Homodimeric fusion proteins for treating atopic dermatitis - Patents.com

Abstract

The present invention provides a composition of a homodimer of canine interleukin-4 receptor alpha fusion protein and canine interleukin-13 receptor alpha 2 fusion protein for treating canine atopic dermatitis. The composition may further comprise a caninized antibody against canine IL-31 or a caninized antibody against canine IL-31Rα.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The filename of the XML file generated on July 13, 2022 will be "25269 SEQ Listing.xml". This Sequence Listing, submitted via EFS-Web, is a part of the present specification and is hereby incorporated by reference in its entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 235,259 (filed August 20, 2021) and U.S. Patent Application No. 63 / 235,261 (filed August 20, 2021).

[0003] FIELD OF THEINVENTION The present invention relates to a composition for treating atopic dermatitis in dogs, comprising a fusion protein that binds to canine interleukin-4 or canine interleukin-13. The composition can be used to treat canine atopic dermatitis. [Background technology]

[0004] The immune system comprises a network of resident and recirculating specialized cells that work in concert to protect the host against infections and cancer. The ability of the immune system to perform this function depends largely on the biological activity of a group of proteins secreted by white blood cells and collectively called interleukins. Among the well-studied interleukins, there are three important molecules identified as interleukin-4 (IL-4), interleukin-13 (IL-13), and interleukin-31 (IL-31). IL-4 and IL-13 are key cytokines in related signaling pathways involved in the generation of immune responses required for defense against certain pathogens (e.g., parasites present in tissues or lumina). However, these two cytokines, together with IL-31, are also involved in the pathogenesis of allergic diseases in humans and animals, including atopic dermatitis.

[0005] Atopic dermatitis (AD) is a recurrent, pruritic and chronic inflammatory skin disease characterized by immune system dysregulation and epidermal barrier abnormalities in humans. The pathological and immunological attributes of atopic dermatitis have been the subject of extensive investigation [reviewed in Rahman et al. Inflammation & Allergy-drug target 10:486-496(2011)and Harskamp et al.,Seminar in Cutaneous Medicine and Surgery 32:132-139(2013)]. Atopic dermatitis is also a common condition in companion animals, especially dogs, with its prevalence estimated to be approximately 10-15% of the canine population. The pathogenesis of atopic dermatitis in dogs and cats [reviewed in Nuttall et al., Veterinary Records 172(8):201-207 (2013)] shows significant similarities to the pathogenesis of atopic dermatitis in humans, including skin infiltration by various immune cells and CD4+ receptors, including a predominance of IL-4, IL-13, and IL-31. + Contains a Th2 polarizing cytokine environment.

[0006] IL-4 and IL-13 are closely related proteins that can be secreted by many cell types, including CD4+Th2 cells, natural killer T cells (NKT), macrophages, mast cells and basophils. IL-4 and IL-13 exhibit many overlapping functions and are important in the development of T cell-dependent humoral immune responses. Both IL-4 and IL-13 are part of signaling pathways involved in atopic dermatitis. IL-4 binds to a heterodimeric receptor that contains a monomer of the common γc chain (γc) and a monomer of the IL-4 receptor alpha (IL-4Rα), respectively, and IL-13 binds to a heterodimeric receptor that contains a monomer of the IL-13 receptor alpha1 (IL13Rα1) and a monomer of IL-4Rα, respectively.

[0007] Therefore, Th2 cytokines IL-4, IL-13 and IL-31 have been the subject of therapeutic intervention to develop better treatment.Medicines that have been proven to assist in the treatment of atopic dermatitis and / or have been proven to be promising include Janus kinase (JAK) inhibitors (see, for example, U.S. Patent No. 8,133,899; U.S. Patent No. 8,987,283; International Publication No. 2018 / 108969; U.S. Patent Application Publication No. 2020 / 0339585), spleen tyrosine kinase (SYK) inhibitors (see, for example, U.S. Patent No. 8,759,366), and antagonists against chemoattractant receptor homologous molecules expressed on TH2 cells (see, for example, U.S. Patent No. 7,696,222, U.S. Patent No. 8,546,422, U.S. Patent No. 8,637,541, and U.S. Patent No. 8,546,422). Furthermore, US Patent Publication No. 2020 / 0048325 discloses adjacent IL-13 / IL-4 receptor fusion proteins. The design of these fusion proteins brings together IL-13Rα1 and IL-4Rα in an adjacent arrangement in which IL-13Rα1 is linked to IL-4Rα by a non-self amino acid sequence called a linker, and the adjacent receptor can also be linked to a fusion partner with a second non-self amino acid linker. Notably, the linker used also has the potential to undergo post-translational modifications, such as glycosylation.

[0008] The therapeutic use of monoclonal antibodies to block signaling in specific pathways by binding to either a protein ligand or its protein receptor has proven to be widely successful. Indeed, such monoclonal antibodies have played a key role in the rapid growth of human biologics, claiming over 25% of the human biologics market as of 2017. Of the 20 highest-selling drugs in 2014, six were monoclonal antibodies [Chung, Experimental & Molecular Medicine 49:e304;doi:10.1038 / emm.2017.46(2017)]. This trend continues to grow. Monoclonal antibodies raised against the human IL-4 receptor alpha (IL-4Rα) have been developed, and several of these antibodies have been extensively tested for their therapeutic efficacy to treat atopic dermatitis in humans [see, e.g., U.S. Patent Application Publication No. 2015 / 0017176 A1]. One such antibody, dupilumab, was produced by immunization of a transgenic mouse in which the mouse antibody genes have been replaced with human antibody genes; therefore, the resulting antibody is a human antibody, as opposed to, for example, a humanized mouse antibody.

