10 -13 M or less) in canine IL-4R α a mammalian antibody or antigen-binding fragment thereof that binds to In certain embodiments, the mammalian antibody or antigen-binding fragment thereof is 1 x 10 -5 M~1×10 -12 Dissociation constant of canine IL-4R by M α For more information, In a specific embodiment, the mammalian antibody or antigen-binding fragment thereof is 1×10 - 7 M~1×10 -11 Dissociation constant of canine IL-4R by M α Further detailed implementation In the form, the mammalian antibody or antigen-binding fragment thereof is 1 x 10 -8 M~1 x10 -11 Dissociation constant of canine IL-4R by M α In a more detailed embodiment, In this case, the mammalian antibody or antigen-binding fragment thereof is 1×10 -8 M~1×10 - 10 Dissociation constant of canine IL-4R by M α Combine with.

[0053] The present invention also provides a method for producing a 1×10 7 M-1 s -1 Larger on-speed (k on ) and dogs IL-4R α The present invention provides a mammalian antibody or antigen-binding fragment thereof that binds to a specific In one embodiment, the mammalian antibody or antigen-binding fragment thereof is 1×10 2 M -1 s -1 ~1×10 7 M -1 s -1 On speed (k on ) in canine IL-4R α Bind to In more particular embodiments, the mammalian antibody or antigen-binding fragment thereof is 1×10 3 M -1 s -1 ~1×10 6 M -1 s -1 On-rate of canine IL-4R α Combined into In even more particular embodiments, the mammalian antibody or antigen-binding fragment thereof The value is 1 x 10 3 M -1 s -1 ~1×10 5 M -1 s -1 On-rate of canine IL-4R α In even more particular embodiments, the mammalian antibody or its antigen-binding fragment binds to Fragment is 1×10 4 M -1 s -1 ~1×10 5 M -1 s -1 On-rate of canine IL- 4R α Combine with.

[0054] The present invention further provides a method for producing a 1×10 -7 s -1 Slower off speed (koff ) and Dog I L-4R α The present invention provides a mammalian antibody or antigen-binding fragment thereof that binds to a particular In embodiments, the mammalian antibody or antigen-binding fragment thereof is 1×10 -3 s -1 ~1×10 -8 s -1 Off-rate of canine IL-4R α More detailed embodiments In some embodiments, the mammalian antibody or antigen-binding fragment thereof is 1×10 -4 s -1 ~ 1×10 -7 s -1 Off-rate of canine IL-4R α Further more detailed embodiments In the case of a mammalian antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment is 1×10 -5 s -1 ~1 x10 -7 s -1 Off-rate of canine IL-4R α Combine with.

[0055] In certain embodiments, the mammalian antibodies (including chimeric antibodies) of the present invention bind to IL-4R α In a more particular embodiment, the antibody blocks the binding of canine IL-4 to 1 x 10 -8 M~1×10 -9 IL-4R at a minimum EC50 of 1 M or even lower α Dogs In an even more particular embodiment, the EC50 is 5×10 - 9 M~5×10 -13 In even more particular embodiments, the EC50 is 5x1 0 -9 M~5×10 -11 It's M.

[0056] In related embodiments, the mammalian antibody or antigen-binding fragment thereof is Type I and and / or type II IL-4 receptors, and / or IL-13. Negatively attenuate (e.g., inhibit) cell signaling pathways mediated by the In one embodiment, the mammalian antibody or antigen-binding fragment thereof is It improves itchy inflammatory skin diseases, such as atopic dermatitis. In some embodiments, the animal subject is a dog. In related embodiments, the animal subject is a cat.

[0057] Therefore, any antibody of the present invention may have these properties, i.e., canine IL-4R α Regarding The dissociation constant of canine IL-4R α the on-rate for binding to the antibody-dog IL4R α the aforementioned off-rates for dissociation from the binding complex, Type I and / or Type II Cellular signaling mediated by IL-4 and / or IL-13 binding to the IL-4 receptor Inhibition of signaling pathways, or the treatment of pruritic inflammatory skin diseases in animal subjects, e.g., atopic dermatitis One, two, three, four or all of the improvements in dermatitis may be noted.

[0058] As described above, the antibodies (and antigen-binding fragments thereof) of the present invention ( and antigen-binding fragments thereof) are monoclonal antibodies (and their antigen-binding fragments). fragments), mammalian antibodies (and antigen-binding fragments thereof), e.g., murine (mouse) ) antibodies (and antigen-binding fragments thereof), caninized antibodies (and antigen-binding fragments thereof) fragment) [including caninized mouse antibodies (and antigen-binding fragments thereof)] In certain embodiments, the antibodies (and antigen-binding fragments thereof) ) has been isolated.

[0059] The present invention further provides a nucleic acid encoding any one of the light chains of the caninized antibodies of the present invention (isolated Similarly, the present invention provides a nucleic acid encoding any one of the heavy chains of the caninized antibodies of the present invention. The present invention provides isolated nucleic acids encoding one of the

[0060] The present invention further provides expression vectors comprising one or more of the nucleic acids (including isolated nucleic acids) of the present invention. The present invention further provides host cells comprising one or more of the expression vectors of the present invention.

[0061] In certain embodiments, the antibody is a recombinant antibody or antigen-binding fragment thereof. In a related embodiment, the variable heavy and light domains are single chain antibodies. They are linked by a flexible linker to form a body.

[0062] In certain embodiments, the antibody or antigen-binding fragment comprises a Fab fragment. In other embodiments, the antibody or antigen-binding fragment is a Fab' fragment. In other embodiments, the antibody or antigen-binding fragment is In yet another embodiment, the antibody or antigen-binding In certain embodiments, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a domain antibody. The synthetic fragment is a single domain antibody.

[0063] In a specific embodiment, caninized mouse anti-canine IL-4R α Antibody or antigen-binding fragment The fragments are used to detect type I and / or type II leukemia in the treated animal subject (e.g., dog). Cell signaling mediated by IL-4 and / or IL-13 binding to the IL-4 receptor In a more specific embodiment, the caninized mouse of the present invention negatively attenuates the null signaling pathway. Anti-canine IL-4R α Administration of the antibody or antigen-binding fragment is performed in the animal subject to be treated ( For example, it acts to improve one or more symptoms of atopic dermatitis in animals (e.g., dogs).

[0064] The present invention further provides a caninized mouse anti-canine IL-4R antibody. α Isolation encoding an antibody or portion thereof In related embodiments, such antibodies or antigen-binding fragments are provided. The segment is used for the manufacture of a medicament for treating atopic dermatitis in a canine subject. Alternatively or in combination, the present invention provides an antibody or a method for the treatment of a disease or condition of the present invention for diagnostic use. In yet further embodiments, the present invention provides for the use of any of the antibody fragments. Kits containing any of the caninized antibodies or antigen-binding fragments disclosed herein are also provided. provide.

[0065] In yet a further embodiment, the caninized murine anti-canine IL-4R antibody of the present invention α Antibodies or and an expression vector comprising an isolated nucleic acid encoding any of the antigen-binding fragments. The present invention also provides a host cell comprising any of the expression vectors described herein. In particular embodiments, the nucleic acids, expression vectors or The polypeptides are useful in the production of antibodies. is SEQ ID NO: 126 or SEQ ID NO: 127 or SEQ ID NO: 128 or SEQ ID NO: 154 or is SEQ ID NO: 155 or SEQ ID NO: 156 or SEQ ID NO: 157 or SEQ ID NO: 158 or is SEQ ID NO: 159 or SEQ ID NO: 160 or SEQ ID NO: 161 or SEQ ID NO: 162 A peptide consisting of 80 or fewer amino acid residues containing the amino acid sequence (isolated antigenic peptide) In a related embodiment, the peptide (including an isolated antigenic peptide) is provided. (including) SEQ ID NO: 125 or SEQ ID NO: 126 or SEQ ID NO: 127 or SEQ ID NO: 1 28 or SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: 156 or SEQ ID NO: 1 57 or SEQ ID NO: 158 or SEQ ID NO: 159 or SEQ ID NO: 160 or SEQ ID NO: 1 61 or SEQ ID NO: 162. In certain embodiments, the peptide (including the isolated antigenic peptide) is SEQ ID NO: 12. 5 or SEQ ID NO: 126 or SEQ ID NO: 127 or SEQ ID NO: 128 or SEQ ID NO: 15 4 or SEQ ID NO: 155 or SEQ ID NO: 156 or SEQ ID NO: 157 or SEQ ID NO: 15 8 or SEQ ID NO: 159 or SEQ ID NO: 160 or SEQ ID NO: 161 or SEQ ID NO: 16 In still another embodiment, the amino acid sequence of For example, the peptide (including the isolated antigenic peptide) may be SEQ ID NO: 125 or SEQ ID NO: 126 or SEQ ID NO: 127 or SEQ ID NO: 128 or SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: 156 or SEQ ID NO: 157 or SEQ ID NO: 158 or SEQ ID NO: 159 or the amino acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161 or SEQ ID NO: 162 It consists of 5 to 25 amino acid residues from or including:

[0066] The present invention further relates to SEQ ID NO: 125 or SEQ ID NO: 126 or SEQ ID NO: 127 or SEQ ID NO: No. 128 or SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: 156 or SEQ ID NO: No. 157 or SEQ ID NO: 158 or SEQ ID NO: 159 or SEQ ID NO: 160 or SEQ ID NO: 80%, 85%, 90%, 95% of the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 162 % or 100% identical to the isolated mammalian antibody or antigen-binding fragment thereof of the present invention. An antigenic peptide consisting of 80 or less amino acid residues containing the amino acid sequence that binds to the target segment. In a related embodiment, the antigenic peptide ( The isolated antigenic peptide is SEQ ID NO: 125 or SEQ ID NO: 126 or SEQ ID NO: No. 127 or SEQ ID NO: 128 or SEQ ID NO: 154 or SEQ ID NO: 155 or SEQ ID NO: No. 156 or SEQ ID NO: 157 or SEQ ID NO: 158 or SEQ ID NO: 159 or SEQ ID NO: 80%, 8% or 8% of the amino acid sequence of SEQ ID NO: 160, SEQ ID NO: 161, or SEQ ID NO: 162 5%, 90%, 95% or 100% identical isolated mammalian antibodies or their antigens It consists of 60 or less amino acid residues that contain the amino acid sequence that binds to the binding fragment. In other embodiments, the peptide is SEQ ID NO: 125 or SEQ ID NO: 126 or SEQ ID NO: 127. Sequence number 127 or sequence number 128 or sequence number 154 or sequence number 155 or sequence number Sequence number 156 or sequence number 157 or sequence number 158 or sequence number 159 or sequence number 80% of the amino acid sequence of sequence number 160 or sequence number 161 or sequence number 162 , 85%, 90%, 95% or 100% identical to an isolated mammalian antibody or 5 to 25 amino acids from or including the amino acid sequence that binds to the antigen-binding fragment In a specific embodiment, the mammalian antibody comprises the CDRs of 4D8. In another embodiment, the mammalian antibody comprises the CDRs of 11H2. In one embodiment, the mammalian antibody comprises the CDRs of 4H3. The mammalian antibody comprises the CDRs of 11B6. In yet another embodiment, the mammalian antibody comprises In yet another embodiment, the mammalian antibody comprises the CDRs of 2E2. Includes:

[0067] The present invention further provides a fusion protein comprising any of the above peptides. In one embodiment, the fusion protein comprises a fusion protein comprising the antigen peptide and a non-canine mammalian IgG antibody. and an Fc region of a non-canine mammal. In one embodiment, the non-canine mammalian IgG antibody comprises the Fc region of an IgG antibody. In an alternative embodiment, the non-canine mammalian IgG antibody is human IgG. In other embodiments, the non-canine mammalian IgG antibody is equine IgG. In other embodiments, the non-canine mammalian IgG antibody is porcine IgG. In one embodiment, the non-canine mammalian IgG antibody is bovine IgG.

[0068] In certain embodiments, the non-canine mammalian IgG antibody is an IgG1. In another embodiment, the non-canine mammalian IgG antibody is IgG2a. In some embodiments, the non-canine mammalian IgG antibody is an IgG3. The canine mammalian IgG antibody is IgG4. In other embodiments, the fusion protein is The antigenic peptides include any of the above antigenic peptides and maltose binding protein. In the present invention, the fusion protein comprises any one of the antigen peptides and beta-galactosidase. In yet other embodiments, the fusion protein comprises any of the antigenic peptides. and glutathione S-transferase. The fusion protein comprises any of the above antigenic peptides and thioredoxin. In one embodiment, the fusion protein comprises any one of the antigen peptides and Gro EL. In yet other embodiments, the fusion protein comprises any of the above antigenic peptides and N. Includes usA.

[0069] The present invention further provides nucleic acids (single or multiple) encoding the antigenic peptides and corresponding fusion proteins of the present invention. The present invention also provides expression vectors containing these nucleic acids, and and a host cell comprising one or more of the expression vectors of the invention.

[0070] The present invention also provides an anti-canine IL-4R antibody of the present invention. α Antibodies or antigen-binding fragments thereof , canine IL-4R α antigenic peptides (including isolated antigenic peptides) from the NuIL-4R α fusion proteins comprising antigenic peptides from the antigenic fragments and / or Nucleic acids (including isolated nucleic acids) encoding the nucleic acid or fusion protein, an expression vector or any combination thereof, and a pharmaceutically acceptable carrier or diluent. and a pharmaceutical composition comprising:

[0071] The present invention also provides a method for negatively attenuating the activity of IL-4 and / or IL-13. and administering to an animal in need thereof a therapeutically effective amount of such a pharmaceutical composition. In one embodiment, the method is for treating atopic dermatitis in a dog. Used for.

[0072] These and other aspects of the present invention are described in the "Brief Description of the Drawings" and "Detailed Description" sections below. This will be better understood by reference to

[0073] Detailed Description Various approaches for the treatment of human AD are currently being investigated in multiple clinical trials. [Malajian et al., New pathogenic and therapeutic ic paradigms in atopic dermatitis Cytoki Some of these approaches are Th2 The aim is to interfere with one or more of the signaling molecules / events that lead to cell development and activation. One direction of research in this area is to identify key interleukin-driven factors in the Th2 pathway. This includes approaches to block the action of AD. AD is a disease that is primarily dominated by Th2. The observation that both IL-4 and IL-13 are key drivers of Th2 cell development Based on accumulating data supporting the central role of the combined effects of IL-4 receptors, The data show that the α-chain of the thyroid gland is an essential receptor for signaling from both cytokines. Based on this data, the present invention provides canine IL-4 and IL-1 receptors. Blocks the binding of canine IL-13 and subsequently signals from both canine IL-4 and IL-13 We describe the generation and characterization of monoclonal antibodies that inhibit transmission. As disclosed herein, the present invention provides a method for treating atopic dermatitis and other diseases in companion animals. It has therapeutic utility.

[0074] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention:

[0075] ADCC antibody-dependent cytotoxicity; CDC complement-dependent cytotoxicity; CDRs are immunoglobulin variable region numbers, as determined using the Kabat numbering system. Complementarity-determining regions within the region; CHO Chinese hamster ovary; EC50 concentration resulting in 50% efficacy or binding; ELISA enzyme-linked immunosorbent assay; FR antibody framework region: immunoglobulin variable region excluding the CDR regions; HRP horseradish peroxidase; IFN interferon; IC50: concentration that produces 50% inhibition; IgG immunoglobulin G; Kabat Elvin A. Kabat[Sequences of Protei ns of Immunological Interest, 5th Ed.Pub lic Health Service, National Institutes o Immunoglobulin G1000 developed by the National Institute of Health, Bethesda, Md. (1991) Blinn alignment and numbering system; mAb monoclonal antibody (also Mab or MAb); PCR polymerase chain reaction; MES 2-(N-morpholino)ethanesulfonic acid; MOA mechanism of action; NHS normal human serum; PCR polymerase chain reaction; PK Pharmacokinetics; TT tetanus toxoid; V region The segment of the IgG chain whose sequence is variable among different antibodies. It is the K region in the light chain. abat residues 109 and extending to 113 within the heavy chain; VH immunoglobulin heavy chain variable region; VL immunoglobulin light chain variable region; VK Immunoglobulin kappa light chain variable region.