[0009] The high cost of monoclonal antibody therapeutics initially limited them to human biologics, but recently, significant reductions in production costs have made canine monoclonal products available. Early indications suggest that such monoclonal antibodies will also likely become major therapeutics in the companion animal market. For example, antibodies against human IL-31 receptor alpha (IL-31RA) have been tested and found to have significant effects on pruritus associated with atopic dermatitis in humans [Ruzicka, et al., New England Journal of Medicine, 376(9), 826-835(2017)]. Furthermore, antibodies against canine IL-31 have been shown to have significant effects on pruritus associated with atopic dermatitis in dogs [U.S. Patent No. 8,790,651; U.S. Patent No. 10,093,731]. This caninized antibody blocks the binding of cIL-31 to the canine IL-31 receptor (cIL-31R), thereby blocking the cIL-31 / cIL-31R signaling pathway. Thus, blocking the binding of IL-31 to its receptor IL-31RA reduces the pruritus associated with atopic dermatitis. However, simply blocking the cIL-31 / cIL-31R signaling pathway only improves the pruritus effect of atopic dermatitis, but does not stop the associated skin inflammation caused by the canine IL-4 (cIL-4) or canine IL-13 (cIL-13) / canine IL-4 receptor alpha (cIL-4Rα) signaling pathway.

[0010] More recently, caninized antibodies against canine IL-4Rα have also been disclosed that block the binding of canine IL-4 to canine IL-4Rα [US Patent Application Publication No. 2018 / 0346580 A1, which is incorporated herein by reference in its entirety]. These antibodies were produced by immunization of conventional, i.e., non-transgenic, mice with the canine IL-4Rα extracellular domain (ECD). Since the type II IL-4 receptor consists of the IL-4Rα chain and the IL-13Rα1 chain, antibodies against canine IL-4Rα have been obtained that can block both canine IL-4 and canine IL-13 from binding to the type II canine IL-4 receptor, thereby helping to block inflammation associated with atopic dermatitis.

[0011] However, despite recent success in treating pruritus associated with atopic dermatitis and recent promising disclosures on treating associated inflammation, many subjects suffering from this condition do not experience the rapid onset of antipruritic action with significant effect on skin inflammation.Therefore, it is necessary to design alternative therapies to address this unmet medical need.

[0012] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent No. 8,133,899 [Patent Document 2] U.S. Patent No. 8,987,283 [Patent Document 3] International Publication No. 2018 / 108969 [Patent Document 4] US Patent Application Publication No. 2020 / 0339585 [Patent Document 5] U.S. Patent No. 8,759,366

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Summary of the Invention

[0015] The present invention provides a composition that can be used to treat atopic dermatitis. The composition may comprise a fusion protein that binds canine IL-4 together with a fusion protein that binds canine IL-13. In a specific embodiment, the composition comprises a homodimer comprising a pair of canine interleukin-4 receptor alpha-canine fragment crystallizable domain fusion proteins (cIL-4Rα-cFc fusion proteins) and a homodimer comprising a pair of canine interleukin-13 receptor alpha2-canine fragment crystallizable domain fusion proteins (cIL-13Rα2-cFc fusion proteins), each of the pair of cIL-4Rα-cFc fusion proteins binding to canine interleukin-4 (cIL-4). Each of the pair of cIL-13Rα2-cFc fusion proteins comprises the extracellular domain (ECD) of canine interleukin-4 receptor alpha (cIL-4Rα) or a fragment thereof and a cFc (referred to herein as a first cFc), and each of the pair of cIL-13Rα2-cFc fusion proteins comprises the extracellular domain (ECD) of canine interleukin-13 receptor alpha 2 (cIL-13Rα2) or a fragment thereof that binds canine interleukin-13 (cIL-13) and a cFc (referred to herein as a second cFc). In certain embodiments, the first cFc and the second cFc are the same. In other embodiments, the first cFc and the second cFc are different.

[0016] In specific embodiments of the composition, the first cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1. In other embodiments, the first cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2. In yet other embodiments, the first cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 51. In yet other embodiments, the first cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 3. In yet other embodiments, the first cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 4.

[0017] In certain embodiments of the composition, the second cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1. In other embodiments, the second cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2. In yet other embodiments, the second cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 51. In yet other embodiments, the second cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 3. In yet other embodiments, the second cFc comprises an amino acid sequence having at least 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 4. In a specific embodiment, the first cFc and the second cFc are the same. In other embodiments, the first cFc and the second cFc are different.

[0018] In certain embodiments of the composition, the cIL-4Rα-cFc fusion protein further comprises a canine hinge region (referred to herein as the first canine hinge region). In related embodiments, the cIL-13Rα2-cFc fusion protein further comprises a canine hinge region (referred to herein as the second canine hinge region). In specific embodiments, the first canine hinge region and the second canine hinge region are the same. In other embodiments, the first canine hinge region and the second canine hinge region are different. The canine hinge region can act as a linker between the ECD of cIL-4Rα and the first cFc, and between the ECD of cIL-13Rα2 and the second cFc.

[0019] In specific embodiments of the composition, the first canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 21. In other embodiments, the first canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 22. In yet other embodiments, the first canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 23. In yet other embodiments, the first canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 24.

[0020] In certain embodiments of the composition, the second canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 21. In other embodiments, the second canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 22. In yet other embodiments, the second canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 23. In yet other embodiments, the first canine hinge region comprises an amino acid sequence having at least 85%, 90%, 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 24. In specific embodiments, the first canine hinge region and the second canine hinge region are the same. In other embodiments, the first canine hinge region and the second canine hinge region are different.

[0021] In specific embodiments, the canine hinge region and cFc are both from IgGA. In other embodiments, the canine hinge region and cFc are both from IgGB. In yet other embodiments, the canine hinge region and cFc are both from IgGC. In yet other embodiments, the canine hinge region and cFc are both from IgGD.

[0022] In certain embodiments of the composition, the ECD of cIL-4Rα comprises at least 85%, 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 48. In other embodiments, the ECD of cIL-13Rα2 comprises at least 85%, 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 50. In yet other embodiments, the ECD of cIL-4Rα comprises at least 85%, 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 48, and the ECD of cIL-13Rα2 comprises at least 85%, 90%, 95% or 100% identity to the amino acid sequence of SEQ ID NO: 50.

[0023] In a specific embodiment of the composition, the only linker between the ECD of cIL-4Rα and the first cFc comprises an amino acid sequence identical to an amino acid sequence in a protein naturally found in dogs, including naturally occurring variants thereof. In a related embodiment, the first canine hinge region acts as the only linker between the ECD of cIL-4Rα and the first cFc. In another specific embodiment, the only linker between the ECD of cIL-13Rα2 and the second cFc comprises an amino acid sequence identical to an amino acid sequence in a protein naturally found in dogs, including naturally occurring variants thereof. In a related embodiment, the second canine hinge region acts as the only linker between the ECD of cIL-13Rα2 and the second cFc.

[0024] In more specific embodiments of the composition, the only linker between the ECD of cIL-4Rα and the first cFc comprises an amino acid sequence identical to an amino acid sequence in a protein naturally found in dogs (including naturally occurring variants thereof), and the only linker between the ECD of cIL-13Rα2 and the second cFc comprises an amino acid sequence identical to an amino acid sequence in a protein naturally found in dogs (including naturally occurring variants thereof). In a related embodiment, the first canine hinge region acts as the only linker between the ECD of cIL-4Rα and the first cFc, and the second canine hinge region acts as the only linker between the ECD of cIL-13Rα2 and the second cFc.

[0025] In a specific embodiment of the composition, the cIL-4Rα-cFc fusion protein consists only of an amino acid sequence identical to the amino acid sequence of a protein naturally found in dogs, including naturally occurring variants thereof. In a related embodiment, the cIL-13Rα2-cFc fusion protein consists only of an amino acid sequence identical to the amino acid sequence of a protein naturally found in dogs, including naturally occurring variants thereof. In a specific embodiment, both the cIL-4Rα-cFc fusion protein and the cIL-13Rα2-cFc fusion protein consist only of an amino acid sequence naturally found in dogs, including naturally occurring variants thereof.

[0026] In certain embodiments of the composition, the cIL-4Rα-cFc fusion protein comprises an amino acid sequence having at least 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 5. In specific embodiments, the cIL-4Rα-cFc fusion protein comprises the amino acid sequence of SEQ ID NO: 5. In other embodiments, the cIL-4Rα-cFc fusion protein comprises the amino acid sequence of SEQ ID NO: 8. In yet other embodiments, the cIL-4Rα-cFc fusion protein comprises the amino acid sequence of SEQ ID NO: 11. In yet other embodiments, the cIL-4Rα-cFc fusion protein comprises the amino acid sequence of SEQ ID NO: 12.

[0027] In specific embodiments of the composition, the cIL-13Rα2-cFc fusion protein comprises an amino acid sequence having at least 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 7. In specific embodiments, the cIL-13Rα2-cFc fusion protein comprises the amino acid sequence of SEQ ID NO: 7. In other embodiments, the cIL-13Rα2-cFc fusion protein comprises the amino acid sequence of SEQ ID NO: 10. In yet other embodiments, the cIL-13Rα2-cFc fusion protein comprises the amino acid sequence of SEQ ID NO: 13.

[0028] Any of the compositions of the present invention can further comprise an anti-pruritus antibody. In a specific embodiment, the anti-pruritus antibody is a canine antibody. In a more specific embodiment, the anti-pruritus antibody is a canine antibody against canine interleukin-31 (cIL-31). In other embodiments, the anti-pruritus antibody is a caninized antibody. In a specific embodiment, the caninized anti-pruritus antibody is an antibody against cIL-31. In a more specific embodiment, the caninized antibody against cIL-31 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15. In an alternative embodiment, the caninized antibody against cIL-31 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0029] In another embodiment of the composition, the anti-pruritus antibody is a canine antibody against canine interleukin-31R (cIL-31R). In a particular embodiment, the anti-pruritus antibody is a caninized antibody against cIL-31R. In yet another embodiment, the caninized antibody against cIL-31R comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:27, and a light chain comprising the amino acid sequence of SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. In yet another embodiment, the caninized antibody against cIL-31R comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:34, and a light chain comprising the amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In yet another embodiment, the caninized antibody against cIL-31R comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43, and a light chain comprising the amino acid sequence of SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47.

[0030] Any of the compositions of the present invention can also further comprise one or more additional therapeutic components. In a specific embodiment, the additional therapeutic component is a Janus kinase (JAK) inhibitor. In another embodiment, the additional therapeutic component is a spleen tyrosine kinase (SYK) inhibitor. In yet another embodiment, the additional therapeutic component is an antagonist to a chemoattractant receptor homologous molecule expressed on TH2 cells.

[0031] In a specific embodiment, the JAK inhibitor is: [ka] (In the formula, R 1 is optionally substituted with hydroxy 1~4 alkyl), and pharma- ceutically acceptable salts thereof.

[0032] In an alternative embodiment, the JAK inhibitor is: [ka] and pharma-ceutically acceptable salts thereof.

[0033] In yet other embodiments, the JAK inhibitor is: [ka] and pharma-ceutically acceptable salts thereof.

[0034] The present invention further includes a method of treating atopic dermatitis comprising administering any of the compositions of the present invention to a dog having atopic dermatitis.