[0076] definition In order that the present invention may be more readily understood, certain scientific and technical terms are specifically defined below. Unless specifically defined elsewhere in this specification, all other terms used herein are All technical and scientific terms have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0077] As used in this specification, including the appended claims, the singular forms "a," "the," and "the" shall be used unless clearly contradicted by the context. Unless shielded, includes its corresponding multiple objects.

[0078] "Activation" as applied to cells or receptors is contextually or explicitly specified. Unless otherwise indicated, activation or treatment of cells or receptors with ligands is not intended. "Ligand" refers to natural and synthetic ligands, e.g., cytokines, cytokine This includes antibody variants, analogs, muteins, and antibody-derived binding compounds. "Bands" also include small molecules, e.g., peptide mimetics of cytokines, and peptide mimetics of antibodies. "Activation" includes both internal mechanisms and external or environmental factors. This may mean controlled cell activation.

[0079] The "activity" of a molecule can be determined by its binding to a ligand or to a receptor, or by catalytic activity; Ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, other molecules The "activity" of a molecule can refer to or mean the modulation of the activity of a molecule, etc. interactions, e.g., activity in modulating or maintaining adhesion or cell structure. It can also mean activity in maintaining a structure, for example, a cell membrane or cytoskeleton. Activity, e.g., [catalytic activity] / [mg protein], or [immunological activity] / [mg protein] "Activity" can also mean the activity of the innate or adaptive immune system. This may mean modulation of a component of

[0080] "Administration" and "treatment" refer to the administration of a substance to an animal, e.g., a canine experimental subject, a cell, a tissue, When applied to an organ or biological fluid, the animal, e.g., canine subject, cell, tissue, refers to contact of an exogenous drug, therapeutic substance, diagnostic agent or composition with an organ or biological fluid. Treatment of cells involves contacting the cells with a reagent and with a fluid in contact with the cells. "Administration" and "treatment" include contacting with a reagent, diagnostic agent, binding compound or In vitro and ex vivo treatment of cells with other cells, e.g. The term "subject" also refers to any living organism, preferably an animal, more preferably a human. includes mammals (eg, dogs, cats, or humans), most preferably dogs.

[0081] As used herein, "antibody" refers to a sequence of amino acids at different amino acid residues in, for example, an antibody amino acid sequence. "Substitution of an amino acid residue" is synonymous with "substitution of an amino acid residue" with another amino acid residue. and a specific amino acid residue at a specific position in the amino acid sequence is a different amino acid residue. Such substitutions can be specifically designed, i.e., For example, recombinant DNA technology can be used to replace alanine with serine at specific positions in an amino acid sequence. Alternatively, a more natural selection process could result in, for example, The antibody produced by the cell binds to the antigen (e.g., an epitope or a portion thereof). based on their ability to bind to a given region on the CDR(s) and / or to be identical to the CDR(s) they are being substituted for. Certain amino acid residues or CDRs of an antibody are modified so that the antibody contains certain CDRs that have the same canonical structure. It is possible to replace a chain of amino acid residues with one or more amino acid residues. Such substitutions may result in "variant" CDRs and / or variant antibodies.

[0082] "Treat" or "treatment" refers to administering a therapeutic agent (e.g., a compound of the present invention) to a subject. a composition containing either an antibody or an antigen-binding fragment thereof, Intravenous administration to a canine subject or patient having or suspected of having one or more disease symptoms indicative of Or it means to administer externally.

[0083] Typically, the substance will cause the regression of such symptoms to any clinically measurable degree. Induction of atrophy or inhibition of progression of such symptoms, resulting in one or more The compound is administered in an amount effective to reduce and / or ameliorate the symptoms of any particular disease. The amount of a therapeutic agent that is effective in alleviating disease symptoms (also called a "therapeutically effective amount") is The condition, age and weight of the patient (e.g., dog) and the type of compound that will elicit the desired response in the subject. The effectiveness of the pharmaceutical composition may vary depending on factors such as the ability of the pharmaceutical composition to reduce or ameliorate disease symptoms. Whether or not to consult a veterinarian or other skilled medical professional to assess the severity or progression of the condition It can be assessed by any clinical measurement typically used by providers. In certain embodiments (e.g., methods of treatment or articles of manufacture), the method of treatment may provide relief of target disease symptoms in all subjects. Although it may not necessarily be effective in any of the methods known in the art, Statistical tests, such as Student's t-test, chi-square 2 Mann and Hui test Whitney U test, Kruskal-Wallis llis test (H test), Jonckheere-Terpstra Statistics determined by the pstra test and Wilcoxon test The therapeutic effect should reduce the target disease symptoms in a meaningful number of subjects.

[0084] "Treatment" means a treatment, whether it is administered to a human, veterinary subject (e.g., a dog), or research subject. As used herein, "treatment" refers to therapeutic treatment, research, and diagnostic applications. whether it is a human, veterinary subject (e.g., dog) or research subject or a cell, tissue or When applied to an organ, dog or other animal subject, cell, tissue, physiological compartment or involves contacting an antibody or antigen-binding fragment of the invention with a physiological fluid.

[0085] As used herein, the term "dog" refers to all domestic dogs, cats, and other domesticated animals, unless otherwise indicated. Canis lupus familiaris or Canis familiaris.

[0086] As used herein, the term "cat" refers to any member of the family Felidae. Members of this family include wild, zoo and domestic members, e.g., cats (Feli any member of the subfamily nae, e.g., cat, lion, tiger, puma, jaguar, leopard, snow leopard, panther, North American mountain n) Lion, cheetah, lynx, bobcat, caracal or any of these Cats also include domestic cats, purebred and / or mixed breed companion cats, show cats, and crossbreed cats. This includes domestic cats, laboratory cats, cloned cats and wild or feral cats.

[0087] As used herein, the term "canine frame" refers to the CDR residues defined herein. The amino acid sequences of the heavy and light chains of a canine antibody excluding the hypervariable region residues identified as In many embodiments, for caninized antibodies, the amino acid sequences of the native canine CDRs are both In the nucleotide sequence of ... Therefore, for example, as exemplified below, the conformation of the foreign CDR in a canine antibody can be The heavy and / or Fc chains of the canine antibody are modified to maintain the Fc function and / or modify the Fc function. The heavy and / or light chains may contain some foreign non-CDR residues.

[0088] Canine IL-4R α The sequence of SEQ ID NO: 2 [the sequence without a signal sequence is SEQ ID NO: 4] In a specific embodiment, canine IL-4R is found to contain the amino acid sequence α teeth , comprising the nucleotide sequence of SEQ ID NO: 1 [SEQ ID NO: 3 if not containing a signal sequence] Encoded by a nucleic acid. Canine IL-4Rα For example, a gene with conservative mutations in non-conserved regions Although the differences may be due to the presence of canine IL-4R α is SEQ ID NO: 2 [without signal sequence] is a canine IL-4R comprising the amino acid sequence of SEQ ID NO: 4 α have substantially the same biological function as .

[0089] The cytokines IL-4 and IL-13 are involved in various allergic diseases in humans and animals. IL-4 receptors have been implicated in the pathogenesis of various diseases (including asthma and atopic dermatitis). The receptor α chain is an essential receptor for signal transduction from these cytokines. The invention relates to IL-4R α Blocks the binding of canine IL-4 and canine IL-13 to canine IL-13. Generation of monoclonal antibodies that inhibit signaling from both IL-4 and IL-13 The identification and characterization of these antibodies are described. Thus, these antibodies may be used in combination with other antibodies as disclosed herein. and has utility in the treatment of atopic dermatitis and other diseases in companion animals. In addition, canine IL-4R α The biological function of, for example, The antigen-binding domain may have an epitope within the extracellular domain that is conserved.

[0090] Individual canine IL-4R α The amino acid sequence is generally an amino acid sequence comprising the amino acid sequence of SEQ ID NO:4. NuIL-4R α In some cases, the canine IL-4R is at least 90% identical to α is a canine IL-4R comprising the amino acid sequence of SEQ ID NO: 4 α At least 95% or Furthermore, they are at least 96%, 97%, 98% or 99% identical. In particular, canine IL-4Rα The amino acid sequence is a canine IL- 4R α In one embodiment, the canine IL- 4R α The amino acid sequence of canine IL-4R comprises the amino acid sequence of SEQ ID NO: 4. α compared to 5 It may refer to differences of no more than an amino acid, or even no more than 4, 3, 2 or 1 amino acid. The percentage can be determined as described below.

[0091] The term "immune response" refers to, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and progenitor cells. Soluble macromolecules produced by the liver or by the inflammatory cells (including antibodies, cytokines, and complement) (including the action of the agonist) on cancer cells, pathogen-infected cells or tissues, or invasive pathogens. selective damage, destruction, or elimination of the mitochondrial proteins from the mammalian body (e.g., the dog's body) It means the effect it has.

[0092] Anti-canine IL-4R α antibody The present invention relates to canine IL-4R α Isolated antibodies (particularly mouse anti-canine IL-4R) that bind to α antibodies and caninized antibodies thereof) or antigen-binding fragments thereof, and such antibodies In a specific embodiment, the present invention provides a method for the treatment of canine IL-4 R α binds to canine IL-4R α of its ligands, canine IL-4 or IL-13 a murine anti-canine IL-4R antibody that has been shown to block binding to one or more of the following: α from antibodies Mouse anti-canine IL-4R α These CDRs are used to prepare modified canine antibodies. Inserted into the frame, caninized mouse anti-canine IL-4R α It is possible to obtain antibodies.

[0093] As used herein, "anti-canine IL-4R α "Antibody" refers to a human antibody (e.g., mouse or rabbit) Canine IL-4R (in mammals such as α Canine IL-4R produced against α Especially "Canine IL-4R" refers to an antibody that specifically binds to canine IL-4R. α (and especially canine IL-4R α ) "specifically binds" or "canine IL-4R α A polypeptide comprising the amino acid sequence The antibody that specifically binds to canine IL-4R is α Shows preferential binding to Although the antibody has specificity, absolute binding specificity is not required. α antibody Canine IL-4R α A protein is considered "specific" for a given antibody to be a protein that binds to a specific antibody in a dog sample. without inappropriately interfering with the activity of other molecules in the The ability to detect and treat enzymes in samples without causing unwanted consequences such as side effects in therapeutic settings. NuIL-4R α or if it determines the presence of canine IL-4R α The activity of Anti-canine IL-4R α The degree of specificity required for an antibody depends on the It may depend on the intended use, but in any event its suitability for the intended purpose use. The antibody of the expected method or the antigen-binding site of the antibody is determined by the combination. The binding compound is either the affinity for other antigens is at least 2-fold greater, preferably at least 10 times larger, more preferably at least 20 times larger, and most preferably at least 100 times larger binds with high affinity.

[0094] Given an antigen sequence (in this case, canine IL-4R α a portion of the amino acid sequence of The antibody specifically binds to the polypeptide because it binds to the canine IL-4R α Amino It binds to a polypeptide containing that portion of the amino acid sequence, but not to canine IL-4R. α That part of the sequence For example, canine IL-4R does not bind to other canine proteins that lack the required amino acid sequence. α Including The antibody that specifically binds to the polypeptide containing canine IL-4R α FLAG (registered trademark) tag can bind to the FLAG®-tagged form but not to other FLAG®-tagged canine proteins The antibody or a binding compound derived from the antigen-binding site of the antibody binds to the canine antigen or is said to bind "specifically" to a mutant or mutated protein if it , at least 10 times greater than its affinity for any other tested canine antigen, More preferably at least 20 times larger, even more preferably at least 100 times larger and having affinity for the canine antigen or a variant or mutein thereof. This is the case.

[0095] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. It is therefore used in the broadest sense and specifically refers to a monoclonal antibody that exhibits a desired biological activity. monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies, and camelized antibodies "Parent antibody" is intended to mean any antibody that is a human or humanized antibody, including, but not limited to, single domain antibodies. Modification of antibodies for use in canine therapeutics (e.g., caninization of antibodies for use as canine therapeutic antibodies). ) are antibodies obtained by exposure of the immune system to an antigen.

[0096] As used herein, an "antibody fragment" or "antigen-binding fragment" refers to an antibody an antigen-binding fragment of the antibody, i.e., a fragment that specifically binds to the antigen bound by the full-length antibody; Antibody fragments that retain the ability to bind to the CDRs, e.g., fragments that retain one or more CDR regions. Examples of antigen-binding fragments include Fab, Fab', and F(ab')2. and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as scFv; as well as multispecific antibodies and nanobodies formed from antibody fragments, However, the present invention is not limited to these.

[0097] A "Fab fragment" is a fragment of one light chain and one heavy chain. H 1 and variable regions The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab fragment" may be the product of papain cleavage of an antibody.

[0098] The "fragment crystallizable" ("Fc") region of an antibody H 3 and C H 2 Domains The two heavy chain fragments comprise two or more heavy chain fragments each comprising by disulfide bonds, and C H The three domains are held together by hydrophobic interactions.

[0099] A "Fab' fragment" contains one light chain and one V H Domain and C H 1 domain and a portion or fragment of one heavy chain containing C H 1 and C H The area between the two domains , resulting in interchain disulfide bonds between the two heavy chains of the two Fab' fragments. A bond can be formed to form an F(ab')2 molecule. "F(ab')2 fragment" means , two light chains, and C H 1 and C H Two domains containing portions of the constant region between the two domains 1 heavy chain, resulting in the formation of an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment has a disulfide bond between its two heavy chains. It consists of two Fab' fragments linked by a bond. "Fragments" may be the product of pepsin cleavage of antibodies.

[0100] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions. .

[0101] The term "single chain Fv" or "scFv" antibody refers to the V of an antibody. H and V L domain Antibody fragments containing these domains, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises an scFv polypeptide, which has the desired structure for antigen binding. allows for the formation of V H and V L Contains an inter-domain polypeptide linker [Pluckthun,THE PHARMACOLOGY OF MONOCLONA L ANTIBODIES, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp.269-315(1994 );WO 88 / 01649; and US 4,946,778 and US 5, 260, 203].

[0102] As used herein, the term "canonical structure" refers to an antibody Each of the hypervariable regions of the heavy and light chains of For each hypervariable region, a small number of canonical structures (general There are corresponding superpositions (denoted by simple integers such as 1 or 2) The amino acid sequence of the variable region [particularly the corresponding anti-canine IL-4R α More on variable domains in the context of its framework amino acid sequence shown in Table 3 below. These standard structures can be predicted with high accuracy from the Whether modifications to the amino acid sequence result in retention or loss of the ability to bind to its antigen-binding partner [Chothia and Lesk, Canonic al Structures for the hypervariable regi ons of immunoglobulins,J.Mol.Biol.196:90 1-917 (1987); Chothia et al., Conformation of immune unoglobulin hypervaribale regions,Nature , 34:877-883 (1989); and Al-Lazikani et al., Stand ard Conformations for the canonical stru ctures of immunoglobulins,J.Mol.Biol.273 :927-948(1997)].

[0103] "Domain antibodies" are immunological antibodies that contain only the variable region of a heavy chain or the variable region of a light chain. A functional immunoglobulin fragment. In some cases, two or more V H The area is The domains are covalently linked by a peptide linker to form a bivalent domain antibody. , two V H The regions may target the same or different antigens.

[0104] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites However, bivalent antibodies may be bispecific (see below). I want to be.

[0105] In certain embodiments, the monoclonal antibodies herein are camelized single domain antibodies. Also included are antibodies [see, e.g., Muyldermans et al., Trends Biochem. Sci.26:230(2001);Reichmann et al., J.Immunol.Me thods 231:25(1999);WO 94 / 04678;WO 94 / 255 91; see US 6,005,079]. In one embodiment, the present invention The aim is to create a single domain antibody by combining two V domains with modifications. H Single containing domain Domain antibodies are provided.