[0035] These and other aspects of the present invention will be better understood by reference to the following brief description and detailed description of the drawings. [Brief description of the drawings]

[0036] [Figure 1] Figure 1 shows the binding activity of chimeric and caninized anti-canine IL-31Rα antibodies as assessed by ELISA. Chimeric rat / canine 44E2 [●]. Caninized 44E2: H2k1 [■], H2k2 [▲], H5k1 [▼], and H5k2 [◆]. [Diagram 2] Figure 2 shows the binding activity of chimeric and caninized anti-canine IL-31Rα antibodies assessed by ELISA. Chimeric rat / dog: 10A12 [●]. Caninized 10A12: H1L5 [■] and H2L6 [▲]. [Diagram 3] Figure 3 shows the binding activity of chimeric anti-canine IL-31Rα antibodies and corresponding caninized anti-canine IL-31Rα antibodies as assessed by ELISA. Chimeric rat / canine 28F12. Caninized 28F12: H1k3 [■], H2k2 [▲], and H2k3 [▼]. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The successful therapeutic use of monoclonal antibodies to block signaling in specific pathways by binding to either the protein ligands or their protein receptors described above was the impetus to generate a caninized antibody against canine IL-4 receptor alpha. Thus, a murine antibody was generated against cIL-4Rα, then caninized and shown in vitro to effectively block the binding of cIL-4Rα to either of its two natural ligands, namely cIL-4 or cIL-13. Surprisingly, however, after administration of the caninized murine cIL-4Rα antibody to dogs, abnormally high amounts of so-called anti-drug antibodies (ADA) were detected in the treated dogs. Even more unexpectedly, this problem arose for several different caninized murine cIL-4Rα antibodies tested.

[0038] Induction of ADA is a substantial obstacle in the development of monoclonal antibodies as therapeutic agents. ADAs are antibodies formed by animal subjects against therapeutic antibodies (i.e., drugs) administered to the animal subjects. They typically neutralize the biological activity of therapeutic antibodies and / or cause rapid clearance of therapeutic antibodies from the general circulation of the animal subjects to which they are administered. The problem of ADA becomes more serious when antibodies are first generated in one species, e.g., mouse or rat, but are used to generate therapeutic antibodies in a second species, e.g., dog, which is how caninized mouse or rat antibodies are constructed.

[0039] Furthermore, in order to retain the strong binding affinity of the selected rat antibody to the target canine protein in the corresponding caninized rat antibody, it is generally necessary to include not only the amino acid sequence of the mouse or rat CDR, but also additional amino acid residues from the amino acid sequence of the mouse or rat antibody. These additional amino acids are called back mutations. Back mutations help maintain the three-dimensional structure of the CDR, thereby promoting the retention of the strong binding affinity of the mouse or rat antibody to the canine target protein in the caninized mouse or rat antibody. However, increasing the number of mouse or rat amino acid residues in a therapeutic caninized mouse or rat antibody, i.e., the addition of mouse or rat CDRs and associated back mutations, also increases the possibility that the antibody will be recognized as foreign by the immune system of the treated dog, resulting in ADA.

[0040] As mentioned above, the occurrence of ADA is a common problem for most therapeutic antibodies, but since the occurrence of ADA usually occurs in a relatively small subpopulation of antibodies being treated, it is generally considered a manageable problem. However, surprisingly, it was found that the number of dogs treated with caninized mouse cIL-4Rα antibodies that showed ADA was unexpectedly high. Without limiting the explanation of this surprising result to any particular molecular mechanism, in retrospect, the fact that cIL-4Rα is expressed on antigen-presenting cells (APCs) may be an important factor. Thus, binding of therapeutically caninized cIL-4Rα antibodies to cIL-4Rα of APCs may result in internalization of the bound cIL-4Rα. Following this, a protein fragment having a sequence containing the mouse CDR (or mouse CDR and mouse back mutation) of the caninized antibody is then presented to the canine T cells, which allows a higher induction of ADA to be observed in treated animals.

[0041] Regardless of the cause of the increased number of dogs treated with caninized murine cIL-4Rα antibodies exhibiting ADA, the discovery led to the evaluation of alternative strategies to block the cIL-4 or cIL-13 / cIL-4Rα signaling pathways. One possible alternative strategy would be to directly block cIL-4 and cIL-13 rather than cIL-4Rα, which is part of both the canine IL-4 receptor and the canine IL-13 receptor as described above. A possible methodology to achieve this goal would be through the use of the extracellular domains (ECDs) of the two naturally occurring binding partners of IL-4 and IL-13, namely, the ECD of IL-4Rα and the ECD of IL-13Rα1, respectively.

[0042] A currently common methodology that can be used would be the use of adjacent bispecific fusion proteins that contain both the ECD of IL-4Rα and the ECD of IL-13Rα1. Adjacent bispecific fusion proteins have distinct advantages, such as allowing the synthesis of a single therapeutic protein molecule, rather than requiring the synthesis of two separate protein molecules. Furthermore, when the two functional components of the bispecific fusion protein are functionally related, as in the case of adjacent bispecific cIL-13Rα1 and cIL-4Rα fusion proteins, synergistic effects would be expected, since binding of the first functional component (e.g., cIL-13Rα1) would be expected to promote binding of the second functional component (e.g., cIL-4Rα). One such strategy has been proposed using adjacent IL-13 / IL-4 receptor fusion proteins [see US Patent Application Publication No. 2020 / 0048325]. The design of such fusion proteins brings together IL-13Rα1 and IL-4Rα in a contiguous arrangement, with IL-13Rα1 linked to IL-4Rα. Furthermore, the contiguous receptors may be linked to the fusion partner with a second linker. However, a significant drawback of such methodology is the use of such linkers, which are generally non-natural components of the fused receptor, and thus may result in potential neoepitopes that may induce ADA formation. Furthermore, the linkers used may also be subject to post-translational modifications (e.g., glycosylation), which may create mutant molecules with potentially altered structures, which in turn may further result in ADA formation.