[0106] As used herein, the term "diabody" refers to a small antibody that has two antigen-binding sites. A fragment of a light chain variable domain (e.g., a ... V L ) linked to a heavy chain variable domain (VH) (V H -V L or V L -V H ) is included. Using a linker that is too short to allow pairing between the two domains on the same chain This forces those domains to pair with complementary domains on another strand, Generate two antigen-binding sites [EP 0 404 097 B1; WO 93 / 111 61; and Holliger et al., Proc. Natl. Acad. Sci. USA 9 0:6444-6448 (1993)]. See generally Holliger and Hudson, Nat. Biotechnol. ol.23:1126-1136(2005).

[0107] Typically, the antibodies or antigen-binding fragments of the present invention have their activity expressed on a molar basis. If so, the canine IL-4R (compared to the parent antibody) α At least 1% of binding activity Preferably, the antibody or antigen-binding fragment of the present invention has a parent antibody Canine IL-4R α At least 20%, 50%, 70%, 80%, 90% of the binding activity 95% or 100% or more of the antibody or antigen-binding activity of the present invention. A fragment may contain conservative or non-conservative amino acid substitutions that do not substantially alter its biological activity. (referred to as "conservative variants" or "function-conservative variants" of antibodies) do.

[0108] "Isolated antibody" refers to a purified state, in which the molecule is different from other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other substances, e.g., cells Generally, "isolated" means that the cells are substantially free of cell debris and growth medium. " refers to the complete absence of such substances, or the absence of water, buffers or salts. They do not imply any experimental evidence for the binding compounds described herein. Or it should not be present in an amount that substantially interferes with therapeutic use.

[0109] As used herein, a "chimeric antibody" refers to a chimeric antibody that contains the variable domains from a first antibody and the variable domains from a second antibody. and a constant domain, wherein the first antibody and the second antibody are from different species. and Morrison et al., Proc. Natl. Acad. Sci. tl. Acad. Sci. USA 81:6851-6855(1984)]. Typically In the present study, the variable domains are derived from antibodies from laboratory animals such as rodents ("parent antibodies") and are used to identify specific antibodies. The common domain sequences are obtained from animal subject (e.g., human or canine) antibodies, resulting in Chimeric antibodies may elicit adverse immune responses in dogs or human subjects, respectively. will be lower than the parental (e.g., rodent) antibody.

[0110] As used herein, the term "caninized antibody" refers to both canine and non-canine (e.g., murine) antibodies. (i.e., a form of an antibody that contains sequences from both a caninized and a caninized antibody.) Generally, a caninized antibody contains at least two sequences from both a caninized and a caninized antibody. and comprising substantially all of at least one, and more typically two, variable domains, wherein the hypervariable All or substantially all of the loops are from a non-canine immunoglobulin (e.g., the exemplified As shown, six mouse anti-canine IL-4R antibodies were used.α (including CDR) and frame All or substantially all of the work (FR) area (and typically all of the remaining frames) or substantially all) are canine immunoglobulin sequences. As mentioned above, caninized antibodies are produced by combining mouse anti-canine I with a canine frame or modified canine frame. L-4R α It contains both the three heavy chain CDRs and the three light chain CDRs from the caninized antibody. Further optimizing the effectiveness of the canine IL-4R α enhances its binding to and / or type I and / or type II IL-4 receptors. and the IL-13 binding inhibitors exemplified herein, which enhance their ability to block the binding of canine IL-13. The modified canine frame contains the above amino acid changes.

[0111] The term "full canine antibody" refers to an antibody that contains only canine immunoglobulin protein sequences. Whole canine antibodies are produced in mice, in mouse cells, or derived from mouse cells. When produced in a resulting hybridoma, the antibody may contain murine carbohydrate chains. Similarly, a "murine antibody" refers to an antibody that contains only murine immunoglobulin sequences. The complete canine antibody is produced in rats, in rat cells, or derived from rat cells. If produced in a hybridoma containing the same, it may contain rat carbohydrate chains. Additionally, "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0112] There are four known IgG heavy chain subtypes of canine IgG: IgG-A, IgG-B, IgG-C, IgG-D, IgG-E, IgG-F, IgG-G, IgG-H, IgG-I ... The two known light chain subtypes are lambda and IgG. They are called Yobi and Kappa.

[0113] The variable regions of each light / heavy chain pair form the antibody binding site. has two binding sites. Except in the case of bifunctional or bispecific antibodies, the two The binding sites for these are generally the same.

[0114] Typically, the variable domains of both the heavy and light chains share a relatively conserved framework. Three hypervariable regions, also called complementarity-determining regions (CDRs), located within the complementarity-determining region (FR) The CDRs typically comprise a framework that enables binding to a specific epitope. Generally, from the N-terminus to the C-terminus, the light and heavy chain variable domains are aligned. Both main sequences contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is generally based on the Sequences of Protein eins of Immunological Interest, Kabat et al.;Na tional Institutes of Health,Bethesda,Md. ;5th ed.;NIH Publ.No.91-3242(1991);Kabat , Adv. Prot. Chem. 32:1-75 (1978); Kabat et al., J. Bi ol.Chem.252:6609-6616(1977);Chothia et al., JM ol. Biol. 196:901-917 (1987) or Chothia et al., Nat. Based on the definition in J. Med. 342:878-883 (1989).

[0115] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable regions are "complementarity determining regions" or "CDRs" (i.e., light chain variable regions). CDRL1, CDRL2, and CDRL3 in the domain and C in the heavy chain variable domain The sequence of the antibody contains amino acid residues from CDRH1, CDRH2, and CDRH3 Defining CDR regions, Kabat et al., Sequences of Proteins o f Immunological Interest,5th Ed.Public H health service,National Institutes of Hea See, e.g., Ith, Bethesda, Md. (1991); also see, e.g., antibodies by structure. Determining the CDR regions of Chothia and Lesk, J. Mol. Biol. 196 :901-917 (1987)]. As used herein, "framework" refers to a The terms "FR" or "FR" residues refer to hypervariable region residues defined herein as CDR residues. The term "variable domain residues" refers to variable domain residues excluding the nucleotide sequence residues.

[0116] IL-4R in addition to binding and activation of canine immune cells α Canine or canine-derived antibodies against The body optimally has two properties: 1. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) Lacking effector functions; and 2. Large-scale purification using industry-standard techniques such as Protein A chromatography-based techniques It is easily purified on a large scale.

[0117] No naturally occurring canine IgG isotype meets both criteria. For example, IgG-B can be purified using Protein A, but with high levels of A. It has DCC activity. On the other hand, IgG-A binds weakly to Protein A, which is undesirable. Furthermore, IgG-D does not exhibit ADCC activity (although IgG-C does). Both IgG-C and IgG-D were detected by Protein A column. One way in which the present invention overcomes this problem is by using IL-4R α to by obtaining specific mutant canine IgG-B antibodies, such antibodies being A It lacks effector functions like DCC and does not perform industry-standard Protein A chromatography. It can be easily purified using

[0118] "Homology" is the degree of homology between two optimally aligned polynucleotide sequences or indicates the sequence similarity between two polypeptide sequences. If the positions in both are occupied by the same base or amino acid monomer subunit, For example, if the position in each of the two DNA molecules is occupied by an adenine If the two sequences are identical, the molecules are homologous at that position. The number of shared homologous positions divided by the total number of positions compared multiplied by 100 For example, 6 out of 10 positions in two sequences are optimally aligned. If two sequences are matched or homologous when they are joined, The alignment is 60% homologous. Generally, the comparison is performed by aligning the two sequences to give the maximum percent homology. This is done when the alignment is complete.

[0119] An "isolated nucleic acid molecule" is a polynucleotide as it is found in nature. Polynucleotides that are not naturally linked or that are not part of the nucleotide sequence. DNA or DNA of genomic, mRNA, cDNA or synthetic origin, linked to nucleotides For the purposes of this disclosure, specific It should be understood that a "nucleic acid molecule comprising" a nucleotide sequence does not include an intact chromosome. An isolated nucleic acid molecule that "comprises" a specified nucleic acid sequence has all the components in addition to the specified sequence. In addition, up to 10 or even up to 20 or more other proteins or portions thereof. It may contain the coding sequence of a fragment or fragments of the recited nucleic acid sequences. It may contain operably linked regulatory sequences that control expression of the coding region, and and / or vector sequences.

[0120] As used herein, the term "control sequence" refers to a sequence that is operably linked to a particular host organism. Control sequences refer to DNA sequences necessary for the expression of a coding sequence that has been isolated from the host. Control sequences suitable for prokaryotes include, for example, For example, it contains a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells use promoters, polyadenylation signals, and enhancers. It is well known that:

[0121] A nucleic acid is said to be "operably linked" if it is functionally linked to another nucleic acid sequence. For example, the DNA of the presequence or secretory leader is A polypeptide is said to be operably linked to DNA if it is When expressed as a preprotein involved in secretion, A sensor is said to be operably linked to a coding sequence if it affects the transcription of that sequence. or when the ribosome binding site is operably linked to the coding sequence. It is said to be functional if it is positioned in a way that facilitates translation. "Linked" means that the DNA sequences are contiguous and secreted. In the case of leaders, ensure that they are contiguous and in the same reading frame. However, enhancers do not have to be contiguous. Linkage can be achieved by ligation at convenient restriction sites. If no such site exists, then the usual practice is to Synthetic oligonucleotide adaptors or linkers are used.

[0122] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably. and all such designations include progeny. The term "transfected cells" refers to the primary subject cell and any subsequent cells, regardless of the number of transfers. and all progeny, whether due to intentional or unintentional mutations, It is understood that the DNA content may not be exactly the same as in the original transformed cell. Mutant progeny that have the same function or biological activity as screened for are also included. Where a different name is intended, it will be clear from the context.

[0123] As used herein, "germline sequence" refers to the sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequences may be used. The sequences are available from, for example, the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes o JOINSOLVER® Germline Data on the f Health website Mouse germline sequences can be obtained from, for example, Giudicelli et al. cleic Acids Res.33:D256-D261(2005)] can be obtained as is.

[0124] Mouse anti-canine IL-4R α and caninized mouse anti-canine IL-4R α Antibody characteristics The present invention provides an isolated murine anti-canine IL-4R antibody. α providing an antibody and a caninized version thereof; The antibody or its derivatives may also be used in the treatment of diseases, for example, in the treatment of atopic dermatitis in dogs. In dogs, the antigen-binding fragments A, B, C, and D are used. There are four IgG heavy chains, called IgG1 and IgG2. These heavy chains are the four different IgG1 heavy chains of dog IgG. These correspond to subclasses, which are called IgGA, IgGB, IgGC, and IgGD. Each of the two heavy chains contains one variable domain (VH), as well as CH-1, CH-2 and It consists of three constant domains called CH-1 and CH-3. The CH-1 domain is the "hinge" or is connected to the CH-2 domain via an amino acid sequence called the "hinge region." do.

[0125] The DNA and amino acid sequences of these four heavy chains are reported by Tang et al. [Vet. Immuno Immunopathol.80:259-270(2001)] The amino acid and DNA sequences of these heavy chains were also obtained from the GenBank database. For example, the amino acid sequence of the IgGA heavy chain is available under accession number AAL35 301.1, IgGB has accession number AAL35302.1, and IgG C has the accession number AAL35303.1, and IgGD has the accession number A The canine antibody also has two types of light chains: kappa and The DNA and amino acid sequences of these light chains are available in the GenBank database. For example, the kappa light chain amino acid sequence can be obtained from the ABCD database under accession number AB Y 57289.1, and the lambda light chain has accession number ABY 55569.1. Has.

[0126] In the present invention, the amino acid sequences of the four canine IgG Fc fragments are The identified boundaries of the CH1 and CH2 domains as determined by Tang et al. (supra). Based on canine IL-4R α Caninized mouse anti-canine IL-4R binds to α The antibody Anti-canine IL-4R α Canine IgG-A, IgG-B and IgG-D heavy chains along with CDRs Antibodies containing canine kappa light chains and / or canine kappa light chains are also included, but are not limited to these. Therefore, the present invention provides a method for detecting canine IL-4R α binds to type I or type II IL-4 Isolated mouse anti-canine antibodies that block the binding of canine IL-4 and canine IL-13 to their receptors IL-4R α and / or caninized mouse anti-canine IL-4R α Antibodies or their antigen binding to provide synthetic fragments.

[0127] The present invention further provides full-length canine heavy chains that can be balanced with corresponding light chains to provide caninized antibodies. Therefore, the present invention further provides a caninized mouse anti-canine antigen antibody (isolated caninized mouse Mouse anti-canine IL-4R α and the treatment of diseases, e.g., in dogs. and methods for using said antibodies or antigen-binding fragments thereof in the treatment of atopic dermatitis. provide.

[0128] The present invention also provides a caninized mouse antibody containing a canine fragment crystallizable region (cFc region). Canine IL-4R α The present invention provides an antibody, wherein the cFc has one or more enhanced effector functions. In one embodiment of the present invention, The genetically modified cFc reduces or eliminates one or more effector functions. In another embodiment of the present invention, the genetically modified cFc is capable of binding to one or more effectors. In one embodiment, the genetically modified cFc region enhances the function of the In another such embodiment, the modified canine IgGB Fc region is The genetically modified cFc region is a genetically modified canine IgGC Fc region. In certain embodiments, the effector function is antibody-dependent, which is enhanced, reduced or eliminated. In another embodiment, the effector function is increased cytotoxicity (ADCC). In yet another embodiment, the complement dependent cytotoxicity (CDC) is enhanced, reduced, or abolished. In the present invention, the cFc region enhances, reduces, or eliminates both ADCC and CDC. They have been genetically modified to enhance their activity.

[0129] To generate canine IgG variants lacking effector function, several mutant IgGs were isolated. These mutants consisted of the following variants in the Fc portion of the heavy chain amino acid sequence: It may contain one or more of the following substitutions, either singly or in combination: P4A, D31A, N63A, G6 4P, T65A, A93G and P95A. Mutant heavy chains (i.e., those amino acids The light chain-encoding gene was cloned into an expression plasmid containing the nucleotide sequence (containing the substitution). The plasmid containing the vector was transfected into HEK293 cells. Intact antibodies expressed and purified from cells were used as Fc γ Evaluated for binding to RI and C1q , and evaluated their potential for mediating immune effector functions [July 30, 2014 U.S. Provisional Patent Application No. 62 / 030,812, filed December 16, 2014. No. 62 / 092,496 filed on 2006 / 010904, the entire contents of both of which are incorporated herein by reference. See, e.g., the entire disclosure of which is incorporated herein by reference.

[0130] The present invention also provides a method for the preparation of a human IgG1A-derived hinge region comprising substituting a hinge region from: The present invention provides a modified canine IgGD comprising:

[0131] IgGA: FNECRCTDTPPCPVPEP (SEQ ID NO: 101); IgGB: PKRENGRVPRPPDCPKCPAPEM (SEQ ID NO: 102); or IgGC: AKECECKCNCNNCPCPGCGL (SEQ ID NO: 103).

[0132] Alternatively, the IgGD hinge region may be modified by substituting serine residues with proline residues. can be genetically modified: i.e., PKESTCKCI P PCPVPES (SEQ ID NO: 104 ) (Naturally occurring serine residues are shown underlined and in bold with proline residues (P) Such modifications result in canine IgGD lacking Fab arm exchange. Modified canine IgGD can be produced using standard methods of recombinant DNA technology [e.g., Maniati s et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct these mutants, canine IgGD The nucleic acid encoding the amino acid sequence of the present invention can be modified so that it encodes a modified IgGD. The modified nucleic acid sequence is cloned into an expression plasmid for protein expression.

[0133] Canine IL-4R α The antibody or antigen-binding fragment thereof that binds to the Complementarity determining regions (CDRs) 1, 2, 3, 4, 5 or 6 of the mouse anti-dog antibody Such 1, 2, 3, 4, 5, or 6 CDRs may independently be any of the CDRs described below. In another embodiment, the CDR sequences of canine IL-4R can be selected from the CDR sequences of α The isolated antibody or antigen-binding fragment thereof that binds to 1. A canine antibody kappa light chain containing CDR-2 and / or CDR-3 and a mouse heavy chain CD and a canine antibody heavy chain IgG comprising CDR-1, CDR-2 and / or CDR-3.