[0043] An alternative method for making bispecific fusion proteins is the use of bispecific heterodimers of fusion proteins of the ECD of IL-13Rα1 and the ECD of IL-4Rα [WO 2020 / 086886] or the ECD of IL-13Rα2 and the ECD of IL-4Rα. Yet another putative strategy is the use of canine Fc fusion proteins incorporating homodimers of IL-4Rα-cFc fusion proteins combined with homodimers of IL-13Rα1-cFc fusion proteins and / or IL-13Rα2-cFc fusion proteins. In either case, these ECDs can be fused to canine IgG (cFc), i.e., IgGA, IgGB, IgGC or IgGD. More preferably, the fusion protein can include a canine IgG hinge region or a fragment thereof. The binding of cFc to the ECD has two main advantages: (i) it extends the in vivo half-life of the fusion protein, and (ii) it aids in the purification of the fusion protein by affinity chromatography. Thus, the ECD of either IL-4Rα, IL-13Rα1 or IL-13Rα2 can be fused / conjugated to a canine IgG hinge region and canine IgG (cFc). In a particular alternative, the resulting fusion protein comprises, in order from N-terminus to C-terminus, the ECD of cIL-13Rα1, or cIL-13Rα2, or cIL-4Rα, a canine hinge region, and cFc. WO 01 / 77332 discloses Fc fusion proteins containing IL-13Rα2 and canine IgG Fc sequences. However, these proteins contain the insertion of a non-self glycine residue (G) as a linker between the ECD of IL-13Rα2 and the canine IgG Fc, followed by 9 amino acid residues from the CH1 domain of canine IgG. Neither the glycyl linker nor the stretch of 9 amino acid residues from the CH1 domain is present in the cFc fusion proteins of the present invention. The presence of a serine residue after the glycine residue, as in the Fc fusion proteins disclosed in WO 01 / 77332, may create an opportunity for enzymatic glycosylation of the fusion protein when the fusion protein is expressed in a cell culture system, thereby resulting in the generation of mutant molecules with some level of glycosylation on the serine residue.This is undesirable from the standpoint of manufacturability on an industrial scale. Furthermore, the insertion of a glycine residue that is not part of the native canine IgG sequence creates the potential to create neoepitopes that can be recognized by the canine immune system and stimulate the production of antibodies against the fusion protein. Such antibodies may negate the therapeutic utility of the fusion protein.

[0044] Thus, one advantage of certain cFc fusion proteins of the invention is that they do not introduce a non-self amino acid linker, thereby minimizing the potential for ADA in treated animals. Furthermore, the cFc fusion proteins of the invention are maintained as non-contiguous molecules separating the cIL-4Rα Fc fusion protein from the canine IL-13Rα1 or canine IL-13Rα2 Fc fusion protein. With some exceptions, 1 It is generally accepted that the absence of a non-self amino acid linker connecting the fusion domains results in low yield, low potency and / or misfolding of the fusion protein domains, and surprisingly, fusion proteins comprising the ECD and cFc of cIL-4Rα, or cIL-13Rα1, or cIL-13Rα2 with canine hinge region were successfully produced and purified even without the use of a non-self amino acid linker. Thus, as shown below, these fusion proteins proved to have high therapeutic value and reduced ADA risk.

[0045] In contrast, in the studies described below, bispecific heterodimeric fusion proteins were found to result in lower expression levels, lower stability and lower purity. Moreover, as mentioned above, they may also increase the likelihood of ADA formation in animal subjects. Moreover, it is not clear whether it is necessary to use twice the amount of bispecific fusion protein to obtain the same therapeutic effect as achieved from the combination of two individual monospecific molecules (i.e., homodimers). Furthermore, the ability to control the efficacy / safety balance of the two individual functional components, for example, to vary the dosage of one of the individual monospecific proteins and keep the dosage of the other constant, is lost.

[0046] In summary, bispecific Fc fusion proteins have been found to be difficult to express and purify. More importantly, they have been found to be less potent as inhibitors of cIL-4 and cIL-13 activity than the combination of the two homodimers, in particular the cIL-4Rα-cFc homodimer and the cIL-13Rα2-cFc homodimer (see Examples below). Thus, the present invention provides a composition comprising a potent blocker of cIL-4 and cIL-13 activity, namely the combination of the homodimer of cIL-4Rα-cFc and cIL-13Rα2-cFc.

[0047] Furthermore, in response to the need for better treatments for atopic dermatitis, the present invention also provides formulations and methodologies that can achieve simultaneous regulation of the cIL-4 / cIL-13 and cIL-31 signaling pathways involved in atopic dermatitis, resulting in rapid onset of anti-pruritic effects with significant effects on skin inflammation and improved skin barrier function. These formulations combine the use of homodimers of cIL-4Rα-cFc fusion protein and cIL-13Rα2-cFc fusion protein along with a caninized rat antibody that binds to canine IL-31Rα.

[0048] Thus, the present invention provides compositions of homodimers of cFc fusion proteins that bind to either cIL-4 or cIL-13 and block the binding of these cytokines to their respective receptors. Additionally, the present invention provides compositions further comprising a canine or caninized antibody that binds to cIL-31 or cIL-31R and blocks the binding of cIL-31 to the cIL-31 receptor. These compositions can be used to treat atopic dermatitis in dogs.

[0049] 1 Fc fusion proteins including certain human proteins, for example, human TNFR-Fc, known as ENBREL®, and human CTLA-4-Fc, known as BELATACEPT®, do not contain a linker.

[0050] Abbreviation The following abbreviations are used throughout the detailed description and examples of the present invention: ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity CDR Complementarity determining region in an immunoglobulin variable region, as defined using the Kabat numbering system cFc dog fragment crystallizable region CHO Chinese hamster ovary EC50 Concentration that results in 50% efficacy or binding ECD extracellular domain ELISA Enzyme-Linked Immunosorbent Assay FR Antibody framework region: immunoglobulin variable region excluding the CDR regions. HRP Horseradish Peroxidase IC50 Concentration that produces 50% inhibition IgG Immunoglobulin G The immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] mAb Monoclonal antibody (also Mab or MAb) PCR Polymerase Chain Reaction PK Pharmacokinetics V region The segment of an IgG chain that is variable in sequence among different antibodies. VH immunoglobulin heavy chain variable region VL immunoglobulin light chain variable region Vl immunoglobulin lambda light chain variable region Vk immunoglobulin kappa light chain variable region

[0051] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0053] The "activity" of a molecule describes or refers to the binding of the molecule to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation, maturation; antigenic activity, modulation of the activity of other molecules, and the like. The "activity" of a molecule can also refer to activity that modulates or maintains cell-cell interactions, such as adhesion, or activity that maintains the structure of a cell, such as a cell membrane or cytoskeleton. "Activity" can also mean specific activity, such as [catalytic activity] / [mg protein], or [immune activity] / [mg protein], concentration in a biological compartment, and the like. "Activity" can refer to modulation of components of the innate or adaptive immune system.