[0134] In other embodiments, the present invention provides the nucleic acids of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 6 6, 67, 68, 69, 70, 71, 72 and / or 73 amino acid sequences 1 to 3, which contain at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity Canine antibody kappa light chains containing six different CDRs and SEQ ID NOs: 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 8, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99 and / or 100 amino acid sequences and at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity. The canine antibody heavy chain IgG contains 1 to 6 different CDRs, while retaining the desired binding and IL-4R still exhibits functional properties α An antibody or its antigen-binding flag that specifically binds to In another embodiment, the antibody or antigen-binding fragment of the invention is The segment may be 0, 1, 2, 3, 4 or 5 while still exhibiting the desired binding and functional properties. and one or more of the CDR amino acid sequences containing conservative or non-conservative amino acid substitutions of The canine framework contains a combination of IgG heavy chain sequences with kappa light chains.

[0135] Sequence identity is the degree to which the amino acids of two polypeptides are aligned optimally ( It means the degree of similarity at the same position when two amino acid sequences are aligned. As used herein, a sequence is said to be 100% "identical" to a second amino acid sequence. , where the amino acid residues of both sequences are identical. 50% "identical" to an amino acid sequence of another is 50% identical to the amino acid residues of that amino acid sequence. Sequence comparison is performed for a given protein (e.g., A contiguous block of amino acid residues contained in a protein or polypeptide (part of a protein or polypeptide that is In certain embodiments, selected deletions or insertions are considered. This is done to alter the correspondence between two amino acid sequences that might otherwise occur.

[0136] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids that may be shared and interchangeable are considered.

[0137] "Conservatively modified variants" or "conservative substitutions" refer to amino acid substitutions in a protein. However, similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation) It means that the amino acid is substituted with another amino acid having the same functionality (such as functionality and rigidity), where the change can frequently be applied without altering the biological activity of the protein. It is understood by those skilled in the art that single amino acid substitutions in non-essential regions of a gene do not substantially alter biological activity. recognizes [e.g., Watson et al., Molecular Biology of the Gene,The Benjamin / Cummings Pub.Co., p.224 (4th Ed.; 1987). Also, structural or functional Substitution of similar amino acids is unlikely to impair biological activity. As shown in Table 1 below. [Table 1] TIFF2025129151000003.tif93145

[0138] Function-conservative variants of the antibodies of the present invention are also encompassed by the present invention. "Potential variants" are those that have altered desirable properties such as antigen affinity and / or specificity. It refers to an antibody or fragment in which one or more amino acid residues have been altered without modification. Such variants include substitutions of amino acids with amino acids having similar properties, e.g., the conserved amino acids of Table 1 above. These include, but are not limited to, selective amino acid substitutions.

[0139] nucleic acid The present invention further provides the murine anti-canine IL-4R antibodies disclosed herein. α and / or Nucleotide mouse anti-canine IL-4R α Immunoglobulins of antibodies and their antigen-binding fragments It includes nucleic acids encoding purine chains (see Examples below).

[0140] In addition, the BLAST algorithm (in this case, the parameters of the algorithm are The sequences are chosen to give the largest match between each other over the entire length of the reference sequence. When the comparison is performed by the method described above, the amino acid sequences of the CDRs and antibodies provided by the present invention are at least about 70% identical, preferably at least about 80% identical, to Preferably, they are at least about 90% identical, most preferably at least about 95% (e.g., , 95%, 96%, 97%, 98%, 99%, 100%) identical amino acid sequences Nucleic acids encoding immunoglobulin polypeptides are also encompassed by the present invention. AST algorithm (where the parameters of the algorithm are the (chosen to give the largest match between the respective sequences over the entire length of the When compared, the amino acid sequence is at least about 70% similar to one of the reference amino acid sequences. Preferably, the sequence is at least about 80% similar, more preferably at least about 90% similar. Similar, most preferably at least about 95% (e.g., 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, Immunoglobulin polypeptides containing amino acid sequences that are similar (8%, 99%, 100%) Nucleic acids encoding the

[0141] As used herein, nucleotide and amino acid sequence identities (%) are based on the C, MacV ector (MacVector, Inc. Cary, NC 27519), Vecto r NTI (Informax, Inc. MD), Oxford Molecular Alignment using Group PLC (1996) and Clustal W algorithms Determined using default parameters and default parameters for identity These commercially available programs may be used with the same or similar default parameters. Alternatively, the sequence similarity can be determined using a computer. GCG (Genetics Computer Group, Program Manual for the GCG Package,Versi on 7, Madison, Wisconsin) using the pileup program. An Advanced Blast search under default filter conditions can be used.

[0142] The following references concern the BLAST algorithm, which is often used for sequence analysis: BLAST algorithm: Altschul, SF et al., J. Mol. Bi ol.215:403-410(1990); Gish, W. et al., Nature Gen et.3:266-272(1993);Madden, TL et al., Meth.Enz ymol.266:131-141(1996);Altschul, SF et al., Nu Cleic Acids Res.25:3389-3402(1997);Zhang , J. et al., Genome Res. 7:649-656 (1997); Wootton, JC et al., Comput. Chem. 17:149-163 (1993); Hanco ck, JM et al., Comput. Appl. Biosci. 10:67-70 (199 4); Alignment Scoring System: Dayhoff, MO et al., “A m evolutionary change in proteins” ,Atlas of Protein Sequence and Structure ,vol.5,suppl.3.MODayhoff(ed.),pp.345-352 (1978);Natl.Biomed.Res.Found.,Washington , DC; Schwartz, RM et al., “Matrices for detecti ng distant relationships”, Atlas of Prote in Sequence and Structure,vol.5,suppl.3( 1978), MODayhoff (ed.), pp.353-358(1978), Na tl.Biomed.Res.Found.,Washington,DC;Altsc hul, SF, J.Mol.Biol.219:555-565(1991);St ates, DJ et al., Methods 3:66-70 (1991); Henikof f, S. et al., Proc. Natl. Acad. Sci. USA 89:10915-10 919(1992);Altschul, SF et al., J.Mol.Evol.36:2 90-300(1993); Alignment Statistics: Karlin, S. et al., Proc.N atl.Acad.Sci.USA 87:2264-2268(1990);Karl in, S. et al., Proc. Natl. Acad. Sci. USA 90:5873-58 77(1993);Dembo, A. et al., Ann. Prob. 22:2022-2039 (1994); and Altschul, S.F. “Evaluating the s statistical significance of multiple dist. inct local alignments”, Theoretical and C Computational Methods in Genome Research( S. Suhai (ed.), pp.1-14, Plenum, New York (1997).

[0143] The present invention also provides an expression vector comprising the isolated nucleic acid of the present invention, wherein the nucleic acid is a restriction enzyme recognized by a host cell when the host cell is transfected with the vector. The present invention also provides a host cell comprising an expression vector of the present invention. In addition, a host containing an expression vector encoding an antibody or antigen-binding fragment thereof The cells are cultured in a medium, and the antigen or antigen-binding fragment thereof is isolated from the host cells or the medium. and isolating the antibody or antigen-binding fragment thereof disclosed herein. The present invention provides a method for producing the same.

[0144] Epitope binding and binding affinity The present invention further provides a murine anti-canine IL-4R antibody. α Same as antibody, canine IL-4R α epitope of The present invention provides antibodies or antigen-binding fragments thereof that bind to amino acid residues of the In an embodiment, the mouse anti-canine IL-4R α Antibodies or antigen-binding fragments thereof The binding of canine IL-4 and canine IL-13 to type I and / or type II IL-4 receptors was investigated. It can suppress / block the connection.

[0145] Caninized mouse anti-canine IL-4R α The antibodies may be recombinantly produced by methods known in the art. can be used as a host for expression of the antibodies or fragments disclosed herein. Available mammalian cell lines are well known in the art and are available from American Type Numerous immortalized cell lines available from the American College of Cardiovascular Medicine (ATCC) These include, inter alia, Chinese hamster ovary (CHO) cells, NSO , SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS ), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK- 293 cells and many other cell lines. Mammalian host cells include human, mouse, rat , dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used include insect cell lines, e.g., Sf9 cells, amphibian cells, bacterial cells, and the like. The heavy chain or antigen-binding portion thereof or fragment thereof may be expressed in a variety of cells, including cells of bacteria, plants, and fungi. a recombinant expression vector encoding the main chain, light chain and / or antigen-binding fragment thereof; When the target is introduced into a mammalian host cell, the expression (or More preferably, sufficient to allow for secretion of the antibody into the medium in which the host cells are cultured. The antibody is produced by culturing the host cells for a sufficient period of time.

[0146] Antibodies can be recovered from the culture medium using standard protein purification methods. Expression of the antibodies of the invention (or other moieties therefrom) from cellular systems can be accomplished using several known techniques. For example, the glutamine synthetase gene expression system (GS system) can be enhanced under certain conditions. The GS system is a general approach to enhance expression under certain conditions. 846, 0 256 055 and 0 323 997 and European patent applications No. 89303964.4, which are discussed in whole or in part.

[0147] Generally, glycoproteins produced in individual cell lines or transgenic animals is a sequence characteristic of the glycoprotein produced in the cell line or transgenic animal. Therefore, the individual glycosylation patterns of the antibodies The antibody is produced by a particular cell line or transgenic animal. However, the nucleic acid molecules provided herein may be encoded by, or All antibodies containing the amino acids provided by the present invention are intended to be used in conjunction with the glycosylation patterns that the antibodies may have. Similarly, in certain embodiments, non-fucosylated Antibodies with a glycosylation pattern that includes only N-glycans may be advantageous because These antibodies have been shown to mimic their fucosylated counterparts both in vitro and in vivo. These drugs have been shown to typically exhibit higher efficacy than those used in the treatment of rheumatoid arthritis [e.g., Shinkaw a et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent No. See US Pat. Nos. 6,946,292 and 7,214,775.

[0148] The present invention further provides the murine anti-canine IL-4R antibodies disclosed herein. α Antibody Flag The antibody fragments include F(ab)2 fragments, which can be, for example, For example, Fab fragments can be produced by enzymatic cleavage of IgG with pepsin. For example, it can be prepared by reduction of F(ab)2 with dithiothreitol or mercaptoethylamine. Fab fragments can be produced by disulfide bridges. H -C H1 connected to a chain TaV L -C L The F(ab)2 fragment is now a chain bound by two disulfide bridges. The Fab portion of the F(ab)2 molecule is two Fab fragments linked by a dimer. F with a sulfide bridge between them c Contains parts of the region. F v Fragment is V L Also is V H It is an area.

[0149] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region: For example, canine constant regions, e.g., IgG-A, IgG-B, IgG-C, and IgG-D canine In another embodiment, the antibody or The antigen-binding fragment may also comprise a light chain constant region, e.g., a canine light chain constant region, e.g., lambda or or Kappa IgE light chain regions or variants thereof. In this case, the canine heavy chain constant region can be derived from IgG-B and the canine light chain constant region can be derived from IgG-C. It is possible that it is derived from papa.

[0150] Antibody engineering Caninized mouse anti-canine IL-4R of the present invention α Antibodies may be modified, for example, to improve the properties of the antibody. In addition, the canine framework and and / or can be engineered to contain modifications to canine frame residues.

[0151] Laboratory and diagnostic uses Mouse anti-canine IL-4R of the present invention α and / or caninized mouse anti-canine IL-4R α anti The antibody or antigen-binding fragment thereof is a canine IL-4R antibody. α Protein Diagnostics Assays, e.g., detecting expression associated with and / or related to atopic dermatitis It may also be useful in

[0152] For example, such a method may include the following steps:

[0153] (a) Mouse anti-canine IL-4R α An antibody or its antigen-binding fragment is attached to a substrate (e.g., For example, coating a microtiter plate well, e.g., the surface of a plastic plate ing; (b) Canine IL-4R α applying to said substrate a sample to be tested for the presence of thing; (c) washing the plate to remove unbound material in the sample; (d) IL-4R α Detectably labeled antibodies (e.g., antibodies) that are similarly specific for the antigen applying antibodies (e.g., enzyme-linked antibodies); (e) washing the substrate to remove unbound labeled antibody; (f) If the labeled antibody is enzyme-conjugated, it is converted by the enzyme into a fluorescent signal. applying chemicals that (g) detecting the presence of the labeled antibody.

[0154] In another embodiment, the labeled antibody is labeled with peroxidase, which is A BTS [e.g., 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfone)] acid)] or 3,3',5,5'-tetramethylbenzidine, resulting in a detectable color change Alternatively, the labeled antibody may contain a detectable radioisotope (e.g., 3 H) This can be detected by a scintillation counter in the presence of a scintillant. Mouse anti-canine IL-4R of the present invention α Antibodies are used in Western blots or immunoprotein blotting. It can be used in a batch process.

[0155] Such methods form part of the present invention and include, for example:

[0156] (i) Bound canine IL-4R α or a membrane to be tested for the presence of fragments thereof Other solid substrates may be used in conjunction with the murine anti-canine IL-4R α Antibodies or antigen-binding fragments thereof Such membranes are suitable for non-denaturing polyacrylamide gel electrophoresis (PAGE). (electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) Canine IL-4R in electrophoresis gel α If the protein to be tested for the presence of (e.g., After electrophoretic separation in the gel, the transferred nitrocellulose or or vinyl-based [e.g., polyvinylidene fluoride (PVDF)] membranes. The membrane was then incubated with mouse anti-canine IL-4R α Before contacting with the antibody or antigen-binding fragment thereof The membrane may then be optionally coated with, for example, a cellulose acetate solution to bind to non-specific protein binding sites on the membrane. For example, blocking may be performed using non-fat dry milk.

[0157] (ii) Washing the membrane one or more times to remove unbound mouse anti-canine IL-4R α antibody or removing antigen-binding fragments and other unbound material; and (iii) Conjugated mouse anti-canine IL-4R α Antibodies or antigen-binding fragments thereof To detect the

[0158] Detection of bound antibodies or antigen-binding fragments may involve the use of detectably labeled secondary antibodies. The antibody or antigen-binding fragment is bound to the secondary antibody (anti-immunoglobulin antibody), This may be by detecting the presence of an antibody label.

[0159] The murine anti-canine IL-4R disclosed herein α Antibodies and their antigen-binding fragments The materials may also be used in immunohistochemical methods. Such methods form part of the present invention. For example, (1) canine IL-4R α The cells to be tested for the presence of the mouse anti- NuIL-4R α (2) contacting the cells with antibodies and antigen-binding fragments thereof; detecting said antibody or antigen-binding fragment within said cell. If the fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or antigen-binding fragment may be detectably labeled. The antibody can be bound to a secondary antibody.

[0160] Imaging techniques include SPECT imaging (single photon emission computed tomography) PET (positron emission tomography) or PET (positron emission tomography). Labels include, for example, iodine-123 ( 123 I) and technetium-99m ( 99m Tc) (e.g., SPECT images in combination with 11 C. 13 N, 15 O or 18 F (e.g., PET imaging), or indium-111 [e.g., Go rdon et al., International Rev. Neurobiol. 67:385 -440(2005) for more details.

[0161] Cross-blocking antibodies Furthermore, the anti-canine IL-4R antibody of the present invention α Antibodies and antigen-binding fragments thereof include those of the present invention. The same canine IL-4R that is bound by the antibodies and fragments described herein. α to Any antibody or antigen-binding fragment thereof that binds to an epitope in NuIL-4R α For binding, the antibodies or fragments described herein (partial or are cross-blocked (completely) or partially (by the antibodies or fragments described herein) by the antibodies or fragments described herein. Any antibody or antigen-binding fragment that cross-blocks (partially or completely), as well as Any variants of these are included.

[0162] The cross-blocking antibodies and antigen-binding fragments thereof described herein are The antibodies disclosed herein (based on the CDRs described in Example 5), i.e. , 1A3, 1A9, 1B12, 10C12, 10F2, 10E10, 10G8 and / or or 11D3, or more particularly 11B6 and / or 6C12, and more particularly Specifically, their ability to cross-compete with 4D8, 4H3, 2E2, and / or 11H2 Based on this, standard binding assays (e.g., BIACore®, as exemplified below) These can be identified in a variety of assays, including ELISA, or flow cytometry. Standard ELISA assays are available, in this case recombinant canine IL-4R α Tan A protein is immobilized on a plate, one of the antibodies is fluorescently labeled, and a fluorescent antibody is added to compete with the binding of the labeled antibody. Additionally or alternatively, assess the ability of antibodies to cross-compete. For example, 1A3, 1A9, 1B 12, 10C12, 10F2, 10E10, 10G8 and / or 11D3, or More particularly 11B6 and / or 6C12, and even more particularly 4D8, 4H 3, 2E2 and / or 11H2, canine IL-4R α The test antibody can inhibit binding to The test antibody is 1A3, 1A9, 1B12, 10C12, 10F2, 10E1 0, 10G8, 11D3, 11B6, 6C12, 4D8, 4H3, 2E2 and / or 11H2 and canine IL-4R α can compete for binding to That is, 1A3, 1A9, 1B12, 10C12, 10F2, 10E10, 10G8, 1 Same as 1D3, 11B6, 6C12, 4D8, 4H3, 2E2 and / or 11H2, Canine IL-4R α As described above, the antibodies of the present invention can bind to the above epitopes. NuIL-4R α Antibodies and fragments that bind to the same epitope as either the antibody or fragment and fragments thereof also form part of the present invention.

[0163] Pharmaceutical Compositions and Administration Caninized mouse anti-canine IL-4R α Pharmaceutical compositions of antibodies or antigen-binding fragments thereof Alternatively, to prepare a sterile composition, it may be mixed with a pharmaceutically acceptable carrier or excipient. [e.g., Remington's Pharmaceutical Sciences es and US Pharmacopeia:National Formula ry, Mack Publishing Company, Easton, PA (198 See 4).

[0164] Formulations of therapeutic and diagnostic agents may be prepared, for example, as lyophilized powders, slurries, aqueous solutions or It may be prepared in the form of a suspension by mixing with an acceptable carrier, excipient or stabilizer. [e.g., Hardman et al. (2001) Goodman and Gilman's s The Pharmacological Basis of Therapeut ics, McGraw-Hill, New York, NY; Gennaro (2000 ) Remington: The Science and Practice of P. harmacy, Lippincott, Williams and Wilkins, Ne. w York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parental Medications,Marc el Dekker, NY; Lieberman et al. (eds.) (1990) Pharmace utical Dosage Forms:Tablets,Marcel Dekke r, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms:Disperse Systems,Marcel Dek ker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker,Inc. New York, NY]. In one embodiment, the anti-IL-1 antibody of the present invention -4R α The antibody was diluted to an appropriate concentration in sodium acetate solution (pH 5-6) and diluted with NaCl or To enhance stability, additional substances, such as sucrose, may be added to the solution. Polysorbate 20 or polysorbate 80 may be added.

[0165] The toxicity and therapeutic efficacy of the antibody composition administered alone or in combination with another therapeutic agent can be evaluated, e.g. For example, LD 50 (the dose that is lethal to 50% of the population) and ED 50 (50% of the population standard assays in cell cultures or experimental animals to determine the dose that is therapeutically effective The dose ratio between toxic and therapeutic effects is called the therapeutic index (LD 50 / ED 50 In certain embodiments, antibodies that exhibit a high therapeutic index are desirable. Data obtained from cell culture assays and animal studies from

[14] support the use of PEG-1 in dogs. The dosage of such compounds is preferably determined by: ED with little or no toxicity 50 The dosage is within a circulating concentration range that includes: It may vary within this range depending on the dosage form used and the route of administration.

[0166] The mode of administration can be varied. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; Intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, In certain embodiments, the method includes insufflation, topical, cutaneous, transdermal, or intra-arterial administration. Canine IL-4R α Antibodies or antigen-binding fragments thereof can be administered by invasive routes such as injection. In another embodiment of the present invention, a murine anti-canine IL-4R antibody can be administered. α If antibodies or an antigen-binding fragment thereof or a pharmaceutical composition thereof, can be administered intravenously, subcutaneously, intramuscularly, or intraarterially. or by inhalation or aerosol delivery. Also within the scope of the present invention are other suitable formulations (e.g., pills, capsules or tablets).

[0167] The compositions can be administered using medical devices known in the art. The pharmaceutical composition may be administered by hypodermic injection (e.g., a pre-filled syringe or an autoinjector). The pharmaceutical compositions disclosed herein can be administered by injection at 200° C. ..., for example, as described in U.S. Pat. No. 6,620,135, No. 6,096,002, No. 5,399,163, No. 5,3 No. 83,851, No. 5,312,335, No. 5,064,413, No. 4,941,8 80, 4,790,824 or 4,596,556 It may also be administered by needleless hypodermic injection devices such as

[0168] The pharmaceutical compositions disclosed herein can also be administered by injection. Examples of well-known implants and modules for providing such treatment include U.S. Pat. No. 4,448,628. No. 7,603 (which describes an implantable microinjection pump for dispensing medication at a controlled rate) U.S. Pat. No. 4,447,233 (which discloses a method for administering a drug at a precise rate of infusion) 4,447,224 (which discloses a drug infusion pump for delivering which discloses a variable flow rate implantable infusion device for continuous drug delivery), U.S. Pat. No. 4,439,196 (which is a systemic drug delivery system having a multi-chamber compartment) (which discloses a material transport system). Plants, conveying systems and modules are well known to those skilled in the art.

[0169] Alternatively, mouse anti-dog or caninized mouse anti-dog IL-4R α Antibodies are not administered systemically Locally, for example, often with depot or sustained release formulations, The antibody is administered by direct injection into the affected arthritic joint or pathogen-induced lesion. Furthermore, targeted drug delivery systems can be used to treat, for example, immunopathologically characterized liposomes coated with tissue-specific antibodies targeting arthritic joints or pathogen-induced lesions The antibody can be administered in a liposome, which is targeted to the affected tissue. , will be taken up selectively by the diseased tissue.

[0170] Administration may be based on serum or tissue turnover rate of the therapeutic antibody, severity of symptoms, immunogenicity of the therapeutic antibody, and other factors. It depends on several factors, including the availability of target cells in the biological matrix and the Preferably, the administration regimen is sufficient to provide a therapeutic effect that results in an improvement in the target condition. It is therefore possible to administer therapeutic antibodies while at the same time minimizing undesirable side effects. The amount of biological material delivered will depend, in part, on the individual therapeutic antibody and the condition being treated. Dependent on severity. Guidance is available for selecting appropriate doses of therapeutic antibodies [e.g. For example, Wawrzynczak, Antibody Therapy, Bios Sci. entific Pub.Ltd,Oxfordshire,UK(1996);Kre sina (ed.) Monoclonal Antibodies, Cytokines a nd Arthritis,Marcel Dekker,New York,NY(1 991); Bach (ed.) Monoclonal Antibodies and Pe. ptide therapy in Autoimmune Diseases,Mar cel Dekker, New York, NY (1993); Baert et al., New Engl.J.Med.348:601-608(2003);Milgrom et al.,Ne w Engl.J.Med.341:1966-1973(1999);Slamon et al. ,New Engl.J.Med.344:783-792(2001);Beniam inovitz 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 (200 0).

[0171] Determining the appropriate dosage can be accomplished using methods known in the art or which affect, for example, treatment. The dosage is determined by a veterinarian using the parameter or factor being suspected. Start with a dose somewhat less than optimal, then increase the dose until the desired or optimal effect is achieved relative to negative side effects. The dose is increased in small increments until a satisfactory result is achieved. Important diagnostic measures include, for example, measures of inflammatory symptoms, or the level of inflammatory cytokines produced.

[0172] The antibodies or antigen-binding fragments disclosed herein can be administered by continuous infusion, Or for example, daily, 1-7 days a week, weekly, biweekly, monthly, bimonthly, quarterly, twice a year, once a year, etc. Administration can be by any administration at any interval. Administration can be, for example, intravenous, subcutaneous, topical, oral, nasal, It may be administered intracavitary, rectal, intramuscular, intracranial, intraspinal, or by inhalation. The total weekly dose is: Generally, at least 0.05 μg / kg body weight, more generally at least 0.2 μg / kg , 0.5μg / kg, 1μg / kg, 10μg / kg, 100μg / kg, 0.25mg / kg, 1.0mg / kg, 2.0mg / kg, 5.0mg / kg, 10mg / kg, 2 5 mg / kg, 50 mg / kg or more [e.g., Yang et al., New Engl. J Med.349:427-434(2003);Herold et al., New Engl. J.Med.346:1692-1698(2002);Liu et al., J.Neurol. Neurosurg.Psych.67:451-456(1999);Portiel ji et al., Cancer Immunol. Immunother. 52:133-144 (2003)]. Also, caninized mouse anti-canine IL-4R in the subject's serum α Predetermined target concentrations of antibody, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, Administration can be performed to achieve 300 μg / ml or greater. Caninized mouse anti-canine IL-4R of the invention α The antibody is administered subcutaneously or intravenously weekly, every other week, or every four weeks. Every month, every other month or quarterly, 10, 20, 50, 80, 100, 200, 50 0, 1000 or 2500 mg / subject.

[0173] In addition, the binding of canine IL-4 and canine IL-13 to type I and type II IL-4 receptors Anti-canine IL-4R vaccines are being developed to generate antibodies that block the α By mAb Such vaccines can be used to treat diseases such as atopic dermatitis. These antigen peptides may be useful as therapeutic vaccines against such diseases. One or more of these peptides may be synthesized chemically or by recombinant DNA technology for use as a peptide. The immunogenicity of these peptides can be enhanced by conjugating them to other carrier proteins, It is possible to generate specific antibodies. The techniques for vaccinating animals are known to those skilled in the art. Used to vaccinate animals by M, S / C, oral, spray or in ovo route Peptide vaccines can be expressed from bacterial, viral, yeast, or baculovirus systems. Alternatively, such peptide vaccines may be used as subunit proteins. Chin is a physician who has prepared such a peptide vaccine, as can be achieved by methods known to those skilled in the art. The peptide can be delivered following administration of various viral or bacterial vectors that express the peptide. The vaccine can be administered at a dose of 1 to 1000 μg, and may optionally contain an adjuvant and and an acceptable pharmaceutical carrier.

[0174] As used herein, "inhibit" or "treat" or "treatment" refers to the treatment of a disorder. The term includes delaying the onset of symptoms and / or reducing the severity of symptoms of such disorders. The invention further provides for the amelioration of existing uncontrolled or unwanted symptoms, the prevention of additional symptoms, and The term therefore includes the amelioration or prevention of the underlying cause of such a condition. a vertebrate subject having or at risk of developing such a disorder, disease or condition The present invention demonstrates beneficial results in vertebrate subjects with the potential for the treatment of cancer.

[0175] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" mean , which, when administered alone or in combination with additional therapeutic agents to a cell, tissue, or subject, may be effective in treating a disease or one or more of the symptoms of a disease state or a measurable effect in the progression of such disease or condition The caninized murine anti-canine IL-4R antibody of the present invention is effective in causing a significant improvement in α Antibodies or A therapeutically effective amount also refers to the amount of antigen-binding fragment thereof that is effective to alleviate at least some of the symptoms. Partial improvement, such as treatment, cure, prevention or amelioration of an associated medical condition, or and a concentration of said binding compound sufficient to result in an increased rate of treatment, cure, prevention or amelioration of a condition. A therapeutically effective amount, when applied to an individual active ingredient administered alone, means an amount A therapeutically effective amount, when applied to a combination, refers to that component alone. Whether administered in combination, sequentially, or simultaneously, An effective amount of a therapeutic agent is at least 10 %, usually at least 20%, preferably at least about 30%, more preferably at least and most preferably at least 40%, and most preferably at least 50%, improvement in a diagnostic measure or parameter. An effective amount also means that when a subjective scale is used to assess the severity of the disease, It may result in improvements in subjective measures.

[0176] Other combination therapies As previously described, the caninized mouse anti-canine IL-4R of the present invention α Antibody or The antigen-binding fragments and / or antigenic peptides of the Inhibitors described in the next paragraph) and / or mice (or canine mice) ) may be co-administered with anti-canine TSLP antibodies [see US 8,791,242] the antibody may be linked to the substance (as an immunoconjugate), and / or The antibody may be administered separately from the substance or other antibodies. It can be administered before, after, or simultaneously with the agent, or it can be co-administered with other known therapies.

[0177] kit Additionally, a pharmaceutically acceptable carrier and / or an inhibitor, such as Janus ) kinase (JAK) inhibitors, such as oclacitinib [See WO 2013 / 040241], spleen tyrosine kinase (SYK) kinase inhibitors [see, e.g., US 8,759,366], or TH2 cells Antagonists for chemoattractant receptor homologue molecules expressed on the cytoplasm [e.g., W O 2010 / 099039; WO 2010 / 031183; and US8,54 6,422], Along with the ingredients, IL-4R α an antibody or antigen described herein that specifically binds to Binding fragments (e.g., caninized mouse anti-canine IL-4R α Antibodies or their antigen binding Kits containing one or more components, including, but not limited to, The immediately preceding binding compounds and / or inhibitors may be prepared in pure form. It may be formulated as a composition or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition. do.

[0178] In one embodiment, the kit comprises a binding composition of the invention (e.g., caninized mouse). Anti-canine IL-4R α antibody or pharmaceutical composition thereof) into a single container (e.g., a sterile glass or or plastic vial) and the pharmaceutical composition and / or said inhibitor. The target is contained within another container (e.g., a sterile glass or plastic vial).

[0179] When the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit includes a For example, the kit may include one or more of the hypodermic needles or The kit may also include other injection devices. The pharmaceutical compositions and dosage forms in the kit may also include Generally, such information may be included in the enclosed pharmaceutical package insert containing information about the Assisting pet owners and veterinarians in the effective and safe use of compositions and dosage forms For example, the following information regarding the combination of the present invention may be provided in the package insert: May be provided: Pharmacokinetics, Pharmacodynamics, Clinical Trials, Efficacy Parameters, Indications and Dosage, Contraindications , warnings, precautions, adverse reactions, overdosage, proper dosage and administration, feeding method, proper storage Terms, references, manufacturer / distributor information and patent information.