[0054] "Administration" and "treatment," as applied to an animal, e.g., canine subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous medicinal, therapeutic, diagnostic agent or composition with an animal, e.g., canine subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to a cell, as well as contact of a reagent to a fluid where the fluid is in contact with a cell.

[0055] "Administration" and "treatment" also refer to in vitro and ex vivo treatments, for example, of a cell with a reagent, diagnostic, binding compound, or another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a dog, cat, or human), most preferably a dog.

[0056] "Treating" or "treatment" means administering a therapeutic agent, e.g., a composition comprising a cFc-fusion protein of the invention, internally or externally to a canine subject or patient having one or more symptoms or suspected of having a condition for which the agent has therapeutic activity.

[0057] Typically, the therapeutic agent is administered in an amount effective to alleviate and / or improve one or more disease / condition symptoms in the treated subject or population, whether by inducing regression of such symptom(s) to any clinically measurable extent or by inhibiting the progression of such symptoms. The amount of the therapeutic agent effective to alleviate any particular disease / condition symptom (also referred to as a "therapeutically effective amount") may vary according to factors such as the disease state, age and weight of the patient (e.g., dog), and the ability of the pharmaceutical composition to induce a desired response in the subject. Whether a disease / condition symptom has been alleviated or improved can be assessed by any clinical measurement normally used by a veterinarian or other skilled health care provider to assess the severity or progression of the condition. Although an embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the target disease / condition symptom(s) in all subjects, it should alleviate the target disease / condition symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art, e.g., Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonkheel-Terpstra test, and Wilcoxon test.

[0058] "Treatment" as applied to a human, veterinary (e.g., dog), or research subject refers to therapeutic treatment as well as research and diagnostic uses. "Treatment" as applied to a human, veterinary (e.g., dog), or research subject, or cell, tissue, or organ, encompasses contacting an antibody and / or fusion protein of the invention with, for example, a dog or other animal subject, cell, tissue, physiological compartment, or physiological fluid.

[0059] As used herein, the term "cat" refers to any member of the Felidae family. Members of this family include wild, zoo, and domestic members, including domestic cats, purebred and / or mixed-breed companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

[0060] As used herein, the term "dog" includes all domestic dogs, Canis lupus familiaris, or Canis familiaris, unless otherwise specified.

[0061] There are four known IgG heavy chain subtypes and two known light chain subtypes of canine IgG. The four IgG heavy chains are designated A, B, C and D. These heavy chains represent four different subclasses of canine IgG, designated IgG-A (or IgGA), IgG-B (or IgGB), IgG-C (or IgGC) and IgG-D (or IgGD). Each heavy chain consists of one variable domain (VH) and three constant domains, designated CH1, CH2 and CH3. The CH1 domain is connected to the CH2 domain via an amino acid sequence called the "hinge" or alternatively the "hinge region". The DNA and amino acid sequences of these four heavy chain IgGs were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001)]. The amino acid and DNA sequences of these heavy chain IgGs are also available from the GenBank database. For example, the amino acid sequence of the IgG-A heavy chain has the accession number AAL35301.1, IgG-B has the accession number AAL35302.1, IgG-C has the accession number AAL35303.1, and IgG-D has the accession number (AAL35304.1). Dog antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequences of these light chains can be obtained from the GenBank Database. For example, the kappa light chain amino acid sequence has the accession number ABY 57289.1, and the lambda light chain has the accession number ABY 55569.1.

[0062] The "fragment crystallizable region", abbreviated as "Fc region" or simply "Fc", corresponds to the CH2-CH3 portion of an antibody that interacts with a cell surface receptor called the Fc receptor. As used herein, the "canine fragment crystallizable region", abbreviated interchangeably as "cFc region" or simply "cFc", corresponds to the canine fragment crystallizable region from a canine antibody. The canine fragment crystallizable region (cFc) of each of the four canine IgGs was first described by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001); see also, Bergeron et al., Vet. Immunol. Immunopathol. 157:31-41 (2014)].

[0063] As used herein, the "extracellular domain" or "ECD" of a transmembrane interleukin, such as canine interleukin-4 receptor alpha, canine interleukin-13 receptor alpha1, or canine interleukin-13 receptor alpha2, refers to the portion of the interleukin protein that naturally protrudes into the environment surrounding the cell. The ECD does not include the transmembrane portion of the interleukin. The ECD of canine interleukin-4 receptor alpha binds canine IL-4. The ECDs of canine interleukin-13 receptor alpha1 and canine interleukin-13 receptor alpha2 both bind IL-13.

[0064] As used herein, "artificial protein" and "artificial protein molecule" are used interchangeably to refer to a protein (or multimer of a protein, such as a dimer, heterodimer, tetramer, and heterotetramer), such as an artificial fusion protein, that does not occur in nature.

[0065] As used herein, a "fusion protein" is an artificial protein that contains amino acid sequences from two or more different proteins joined together by peptide bonds.