[0180] For convenience, the antibodies or specific binding agents disclosed herein may all be packaged as a kit. That is, a package of predetermined amounts of reagents accompanied by instructions for performing a diagnostic or detection assay. When the antibody is labeled with an enzyme, the kit can be provided as a combined antibody. is a substrate and cofactor (e.g., a detectable chromophore or fluorophore) required by the enzyme. Also, other additives such as stabilizers, buffers (e.g. , blocking buffer or cell lysis buffer). The relative amounts should be large enough to result in concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. In particular, the reagent may contain excipients that, when dissolved, provide a reagent solution having the appropriate concentration. The compound may be provided as a dry powder, typically a lyophilized powder, containing the compound. [Brief explanation of the drawings]

[0181] [Figure 1] Figure 1 shows the reactivity of purified mouse anti-canine IL-4Rα monoclonal antibodies (mAbs) to the extracellular domain of canine IL-4Rα. Various mouse mAbs were tested by ELISA for their binding to the extracellular domain of canine IL-4Rα. The mAbs tested have the following names: [ka]

[0182] The horizontal axis shows the log concentration of the added mAb, and the vertical axis shows the optical density obtained by ELISA. Shows. [Figure 2] Figure 2A shows the dose-response curve for the binding of canine IL-4 to canine IL-4Rα expressed on the surface of CHO cells using a cell-based CHO-cIL-4Rα binding assay. The horizontal axis shows the log concentration of added IL-4, and the vertical axis shows the mean fluorescence intensity (MFI) using FACS. Figure 2B shows the dose-response curve for CHO-cIL-4Rα using the following mouse anti-canine IL-4Rα monoclonal antibodies (mAbs): [ka]

[0183] The horizontal axis shows the log concentration (nM) of the added mAb, and the vertical axis shows the average concentration when FACS was used. The mean fluorescence intensity (MFI) is shown. The half-maximal effective concentration (EC50) for each of the antibodies are shown in Table 2 below. [Figure 3] Figures 3A and 3B show the results of serially diluted individual mouse anti-canine IL-4Rα monoclonal antibodies (mAbs) on IL-4 binding to cell-based CHO-cIL-4Rα. Figure 3A shows the results of individual monoclonal antibodies that individually block IL-4 binding to cell-based CHO-cIL-4Rα. [ka]

[0184] Figure 3B shows the concentration-dependent ability of cell-based CHO-cIL-4R α and IL-4 Monoclonal antibodies that individually block the binding of [ka]

[0185] The horizontal axis shows the log concentration (nM) of the added mAb, and the vertical axis shows the concentration-dependent ability of the Mean fluorescence intensity (MFI) is shown when FACS was used. [Figure 4] Figure 4 shows the binding of chimeric and caninized monoclonal antibodies to canine IL-4Rα as assessed by ELISA. The dose-dependent reactivity of caninized monoclonal antibodies to the canine IL-4 receptor alpha chain is as follows: [ka]

[0186] Example Example 1 stomach Identification and cloning of the IL-4 receptor α chain receptor The cDNA encoding the predicted full-length canine IL-4 receptor alpha chain (SEQ ID NO: 1) was cloned from Ge nbank database (accession number XM 547077.4;US 7,20 This putative cDNA was identified by a search of 25 amino acids (see also 8,579 B2). It encodes 823 amino acids (SEQ ID NO: 2) including the amino acid leader sequence. XP Identified as 547077.3. Mature putative canine IL-4 receptor alpha chain The protein (SEQ ID NO: 4) is the human IL-4 receptor alpha chain (accession number NP 000 409.1) and 65% identity to the porcine IL-4 receptor α chain (accession no. No. NP 999505.1) shares 70% identity with mature putative canine IL-4 The receptor alpha chain protein is encoded by the nucleotide sequence identified as SEQ ID NO:3. Comparison of the predicted mature IL-4 receptor α chain with the known sequence of the human IL-4 receptor α chain reveals The extracellular domain (ECD) of the mature canine IL-4 receptor α chain protein was identified. Referred to as SEQ ID NO: 6. Mature canine IL-4 The DNA sequence encoding the ECD of the receptor alpha chain is identified as SEQ ID NO:5. [ka] TIFF2025129151000010.tif212157TIFF2025129151000011.tif199157TIFF2025129151000012.tif71156

[0187] Example 2 Mouse anti-canine IL-4 receptor alpha chain antibody Production of anti-canine IL-4 receptor α-chain monoclonal antibody : A total of three Balc / c mice were given multiple doses (10 μg per dose) over a 17-day period. The immunization antigen was canine IL-4R alpha chain extracellular domain (ECD)-human F After immunization, serum was collected from each mouse and analyzed using a canine IL-4 receptor antigen (IL-4A). The best sera were tested for reactivity against the ruffian chain ECD HIS-tagged protein. Spleen cells from mice with anti-IL-4 receptor alpha chain ECD titers were cultured in the myeloma P3X63A Approximately two weeks after fusion, the putative hybridoma cells were fused with the g8.653 cell line. IL-4 receptor alpha chain ECD His-tagged protein The ELISA showed a strong positive signal. The hybridoma was subcloned by limiting dilution and expressed as canine IL-4 receptor alpha chain EC D was tested again for reactivity to HIS-tagged proteins.

[0188] Confirmation of reactivity of monoclonal antibodies against the canine IL-4 receptor α chain : Hybridoma-secreted antibodies against the ECD of the canine IL-4 receptor alpha chain The reactivity of the antibody was confirmed by ELISA. Hybridoma cells were cultured for 10 to 30 days using a PBS (ra-biosciences). The first was a mixture of 4mM L-glutamine and 10% Ultra Low IgG bovine fetal bovine serum. Cells were maintained in DMEM supplemented with fetal bovine serum (FBS) (Gibco). In the bioreactor cell chamber, the same medium was cultured with an increased FBS concentration of 20%. Approximately 2 x 10 hybridoma cells in 15 mL 6 The cells were seeded at a cell density of 1000 cells / mL. The chamber was filled with 1 L of nutrient medium (containing 4 mL L-glutamine and 2% standard FBS). The hybridoma cells in the cell chamber were then filled with DMEM containing 100% ethanol (DMEM). 2.5×10 7 The cells were grown to 100 cells / mL. Then, 10 mL of the cell suspension was added to the cell channel. The cells were collected from the hamster and replaced with fresh medium to allow for cell re-growth and subsequent recovery. Repeat this procedure as necessary to obtain adequate amounts of mAb from the hybridoma clone. The collected cell suspension was centrifuged, and the supernatant was filtered through a 0.2 micron filter membrane. For purification, Protein G Sepharose 4 Fast Flow (Protein GS epharose 4 Fast flow) 5 mL column (GE Healthcare The supernatant of each clone was purified by gravity flow using Tris-EDTA (T E) After washing with buffer (pH 8.0), the cells were separated into 0.1 M glycine buffer (pH 2. 7) to elute the bound antibody, and then pH 7.0 was added using 1 M Tris (pH 8.0). Neutralization was performed using a Centriprep YM-10kDa NMWL centrifugal filter. The antibody was concentrated using a centrifuge (Millipore) and the buffer was adjusted to phosphate-buffered saline. The solution was exchanged into water (PBS). The antibody concentration was quantified by spectrophotometry. Purified anti-dog I The canine IL-4 receptor alpha chain mAb was synthesized using the HIS-tagged ECD domain of the canine IL-4 receptor alpha chain. The antibodies were tested for reactivity to the HIS-tagged antibodies by ELISA as follows. Nucleotide IL-4 receptor alpha chain protein in coating buffer (carbonate / bicarbonate , pH 9.0) and plated in a 96-well flat-bottom ELISA plate (NU Dispense 100 μl / well into the plate (NC). Incubate the plate overnight at 4°C. The plates were then washed with phosphate buffered saline containing 0.05% Tween-20 (PBST). Wash three times with buffered saline. Then add 200 μl of blocking buffer (5% in PBST). Skim milk is added to each well and the plate is incubated at 30°C for 60 minutes. The plate is then washed three times with PBST. Add 100 μl of the test mAb to the first well of the appropriate row. The plate was incubated at 37°C for 60 minutes, after which the plates were diluted 3-fold. Wash plates three times with PBST. Then add 100 μl / well of a 1:2,000 dilution of Sladish peroxidase-conjugated goat anti-mouse IgG (KPL) was added to the plate. This is then incubated at 37°C for 60 minutes. The plate is then washed with PBST for 3 Wash the plates twice and add 100 μl / well of 3,3',5,5' tetramethylbenzidine (TMB ) Substrate (obtained from KPL) is added to the plate. The color reaction proceeds at 37°C for 5 to 20 minutes. After allowing to stand, the absorbance at 650 nm is measured.

[0189] Various mouse anti-canine IL-4R α Monoclonal antibodies (mAbs) were administered to canine IL-4R α The antibodies were assayed by ELISA for their ability to bind to the extracellular domain of . As shown in Figure 1, the majority of these mAbs exhibit positive dose-dependent binding.

[0190] Example 3 Heavy and light chain variable domains of anti-canine IL-4 receptor alpha chain monoclonal antibody Identification of DNA and predicted protein sequences mRNA was isolated from each hybridoma using standard molecular biology methods and then transferred to mouse embryos. The DNA sequences of the VH and VL chains from these hybridomas were identified. The DNA and deduced amino acid sequences of VL are shown below. The DNA and amino acids corresponding to the putative signal sequence are underlined, and the CDRs The corresponding parts are shown in bold, and the FRs are neither underlined nor bolded (i.e., S Signal array -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4). [ka] TIFF2025129151000014.tif212157TIFF2025129151000015.tif199156TIFF2025129151000016.tif205156TIFF2025129151 000017.tif216157TIFF2025129151000018.tif209157TIFF2025129151000019.tif211157TIFF2025129151000020.tif15152

[0191] Example 4 Construction of a CHO cell line expressing the canine IL-4 receptor alpha chain and its ligand-blocking assay Use in Sei Full-length canine IL-4 receptor alpha chain (cIL-4R α ;SEQ ID NO: 4) The gene was synthesized and subcloned into a mammalian expression vector. The resulting plasmid was HO DG44 cells. 48 hours after transfection, The cells were diluted into 96-well plates to obtain single cell clones. After 4 weeks of incubation, After screening, approximately 130 clones were obtained. All of the clones were screened for expression of cIL-4Rα by FACS using Three clones were selected for stability evaluation. Stability was monitored over time.

[0192] The binding of canine IL-4 to canine IL-4R alpha expressed on the surface of CHO cells was investigated. To evaluate the ability of monoclonal antibodies specific for IL-4 receptor alpha to block Next, a ligand blocking assay was set up as follows.

[0193] Reagents and equipment: Cell growth medium: CD OptiCHO medium + 8mM L-glutamine + 0 .018% F-68; FACS buffer: BD Pharmingen stain buffer (BD Catalog No. 554657); R-phycoerythin-conjugated streptavidin (Li fe Technologies:SB66); ·Canine IL-4 (R&D system, catalog number 754-CL / CF); Light staining was used to biotinylate canine IL-4 according to the manufacturer's recommendations. Ning-Link Biotin Binding Kit Type A (Novus: 704-0010) Flow cytometer: BD Accuri-C6.

[0194] procedure: 1. CHO-DH44-canIL-4Rα cells with a viability of 96% or more were cultured at 2-4 x 1 0 6 cells / mL.

[0195] 2. Centrifuge the cells, discard the supernatant, and collect 2 x 10 cells. 7 FAC up to cells / mL The cells were suspended in S buffer.

[0196] 3. The cells were dispensed into a U-shaped 96-well plate (50 μl per well).

[0197] 4. Immunoprecipitation of anti-canine IL-4Rα mAb in FACS buffer onto a 96-well plate Three-fold dilutions were made from 50 μg / mL from the top well to the bottom well.

[0198] 5. Transfer 50 μl of each diluted Ab into the cell plate and then incubate on ice for 30 minutes. I bet.

[0199] 6. The cells were washed twice with FACS buffer.

[0200] 7. Cells were incubated with 0.32 µg / mL biotinylated canine IL-4 in FACS buffer Resuspended in 100 μl and incubated on ice for 30 minutes.

[0201] 8. The cells were washed twice with FACS buffer.

[0202] 9. Isolate cells with R-phycoerythrin-conjugated streptavidin (100mg / L) in FACS buffer The cells were resuspended in 100 μl of 1:1000 dilution of PBS and incubated on ice for 30 minutes.

[0203] 10. Cells were washed twice with FACS buffer.

[0204] 11. Cells were loaded in 300 μl of FACS buffer.

[0205] 12. BD Accuri-C6 reads 10,000 cells per sample. I took it.

[0206] 13. The resulting readouts are analyzed by FlowJo to determine the mean fluorescence intensity. The MFI was obtained.

[0207] Canine IL-4R expressed on the surface of CHO cells α Binding of canine IL-4 to Dose-response curves were performed using cell-based CHO-cIL-4R α obtained using a binding assay (Figure 2A (See ). From this curve, a half-maximal effective concentration (EC50) of 25 nM was determined. Next, mouse anti-canine IL-4R α Monoclonal antibodies (mAb): 11B6, 4D8, CHO-cIL-4R with 4H3, 2E2, 11H2, and 6C12 α Regarding the bond Dose-response curves were obtained (see Figure 2B). Half-maximal effective doses for each of the antibodies were The concentrations (EC50) are shown in Table 2 below. [Table 2]

[0208] Then mouse anti-canine IL-4R α Monoclonal antibodies (mAbs) are used in cell-based CH O-cIL-4R α The antibodies were assayed for their ability to block the binding of canine IL-4 to As shown in Figure 3A, five mAbs, namely, 11B6, 4D8, and 4H3 In a supplemental study, a sixth mAb was tested. (6C12) and compared with one of the five mAbs tested (11H2) (Figure 3A). As can be seen from Figure 3B and Table 2, 6C12 mAb was significantly more potent than 11H2 mAb. 3A and 3B and Table 2. As can be seen, anti-cIL-4R α Monoclonal antibodies 4D8, 2E2, 4D8 and and 11H2 showed excellent blocking ability.

[0209] Example 5 Mouse CDR amino acid sequence [ka] TIFF2025129151000023.tif179154TIFF2025129151000024.tif123155 [Table 3] TIFF2025129151000026.tif40146

[0210] Example 6 Epitope mapping of mouse anti-canine IL-4 receptor alpha antibodies The interaction of an antibody with its cognate protein antigen is determined by the specific amino acid sequence of the antibody (paratoxin). It is mediated by the binding of a specific amino acid (epitope) to a specific amino acid (epitope) on the target antigen. An epitope is an antigenic determinant that elicits a specific response by immunoglobulins. It consists of a group of amino acids on the surface of an antigen. The protein of interest is recognized by various antibodies. The epitopes recognized by the antibodies may be linear epitopes. Linear epitopes are classified as protein-specific or conformational epitopes. It is formed by a continuous sequence of amino acids in a protein, while conformational A pitope may be discontinuous (e.g., widely separated) in the primary amino acid sequence, but It is composed of amino acids that come together during three-dimensional protein folding.

[0211] Epitope mapping involves identifying the amino acid sequence on the target antigen that is recognized by an antibody (i.e., This refers to a method for identifying a specific epitope (i.e., a monoclonal antibody (mAb)) on a target antigen. The identification of the epitope recognized by b) has important applications. For example, it may be used to identify novel therapeutics. Epitope mapping can also aid in the development of therapeutic agents, diagnostic agents, and vaccines. This will aid in the selection of therapeutic mAbs and help elucidate their mechanisms of action. Epitope information on body alpha reveals unique epitopes and enhances vaccine protection. Identification of epitopes may also reveal a pathogenic or pathogenic effect on the carrier protein or Chemical or genetic coupling of identified peptide epitopes to other immunostimulatory agents This could lead to the development of subunit vaccines based on

[0212] Epitope mapping can be performed using polyclonal or monoclonal antibodies depending on the predicted nature of the epitope (i.e., linear vs. conformational). Therefore, several methods are used to identify epitopes. For this purpose, linear epitaxy is more straightforward and relatively easier to implement. Commercial services for tope mapping often use peptide scanning. In this case, an overlapping set of short peptide sequences of the target protein is chemically synthesized and The antibodies are tested for their ability to bind to the antibody. This method is rapid and high-throughput. On the other hand, mapping of discontinuous epitopes is technically more difficult. This is difficult and requires more specialized techniques, such as the isolation of monoclonal antibodies against the corresponding target protein. X-ray cocrystallography, hydrogen-deuterium (H / D) exchange, mass spectrometry combined with enzymatic digestion, and Some other method known to those skilled in the art is required.

[0213] Mapping the canine IL-4 receptor alpha epitope using mass spectrometry : To identify the epitope recognized by the anti-canine IL-4 receptor alpha mAb, A method based on chemical cross-linking and mass spectrometry detection was used [CovalX Instrument nt Incorporated]. Epitope mapping of the canine IL-4 receptor alpha chain The application of this technology to immunohistochemistry is demonstrated by the epitopes recognized by the mAbs listed in Table 4. This resulted in the identification of the group.

[0214] Epitope markers of canine IL-4 receptor alpha using the six antibodies shown in Table 4 Results from mapping revealed that specific IL-4 receptors exist within the extracellular domain of the canine IL-4 receptor alpha. This indicates that the mAb recognizes a target peptide epitope. Two to three epitopes were identified for each of the six monoclonal antibodies (mAbs). Interestingly, one of the epitopes identified for mAb 2E2 was mAb 2E2. b) having the exact same amino acid sequence as that of 11B6 (i.e., SEQ ID NO: 158); As shown in Table 4 below, mAbs: 4D8, 11H2 and 11B All 6 recognize the epitope designated "1," which is part of the same linear amino acid sequence. , mAbs: 11H2, 4H3 and 2E2 all contain a portion of another linear amino acid sequence. mAb: 4H3 and 2H2 all recognize the epitope labeled "2" It recognizes the epitope designated "3", which is part of the linear amino acid sequence of 3. This relative agreement in identifying the target pitopes indicates that these six monoclonal antibodies are It recognizes a limited number of regions within the extracellular domain of the IL-4 receptor alpha. It shows. [Table 4]

[0215] In conjunction with the CDRs described in Example 5 for the six antibodies listed in Table 4 above, In this sense, there is a one-to-one correspondence between each set of CDRs and their corresponding epitopes in Table 4. A relationship is established. This relationship is shown in Example 5 for each of the six antibodies in Table 4. The relationship between the set of six CDRs in each gene and the corresponding epitopes to which they bind is determined. Thus, the antibodies described in Example 5 that bind to the corresponding epitopes in Table 4 can be used. Antibodies (e.g., caninized antibodies) having a defined set of six CDRs are also part of the invention. is.

[0216] Example 7 Construction of caninized anti-canine IL-4 receptor alpha monoclonal antibody To perform the caninization procedure, DNA sequences encoding the heavy and light chains of canine IgG were prepared. The DNA and protein sequences of the canine heavy and light chains are known in the art. and can be obtained by searching the NCBI gene and protein databases. Regarding canine antibodies, there are four known IgG subtypes: IgG-A, IgG-B, IgG-C, IgG-D, IgG-E, IgG-F, IgG-G, IgG-H, IgG-I ... -B, IgG-C and IgG-D, and two types of light chains, namely, kappa and Without being limited to any particular approach, Canine IL-4 receptor alpha antibodies can be mixed in various combinations to obtain a canine IL-4 receptor alpha mAb. The overall process for producing heavy and light chains involves the following scheme.

[0217] i) VH and VL domains containing the CDRs of the desired anti-IL-4 receptor alpha mAb Identify the DNA sequence of

[0218] ii) Identifying the heavy and light chain CDRs of the desired anti-IL-4 receptor mAb.

[0219] iii) Identify suitable sequences for the heavy and light chains of canine IgG.

[0220] iv) DNA sequences encoding the endogenous CDRs of the canine IgG H and L chains of said sequences. Identify.

[0221] v) DNA sequences encoding the endogenous canine H and L chain CDRs are transfected into the desired anti-IL-4 receptor. The alpha CDR is replaced with a DNA sequence encoding the receptor alpha CDR. The framework residues are selected from the desired anti-IL-4 receptor mAb framework region. The amino acid sequence may be substituted with a residue that has been modified.

[0222] vi) synthesizing the DNA from step (v) and cloning it into a suitable expression plasmid; The plasmids containing the desired caninized heavy and light chains were transfected into HEK293 cells. Infect.

[0223] vii) Purify expressed caninized antibodies from HEK293 supernatants.

[0224] viii) Testing the purified caninized antibody for binding to the canine IL-4 receptor alpha chain Test.

[0225] The application of the process broadly described above is illustrated in Table 5 below, where SEQ ID NOs are listed. This gave a set of caninized heavy and light chain sequences. [Table 5]

[0226] The present invention provides caninized heavy and light chains formed by combining various caninized heavy and light chains listed in Table 5 above. and caninized antibodies that bind to canine IL-4 receptor alpha. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 164. and the light chain comprises the amino acid sequence of SEQ ID NO: 170. wherein the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 163 and the light chain is encoded by the sequence In another embodiment, the gene is encoded by the nucleotide sequence of The heavy chain comprises the amino acid sequence of SEQ ID NO: 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 172. In more particular embodiments of this type, the heavy chain comprises the nucleotide sequence of SEQ ID NO: 165. The light chain is encoded by the nucleotide sequence of SEQ ID NO: 171. In yet another embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 168. and the light chain comprises the amino acid sequence of SEQ ID NO: 174. wherein the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 167 and the light chain is encoded by the sequence The IL-4 receptors of these canine antibodies are encoded by the nucleotide sequence of nucleotide no. 173. Binding studies to receptor alpha are described infra 8 and shown in FIG.

[0227] As described above, the caninized antibodies are engineered to eliminate ADCC and CDC effector functions. The Fc portions of these antibodies are based on modified sequences of canine IgG-B. and / or can be combined with a surrogate hinge region, as described above, U.S. Provisional Patent Application No. 62 / 030,812, filed July 30, 2014, filed September 3, 2014 U.S. Provisional Patent Application No. 62 / 057,541, filed December 16, 2014 U.S. Provisional Patent Application No. 62 / 092,496 filed June 8, 2015 Patent application No. 62 / 172,511 and WO 2015 / 091910 (all of which (the entire contents of which are incorporated herein by reference). [ka] TIFF2025129151000030.tif214151TIFF2025129151000031.tif213152TIFF2025129151000032.tif43152

[0228] Example 8 Reactivity of caninized antibodies against canine IL-4 receptor alpha The caninized antibody was tested for reactivity to canine IL-4 receptor alpha as follows. I tried it.

[0229] 1. Coat IL-4 receptor alpha at 200 ng / well on an immunoplate. The plate is incubated at 4°C overnight.

[0230] 2. The plate is filled with phosphate buffered saline (PBS) containing 0.05% Tween 20. Wash three times with PBST.

[0231] 3. The plate is incubated with 0.5% bovine serum albumin (BSA) in PBS for 45 minutes at room temperature. Block for ~60 minutes.

[0232] 4. Wash the plate 3 times with PBST.

[0233] 5. Dilute the caninized antibody from 0.3 μg / mL to 3x in each row or column of the dilution plate. Dilute.

[0234] 6. Transfer the diluted caninized antibodies into each row or column of the immunoplate and allow the plate to stand at room temperature. Incubate at RT for 45-60 min.

[0235] 7. Wash the plate 3 times with PBST.

[0236] 8. Add 1:4000 diluted horseradish peroxidase to each well of the plate. Add enzyme-labeled anti-dog IgG Fc and incubate the plate at room temperature for 45 to 60 minutes. do.

[0237] 9. Wash the plate 3 times with PBST.

[0238] 10. Add 3,3',5,5'-tetramethylbenzidine (T) to each well of the plate. MB) substrate is added and the plate is incubated at room temperature for 10-15 minutes to allow color development.

[0239] 11. Add 100 μL of 1.5 M phosphoric acid to each well to stop the reaction. Plates are read at 450 nm with a reference wavelength of 0 nm.

[0240] As shown in Figure 4, the binding of the following five antibodies to IL-4 receptor alpha was investigated. Beta: 4H3 MC, c4H3 H1-L1, c4H3 H2-L2, c4H3 H3 -L3 and 2G9 MC. 2G9 MC was used as a negative control antibody. 4H3 MC is the murine variable heavy chain region and canine constant region of the 4H3 antibody disclosed herein; and a chimeric antibody consisting of the light chain from the mouse 4H3 antibody. c4H3 H2-L2, c4H3 H3-L3 are three canine versions of the mouse 4H3 antibody. 2G9 is a variant of 2G9, which contains specific heavy and light chains as shown in Table 5 above. MC is a murine variable region of a murine antibody against an antigen completely unrelated to IL-4 receptor alpha. from the heavy chain and canine constant region, and the light chain from a mouse antibody against an unrelated antigen Consistently, 2G9 MC does not bind to IL-4 receptor alpha. However, the remaining four antibodies tested, i.e., 4H3 MC, c4H3 H1-L1 , c4H3 H2-L2, and c4H3 H3-L3 all bound relatively tightly (Fig. See 4).

Claims

1. Three light chain complementarity determining regions (CDRs): CDR light chain 1 (CDRL1), CD CDR light chain 2 (CDRL2) and CDR light chain 3 (CDRL3), and three heavy chain CDRs , i.e., CDR heavy chain 1 (CDRH1), CDR heavy chain 2 (CDRH2) and CDR heavy chain Canine interleukin-4 receptor alpha (IL-4R) containing chain 3 (CDRH3) α ) specifically 1. An isolated mammalian antibody or antigen-binding fragment thereof that binds to: (a) CDRL1 is SEQ ID NO: 47, SEQ ID NO: 54, SEQ ID NO: 48, SEQ ID NO: 52, Sequence number 55, sequence number 49, sequence number 50, sequence number 51, sequence number 53, sequence number 12 9, SEQ ID NO: 130, SEQ ID NO: 131; SEQ ID NO: 47, SEQ ID NO: 54, SEQ ID NO: 48, Sequence number 52, sequence number 55, sequence number 49, sequence number 50, sequence number 51, sequence number 53 , a conservatively modified variant of SEQ ID NO: 129, SEQ ID NO: 130 or SEQ ID NO: 131; Variants of SEQ ID NO: 47 or SEQ ID NO: 54, including the structural class; Variants of SEQ ID NO: 48, SEQ ID NO: 52 or SEQ ID NO: 55, including 4 canonical structural classes; Variants of SEQ ID NO: 49; SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 52 containing the three canonical structural classes SEQ ID NO: 53, SEQ ID NO: 129, which contain 6 canonical structural classes; or a variant of SEQ ID NO: 130; (b) CDRL2 is SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, Sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 13 2, SEQ ID NO: 133, SEQ ID NO: 134; SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, Sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64 , conservatively modified variants of SEQ ID NO: 132, SEQ ID NO: 133 or SEQ ID NO: 134; and Sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133 or The amino acid sequence is selected from the group consisting of variants of SEQ ID NO: 134, wherein the variant The body contains one standard structural class; (c) CDRL3 is selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, Sequence number 69, sequence number 70, sequence number 71, sequence number 72, sequence number 73, sequence number 13 5, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139; SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137 , a conservatively modified variant of SEQ ID NO: 138 or SEQ ID NO: 139; SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 7 0, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, Variants of sequence number 137 or sequence number 139; and sequence numbers containing the three canonical structural classes 138 variants; (d) CDRH1 is selected from SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, Sequence number 78, sequence number 79, sequence number 80, sequence number 81, sequence number 82, sequence number 14 0, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143; SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 8 1, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 or SEQ ID NO: 1 Conservatively modified variants of SEQ ID NO: 43; and SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO: The group consisting of variants of SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 or SEQ ID NO: 143 wherein the variant comprises one canonical structural class; (e) CDRH2 is selected from SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 91, SEQ ID NO: 85, Sequence number 88, sequence number 89, sequence number 87, sequence number 90, sequence number 144, sequence number 1 45, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148; SEQ ID NO:83, SEQ ID NO:84 , SEQ ID NO:91, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:87, SEQ ID NO: 90, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 1 48 conservatively modified variants; SEQ ID NO: 83, SEQ ID NO: 144, containing the 3A canonical structural class; Variants of SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NO: 148; canonical structural class of 2A variants of SEQ ID NO: 84, SEQ ID NO: 91 or SEQ ID NO: 145, including the canonical structure class 2B; variants of SEQ ID NO:85, SEQ ID NO:88 or SEQ ID NO:89 containing the canonical structure of SEQ ID NO:1; an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 87 or SEQ ID NO: 90, including the Contains an acid sequence; (f) CDRH3 is SEQ ID NO: 92; conservatively modified variants of SEQ ID NO: 92; 12 standard structures Variants of SEQ ID NO:92, including structural classes; SEQ ID NO:93; Conservatively modified variants of SEQ ID NO:93 ; variants of SEQ ID NO:93 containing 7 canonical structural classes; SEQ ID NO:94, SEQ ID NO:96, sequences SEQ ID NO: 149, SEQ ID NO: 151; SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 149 or sequence Conservatively modified variants of SEQ ID NO: 151; SEQ ID NO: 94, SEQ ID NO: 9, containing 15 canonical structural classes 6. Variants of SEQ ID NO: 149 or SEQ ID NO: 151; SEQ ID NO: 95; Conservation of SEQ ID NO: 95 or variants of SEQ ID NO:95 containing 11 canonical structural classes; SEQ ID NO:97 , SEQ ID NO: 100; SEQ ID NO: 97 or conservatively modified variants of SEQ ID NO: 100; 6 standard structures Variants of SEQ ID NO:97 or SEQ ID NO:100, including structural classes; SEQ ID NO:98; SEQ ID NO:9 8 conservatively modified variants; variants of SEQ ID NO:98 containing 4 canonical structural classes; SEQ ID NO:99 , SEQ ID NO: 153; conservatively modified variants of SEQ ID NO: 99 or SEQ ID NO: 153; 13 standards Variants of SEQ ID NO: 99 or SEQ ID NO: 153, including structural classes: SEQ ID NO: 150; Conservatively modified variants of SEQ ID NO: 150; variants of SEQ ID NO: 150 containing 10 canonical structural classes; SEQ ID NO: 152; and conservatively modified variants of SEQ ID NO: 152; sequences containing 9 canonical structural classes comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 152; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to An isolated mammalian antibody or antigen-binding fragment thereof.

2. (a) CDRL1 is SEQ ID NO: 54, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 13 1; conservative modifications of SEQ ID NO:54, SEQ ID NO:129, SEQ ID NO:130 or SEQ ID NO:131 Variants of SEQ ID NO: 54 containing one canonical structural class; variants of SEQ ID NO: 54 containing three canonical structural classes variants of sequence number 131; and SEQ ID NO: 129 or SEQ ID NO: 132, which contain 6 canonical structural classes 130 variants; (b) CDRL2 is SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 132, SEQ ID NO: 133 , SEQ ID NO: 134; SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 132, SEQ ID NO: 133 or is a conservatively modified variant of SEQ ID NO: 134; and SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 1 32, an amino acid selected from the group consisting of SEQ ID NO: 133 or a variant of SEQ ID NO: 134 sequences, wherein the variants comprise one class of canonical structural classes; (c) CDRL3 is SEQ ID NO: 72, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 13 7, SEQ ID NO: 138, SEQ ID NO: 139; SEQ ID NO: 72, SEQ ID NO: 135, SEQ ID NO: 136 , a conservatively modified variant of SEQ ID NO: 137, SEQ ID NO: 138 or SEQ ID NO: 139; SEQ ID NO: 72, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137 or variants of SEQ ID NO: 139; and variants of SEQ ID NO: 138 containing the three canonical structural classes comprising an amino acid sequence selected from the group consisting of: (d) CDRH1 is SEQ ID NO: 81, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 14 2, SEQ ID NO: 143; SEQ ID NO: 81, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 or conservatively modified variants of SEQ ID NO: 143; and SEQ ID NO: 81, SEQ ID NO: 140, SEQ ID NO: SEQ ID NO: 141, SEQ ID NO: 142 or a variant of SEQ ID NO: 143 amino acid sequence, wherein the variant comprises one of the canonical structural classes; (e) CDRH2 is SEQ ID NO: 90, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 14 6, SEQ ID NO: 147, SEQ ID NO: 148; SEQ ID NO: 90, SEQ ID NO: 144, SEQ ID NO: 145 , SEQ ID NO: 146, SEQ ID NO: 147, conservatively modified variants of SEQ ID NO: 148; SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NO: 148 containing the structural class variants of SEQ ID NO: 145 containing the canonical structural class of 2A; and variants of SEQ ID NO: 145 containing the canonical structural class of 1A; comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 90 containing ras; (f) CDRH3 is SEQ ID NO: 149, SEQ ID NO: 151; SEQ ID NO: 149 or SEQ ID NO: SEQ ID NO: 149 or SEQ ID NO: 151 containing 15 canonical structural classes SEQ ID NO: 99, SEQ ID NO: 153; SEQ ID NO: 99 or SEQ ID NO: 153 conservatively modified variants of SEQ ID NO: 99 or SEQ ID NO: 153, which contain 13 canonical structural classes; Variants; SEQ ID NO: 150; Conservatively modified variants of SEQ ID NO: 150; 10 canonical structural classes Variants of SEQ ID NO: 150 including: SEQ ID NO: 152; conservatively modified variants of SEQ ID NO: 152; and and variants of SEQ ID NO: 152 comprising 9 canonical structural classes. Contains an acid sequence; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to dogs Canine IL-4R to interleukin 4 α The isolated compound of claim 1, which blocks the binding of A mammalian antibody or antigen-binding fragment.