[0066] As used herein, a "cFc fusion protein" is an artificial protein that combines the cFc of an IgG antibody, which may include a hinge region, e.g., IgGB hinge region-CH2-CH3, with another biologically active protein domain to generate a molecule with unique structure and therapeutic utility. For example, a canine IL-13Rα2-cFc fusion protein includes the extracellular domain (ECD) of canine IL-13Rα2 linked to the N-terminus of canine IgG Fc (cFc). The ECD of IL-13Rα2 may be linked to the N-terminus of the cFc by a canine hinge region. The cFc fusion proteins of the present invention are exemplified by the use of an IgGB hinge region and an IgGB cFc, but are in no way limited to such, rather, they include corresponding fusion proteins that have the cFc of IgGA, IgGC, and IgGD, as well as may have the hinge regions of IgGA, IgGC, and IgGD. The canine Fc fusion protein cIL-4Rα-cIgGB-Fc is therefore a member of the cIL-4Rα-cFc genus which also includes cIL-4Rα-cIgGA-Fc, cIL-4Rα-cIgGC-Fc and cIL-4Rα-cIgGD-Fc.

[0067] A particular component of a cFc fusion protein of the invention (e.g., a canine ECD, a canine hinge region, or a cFc) "comprises an amino acid sequence identical to that of a protein naturally found in a canine" means that the component consists of an amino acid sequence identical to the corresponding amino acid sequence of the corresponding region of the protein found in a canine, including naturally occurring variants thereof. For example, when a component of a cFc fusion protein is a cFc itself, and the cFc "comprises an amino acid sequence identical to that of a protein naturally found in a canine", the amino acid sequence of the cFc region of the cFc fusion protein is identical to the amino acid sequence of the naturally occurring canine cFc region of a canine antibody or a variant thereof.

[0068] As used herein, a cFc fusion protein that is "consisting solely of an amino acid sequence identical to that of a protein naturally found in dogs" consists solely of components of the cFc fusion protein that are individually identical to the amino acid sequence of the corresponding region of a protein found in dogs, including naturally occurring variants thereof. For example, if a cFc fusion protein is a cIL-13Rα2-cFc fusion protein that is comprised of three components, namely the ECD of cIL-13Rα2 linked to the N-terminus of cFc by the canine hinge region, and is "consisting solely of an amino acid sequence identical to that of a protein naturally found in dogs," then the individual amino acid sequences of all three components of the cIL-13Rα2-cFc fusion protein: (i) the amino acid sequence of the ECD of cIL-13Rα2, (ii) the amino acid sequence of cFc, and (iii) the amino acid sequence of the canine hinge region are individually identical to the amino acid sequence of the corresponding region of a protein naturally found in dogs, including naturally occurring variants thereof.

[0069] As used herein, the term "sole linker" of a cFc fusion protein of the present invention indicates that the linker is the only linker in the cFc fusion protein. For example, if the canine hinge region is the only linker contained by a cFc fusion protein that includes the ECD of cIL-13Rα2 linked to the N-terminus of cFc by the canine hinge region, then the canine hinge region is the only linker.

[0070] As used herein, "canine interleukin-13 receptor alpha1-canine fragment crystallizable region fusion protein", "canine interleukin-13 receptor alpha1-cFc fusion protein", "canine IL-13Rα1-cFc fusion protein", or "cIL-13Rα1-cFc fusion protein" are all used interchangeably and comprise the extracellular domain (ECD) of cIL-13Rα1 [or a fragment of the ECD that binds canine interleukin-13 (cIL-13)] linked via a peptide bond to canine IgG Fc (cFc). In a specific embodiment, the cIL-13Rα1-cFc fusion protein further comprises a canine hinge region linking the ECD of cIL-13Rα1 (or a fragment of the ECD that binds cIL-13) to the cFc. A cIL-13Rα1-cFc fusion protein can be generated from a chemically synthesized nucleic acid encoding the cIL-13Rα1 ECD (or a fragment of the ECD that binds cIL-13) together with cFc (with or without a linking hinge region) via genetic engineering.

[0071] As used herein, "canine interleukin-13 receptor alpha2-canine fragment crystallizable region fusion protein," "canine interleukin-13 receptor alpha2-cFc fusion protein," "canine IL-13Rα2-cFc fusion protein" or "cIL-13Rα2-cFc fusion protein" are all used interchangeably and comprise the extracellular domain (ECD) of cIL-13Rα2 [or a fragment of the ECD that binds canine interleukin-13 (cIL-13)] linked via a peptide bond to canine IgG Fc (cFc). In a specific embodiment, the cIL-13Rα2-cFc fusion protein further comprises a canine hinge region linking the ECD of cIL-13Rα2 (or a fragment of the ECD that binds cIL-13) to the cFc. A cIL-13Rα2-cFc fusion protein can be generated by genetic engineering from a chemically synthesized nucleic acid that encodes the cIL-13Rα2 ECD (or a fragment of the ECD that binds cIL-13) together with cFc (with or without a linking hinge region).

[0072] As used herein, "canine interleukin-4 receptor alpha-canine fragment crystallizable region fusion protein", "canine interleukin-4 receptor alpha-cFc fusion protein", "canine IL-4Rα-cFc fusion protein" or "cIL-4Rα-cFc fusion protein" are all used interchangeably and comprise the extracellular domain (ECD) of cIL-4Rα [or a fragment of the ECD that binds canine interleukin-4 (cIL-4)] linked via a peptide bond to canine IgG Fc (cFc). In a specific embodiment, the cIL-4Rα-cFc fusion protein further comprises a canine hinge region linking the ECD of cIL-4Rα (or a fragment of the ECD that binds cIL-4) to the cFc. The cIL-4Rα-cFc fusion protein can be generated from a chemically synthesized nucleic acid encoding the cIL-4Rα ECD (or a fragment of the ECD that binds cIL-4) together with the cFc (with or without a linked hinge region) via genetic engineering.