3. (a) CDRL1 is SEQ ID NO: 129, conservatively modified variants of SEQ ID NO: 129, and 6 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 129 comprising the canonical structural class Includes; (b) CDRL2 is SEQ ID NO: 132, a conservatively modified variant of SEQ ID NO: 132, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 132 comprising the canonical structural class Includes; (c) CDRL3 is selected from the group consisting of SEQ ID NO: 135, conservatively modified variants of SEQ ID NO: 135, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 135 comprising the canonical structural class Includes; (d) CDRH1 is selected from the group consisting of SEQ ID NO: 140, conservatively modified variants of SEQ ID NO: 140, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 140 comprising the canonical structural class Includes; (e) CDRH2 is selected from the group consisting of SEQ ID NO: 144, conservatively modified variants of SEQ ID NO: 144, and 3 A variant of SEQ ID NO: 144 that contains the canonical structural class of Contains columns; (f) CDRH3 is selected from the group consisting of SEQ ID NO: 149, conservatively modified variants of SEQ ID NO: 149, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 149 comprising the five canonical structural classes; Contains columns; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to dogs Canine IL-4R to interleukin 4 α The isolated compound of claim 2, which blocks the binding of A mammalian antibody or antigen-binding fragment.

4. (a) CDRL1 is SEQ ID NO: 130, conservatively modified variants of SEQ ID NO: 130, and 6 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 130 comprising the canonical structural class Includes; (b) CDRL2 is SEQ ID NO: 133, a conservatively modified variant of SEQ ID NO: 133, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 133 comprising the canonical structural class Includes; (c) CDRL3 is SEQ ID NO: 136, a conservatively modified variant of SEQ ID NO: 136, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 136 comprising the canonical structural class Includes; (d) CDRH1 is selected from the group consisting of SEQ ID NO: 141, conservatively modified variants of SEQ ID NO: 141, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 141 comprising the canonical structural class Includes; (e) CDRH2 is selected from the group consisting of SEQ ID NO: 145, conservatively modified variants of SEQ ID NO: 145, and 2 A variant of SEQ ID NO: 145 that contains the canonical structural class of Contains columns; (f) CDRH3 is selected from the group consisting of SEQ ID NO: 150, conservatively modified variants of SEQ ID NO: 150, and 1 150, wherein the amino acid sequence is selected from the group consisting of variants of SEQ ID NO: 150 comprising canonical structural classes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, Contains columns; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to dogs Canine IL-4R to interleukin 4 α The isolated compound of claim 2, which blocks the binding of A mammalian antibody or antigen-binding fragment.

5. (a) CDRL1 is SEQ ID NO: 129, conservatively modified variants of SEQ ID NO: 129, and 6 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 129 comprising the canonical structural class Includes; (b) CDRL2 is SEQ ID NO: 134, a conservatively modified variant of SEQ ID NO: 134, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 134 comprising the canonical structural class Includes; (c) CDRL3 is selected from the group consisting of SEQ ID NO: 137, conservatively modified variants of SEQ ID NO: 137, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 137 comprising the canonical structural class Includes; (d) CDRH1 is selected from the group consisting of SEQ ID NO: 140, conservatively modified variants of SEQ ID NO: 140, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 140 comprising the canonical structural class Includes; (e) CDRH2 is selected from the group consisting of SEQ ID NO: 146, conservatively modified variants of SEQ ID NO: 146, and 3 A variant of SEQ ID NO: 146 that contains the canonical structural class of Contains columns; (f) CDRH3 is selected from the group consisting of SEQ ID NO: 151, conservatively modified variants of SEQ ID NO: 151, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 151 comprising the five canonical structural classes; Contains columns; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to dogs Canine IL-4R to interleukin 4 α The isolated compound of claim 2, which blocks the binding of A mammalian antibody or antigen-binding fragment.

6. (a) CDRL1 is SEQ ID NO: 131, conservatively modified variants of SEQ ID NO: 131, and 3 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 131 comprising the canonical structural class Includes; (b) CDRL2 is selected from the group consisting of SEQ ID NO: 60, conservatively modified variants of SEQ ID NO: 60, and the target of 1 comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 60, including parastructural classes ； (c) CDRL3 is SEQ ID NO: 138, a conservatively modified variant of SEQ ID NO: 138, and 3 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 138 comprising the canonical structural class Includes; (d) CDRH1 is selected from the group consisting of SEQ ID NO: 142, conservatively modified variants of SEQ ID NO: 142, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 142 comprising the canonical structural class Includes; (e) CDRH2 is selected from the group consisting of SEQ ID NO: 147, conservatively modified variants of SEQ ID NO: 147, and 3 A variant of SEQ ID NO: 147 that contains the canonical structural class of Contains columns; (f) CDRH3 is selected from the group consisting of SEQ ID NO: 152, conservatively modified variants of SEQ ID NO: 152, and 9 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 152 comprising the canonical structural class Includes; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to dogs Canine IL-4R to interleukin 4 α The isolated compound of claim 2, which blocks the binding of A mammalian antibody or antigen-binding fragment.

7. (a) CDRL1 is SEQ ID NO: 129, conservatively modified variants of SEQ ID NO: 129, and 6 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 129 comprising the canonical structural class Includes; (b) CDRL2 is SEQ ID NO: 132, a conservatively modified variant of SEQ ID NO: 132, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 132 comprising the canonical structural class Includes; (c) CDRL3 is selected from the group consisting of SEQ ID NO: 139, conservatively modified variants of SEQ ID NO: 139, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 139 comprising the canonical structural class Includes; (d) CDRH1 is selected from the group consisting of SEQ ID NO: 143, conservatively modified variants of SEQ ID NO: 143, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 143 comprising the canonical structural class Includes; (e) CDRH2 is selected from the group consisting of SEQ ID NO: 148, conservatively modified variants of SEQ ID NO: 148, and 3 A variant of SEQ ID NO: 148 that contains the canonical structural class of Contains columns; (f) CDRH3 is selected from the group consisting of SEQ ID NO: 153, conservatively modified variants of SEQ ID NO: 153, and 1 an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 153, including the three canonical structural classes; Contains columns; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to dogs Canine IL-4R to interleukin 4 α The isolated compound of claim 2, which blocks the binding of A mammalian antibody or antigen-binding fragment.

8. (a) CDRL1 is SEQ ID NO: 54, conservatively modified variants of SEQ ID NO: 54, and the target of 1 comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 54, including parastructural classes ； (b) CDRL2 is SEQ ID NO: 63, conservatively modified variants of SEQ ID NO: 63, and the target of 1 comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 63, including parastructural classes ； (c) CDRL3 is selected from the group consisting of SEQ ID NO: 72, conservatively modified variants of SEQ ID NO: 72, and the target of 1 comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 72, including the parastructural class ； (d) CDRH1 is selected from the group consisting of SEQ ID NO: 81, conservatively modified variants of SEQ ID NO: 81, and the target of 1 comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 81, including the parastructural class ； (e) CDRH2 is selected from the group consisting of SEQ ID NO: 90, conservatively modified variants of SEQ ID NO: 90, and the target of 1 comprising an amino acid sequence selected from the group consisting of variants of SEQ ID NO: 90, including the parastructural class ； (f) CDRH3 is selected from the group consisting of SEQ ID NO: 99, conservatively modified variants of SEQ ID NO: 99, and 13 Variants of SEQ ID NO: 99 comprising the canonical structural class. nothing; wherein the antibody and antigen-binding fragment thereof are canine IL-4R α Binds to dogs Canine IL-4R to interleukin 4 α The isolated compound of claim 2, which blocks the binding of A mammalian antibody or antigen-binding fragment.

9. The monoclonal antibody of claim 1, 2, 3, 4, 5, 6, 7 or 8, wherein the mammalian antibody is a mouse antibody. An isolated mammalian antibody or antigen-binding fragment thereof.

10. A caninized antibody or a caninized antigen-binding fragment thereof according to claims 1, 2, 3, or 4.

9. An isolated mammalian antibody or antigen-binding fragment thereof according to 5, 6, 7 or 8.

11. From the group consisting of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104 The caninized antibody of claim 10, comprising a hinge region comprising an amino acid sequence selected from the group consisting of: A caninized antigen-binding fragment thereof.

12. The isolated antibody of claim 6, which is a caninized antibody or a caninized antigen-binding fragment thereof. A mammalian antibody or antigen-binding fragment thereof.

13. an amino acid sequence selected from the group consisting of SEQ ID NO: 164, SEQ ID NO: 166 and SEQ ID NO: 168; a heavy chain comprising the amino acid sequence of SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174; or a combination of said heavy chain and said light chain, Item 13. The caninized antibody or antigen-binding fragment thereof according to Item 12.

14. Nucleic acids selected from the group consisting of SEQ ID NO: 163, SEQ ID NO: 165 and SEQ ID NO: 167 The heavy chain encoded by the peptide sequence, SEQ ID NO: 169, SEQ ID NO: 171 and SEQ ID NO: 173, or the light chain encoded by a nucleotide sequence selected from the group consisting of: The caninized antibody or its antigen-binding fragment according to claim 13, comprising a combination of a nucleotide sequence and a light chain. Ment.

15. Canine interleukin-4 receptor α (IL-4R α ) specifically binds to Antibodies or antigen-binding fragments thereof, which are directed against canine IL-4R α When binding to The antibodies are SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 1 54, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 1 59, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162 or any combination thereof and wherein the antibody or its The antigen-binding fragment of canine IL-4R α binds to canine interleukin-4 NuIL-4R α An isolated mammalian antibody or antigen-binding fragment thereof that blocks binding of nt.

16. Canine IL-4R α When the mammalian antibody or antigen-binding fragment thereof binds to or the caninized antibody or antigen-binding fragment thereof is SEQ ID NO: 125, SEQ ID NO: 126 , SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156 , SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161 , SEQ ID NO: 162, or any combination thereof and wherein the antibody or antigen-binding fragment thereof binds to a canine IL- 4th Race α binds to canine IL-4R and binds to canine interleukin-4 α Blocking the connection, claim Item 1, 2, 3, 4, 5, 6, 7 or 8, an isolated mammalian antibody or an antigen-binding or a caninized antibody or fragment thereof according to claim 10, 11, 12, 13 or 14. or an antigen-binding fragment thereof.

17. Canine IL-4R α When the mammalian antibody or antigen-binding fragment thereof binds to or the caninized antibody or antigen-binding fragment thereof is SEQ ID NO: 154, SEQ ID NO: 155 , SEQ ID NO: 156, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161 and any of them At least one amino acid residue in the amino acid sequence selected from the group consisting of any combination thereof and wherein the antibody or antigen-binding fragment thereof is linked to a canine IL-4R α In conclusion Combined, canine IL-4R binds to canine interleukin 4 α 6 or 13. The isolated mammalian antibody or antigen-binding fragment thereof of claim 7, or claim 12.

15. A caninized antibody or antigen-binding fragment thereof according to any one of claims 13 to 14.

18. the mammalian antibody or its antigen-binding fragment, or the caninized antibody or its antigen The binding fragment has the following properties: (i) 1 x 10 －５ M~1 x 10 －１２ The dissociation constant (Kd) of canine IL-4R α In conclusion To unite; (ii) 1 × 10 ２ M －１ s －１ ~1 x 10 ７ M －１ s －１ On speed (k ｏｎ ) NuIL-4R α to bind to; (iii) 1 × 10 －３ s －１ ~1 x 10 －８ s －１ Off-rate (k ｏｆｆ ) and Dog I L-4R α to bind to; (iv) blocking the binding of IL-4 to the type I IL-4 receptor; (v) blocking the binding of IL-13 to the type I IL-4 receptor; (vi) blocking the binding of IL-4 to the type II IL-4 receptor; and (vii) blocking the binding of IL-13 to the type II IL-4 receptor Claims 1, 2, 3, 4, 5, 6 or all of 18. The mammalian antibody or antigen-binding fragment thereof according to any one of claims 6, 7, 8, 15, 16, or 17. or the caninized antibody of claim 10, 11, 12, 13, 14, 16 or 17. or an antigen-binding fragment thereof.

19. Canine IL-4R α With respect to binding to claims 1, 2, 3, 4, 5, 6, 7, 8, 15, 19. One or more mammalian antibodies or antigen-binding fragments thereof according to 16, 17 or 18. or the caninized antibody according to claim 10, 11, 12, 13, 14, 16, 17 or 18. a caninized monoclonal antibody or its antigen that cross-competes with the antigen-binding fragment thereof A canine IL-4R-binding fragment α binds to canine IL-4 4th Race α A caninized monoclonal antibody or antigen-binding fragment thereof that blocks binding of

20. 19. The mammalian antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 15, 16, 17 or 18.

10. The murine antibody or its antigen-binding fragment according to claim 9. a fragment thereof, or a fragment of claim 10, 11, 12, 13, 14, 16, 17, 18 or 20. An isolated antibody encoding the light chain of the caninized antibody or antigen-binding fragment thereof according to 19. Nucleic acid.

21. 19. The mammalian antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 15, 16, 17 or 18.

10. The murine antibody or its antigen-binding fragment according to claim 9. a fragment thereof, or a fragment of claim 10, 11, 12, 13, 14, 16, 17, 18 or 19. An isolated antibody encoding the heavy chain of the caninized antibody or antigen-binding fragment thereof according to 19. Nucleic acid.

22. SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 、５９、６０、６１、６２、６３、６４、６５、６６、６７、６８、６９、７０、７１、 ７２、７３、７４、７５、７６、７７、７８、７９、８０、８１、８２、８３、８４、８ ５、８７、８８、８９、９０、９１、９２、９３、９４、９５、９６、９７、９８、９９ 、１００、１２９、１３０、１３１、１３２、１３３、１３４、１３５、１３６、１３７ 、１３８、１３９、１４０、１４１、１４２、１４３、１４０、１４１、１４２、１４３ , 144, 145, 146, 147, 148, 149, 150, 151, 152 and 1 53.

2. The isolated nucleic acid according to claim 1.

23. 23. An expression vector comprising the isolated nucleic acid of claim 20, 21 or 22.

24. A host cell comprising the expression vector of claim 23.

25. SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 154 , SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159 , SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162 95% identical to the sequence of claims 1, 2, 3, 4, 5, 6, 7, 8, 15, 16, 19. An isolated mammalian antibody or binding fragment thereof according to claim 17 or 18, or Item 10, 11, 12, 13, 14, 16, 17, 18 or 19, or An isolated peptide comprising 5 to 25 amino acid residues that binds to the antigen-binding fragment. Petite.

26. A fusion protein comprising the peptide of claim 25.

27. 27. The fusion protein of claim 26, further comprising an Fc region of a non-canine mammalian IgG.

28. 26. An isolated peptide according to claim 25, a fusion protein according to claim 26 or a 28. An isolated nucleic acid encoding a fusion protein according to 27.

29. 29. An expression vector comprising the isolated nucleic acid of claim 28.

30. 30. A host cell comprising the expression vector of claim 29.

31. The caninized antibody of claim 10, 11, 12, 13, 14, 16, 17, 18, or 19. or the nucleic acid of claim 20, 21, 22 or 28, or the nucleic acid of claim 23 or 29 An expression vector, an isolated peptide according to claim 25, a fusion according to claim 26 or 27. proteins, or any combination thereof, and a pharmaceutically acceptable carrier or diluent. and a pharmaceutical composition comprising:

32. A therapeutically effective amount of the pharmaceutical composition of claim 31 is administered to a subject in need of reduced immune cell activity. A method for reducing the activity of immune cells, comprising administering

33. (i) treating atopic dermatitis, or (ii) the treatment of asthma, or (iii) Treatment of atopic dermatitis and treatment of asthma 33. The method of claim 32, wherein

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