[0073] As used herein, a cIL-4Rα-cFc fusion protein comprising a "fragment of the ECD of cIL-4Rα that binds cIL-4" (or, interchangeably, a "fragment thereof" of the ECD of cIL-4Rα that binds cIL-4) has a binding affinity for cIL-4 that is up to 100-fold lower than the binding affinity of a corresponding cIL-4Rα-cFc fusion protein comprising a full-length ECD, i.e., a dissociation constant of up to 10 2 times higher (e.g., 10 -9 10 compared to M -7M). In certain embodiments, a cIL-4Rα-cFc fusion protein comprising a fragment of the ECD of cIL-4Rα that binds to cIL-4 has a binding affinity for cIL-4 that is up to 10-fold lower than the binding affinity of the corresponding cIL-4Rα-cFc fusion protein comprising the full-length ECD, i.e., the dissociation constant is up to 10-fold higher. In yet other embodiments, a cIL-4Rα-cFc fusion protein comprising a fragment of the ECD of cIL-4Rα that binds to cIL-4 has a binding affinity for cIL-4 that is up to 5-fold lower than the binding affinity of the corresponding cIL-4Rα-cFc fusion protein comprising the full-length ECD, i.e., the dissociation constant is up to 5-fold higher.

[0074] As used herein, a cIL-13Rα2-cFc fusion protein comprising a "fragment of the ECD of cIL-13Rα2 that binds to cIL-13" (or, interchangeably, a "fragment of the ECD of cIL-13Rα2 that binds to cIL-13) has a binding affinity for cIL-13 that is up to 100-fold lower than the binding affinity of a corresponding cIL-13Rα2-cFc fusion protein comprising a full-length ECD, i.e., the dissociation constant is up to 10 2 In certain embodiments, a cIL-13Rα2-cFc fusion protein comprising a fragment of the ECD of cIL-13Rα2 that binds cIL-13 has a binding affinity for cIL-13 that is up to 10-fold lower than the binding affinity of a corresponding cIL-13Rα2-cFc fusion protein comprising a full-length ECD, i.e., the dissociation constant is up to 10-fold higher. In yet other embodiments, a cIL-13Rα2-cFc fusion protein comprising a fragment of the ECD of cIL-13Rα2 that binds cIL-13 has a binding affinity for cIL-13 that is up to 5-fold lower than the binding affinity of a corresponding cIL-13Rα2-cFc fusion protein comprising a full-length ECD, i.e., the dissociation constant is up to 5-fold higher.

[0075] As used herein, a cIL-13Rα1-cFc fusion protein comprising a "fragment of the ECD of cIL-13Rα1 that binds to cIL-13" (or, interchangeably, a "fragment of the ECD of cIL-13Rα1 that binds to cIL-13) has a binding affinity for cIL-13 that is up to 100-fold lower than the binding affinity of a corresponding cIL-13Rα1-cFc fusion protein comprising a full-length ECD, i.e., the dissociation constant is up to 10 2 3-fold higher. In certain embodiments, a cIL-13Rα1-cFc fusion protein comprising a fragment of the ECD of cIL-13Rα1 that binds cIL-13 has a binding affinity for cIL-13 that is up to 10-fold lower than the binding affinity of a corresponding cIL-13Rα1-cFc fusion protein comprising a full-length ECD, i.e., the dissociation constant is up to 10-fold higher. In yet other embodiments, a cIL-13Rα1-cFc fusion protein comprising a fragment of the ECD of cIL-13Rα1 that binds cIL-13 has a binding affinity for cIL-13 that is up to 5-fold lower than the binding affinity of a corresponding cIL-13Rα1-cFc fusion protein comprising a full-length ECD, i.e., the dissociation constant is up to 5-fold higher.

[0076] As used herein, a "homodimer" of the canine interleukin receptor-cFc fusion protein of the present invention is a dimer of two monomeric fusion proteins that minimally have the same ECD (or a fragment of that ECD that binds to the corresponding ligand). The two monomeric fusion proteins also generally have the same cFc and the same hinge region. For example, if the canine interleukin receptor-cFc fusion protein is a cIL-4Rα-cFc fusion protein, the ECD is the IL-4Rα ECD and the ligand is cIL-4. The two monomers of the homodimer are held together by disulfide bonds formed by cysteine ​​residues in the hinge region of each monomer. For example, a homodimer of a cIL-4Rα-cFc fusion protein contains two cIL-4Rα-cFc fusion protein monomers, and a homodimer of a cIL-13Rα2-cFc fusion protein contains two cIL-13Rα2-cFc fusion protein monomers.

[0077] As used herein, a "heterodimer" of the canine interleukin receptor-cFc fusion protein of the present invention is a dimer of two monomeric fusion proteins with different ECDs (or fragments of each ECD that bind to the corresponding ligand of each ECD). The two monomeric fusion proteins generally have the same cFc, but in certain instances may differ slightly due to modifications to keep the two monomers together. For example, a heterodimer of a cIL-4Rα-cFc fusion protein and a cIL-13Rα2-cFc fusion protein contains one cIL-4Rα-cFc fusion protein monomer and one cIL-13Rα2-cFc fusion protein monomer, while a heterodimer of a cIL-4Rα-cFc fusion protein and a cIL-13Rα1-cFc fusion protein contains one cIL-4Rα-cFc fusion protein monomer and one cIL-13Rα1-cFc fusion protein monomer. One such embodiment is cIL-4Rα-13Rα1_ZW1-cFc, which is a heterodimer of cIL-4Rα-cFc-ZW-A and cIL-13Rα1-cFc-ZW-B. Another such embodiment is cIL-4Rα-13Rα2_ZW1-cFc, which is a heterodimer of cIL-4Rα-cFc-ZW-A and cIL-13Rα2-cFc-ZW-B.

[0078] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Antibodies can be monomeric, dimeric, or larger multimeric. It is therefore used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, full canine antibodies, chimeric antibodies, and camelized single domain antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as caninization of the antibody for use as a canine therapeutic antibody.

[0079] As used herein, a cFc fusion protein of the invention or an antibody used in the invention that is "blocking" or "blocking" or "blocking binding" to, for example, its binding partner (ligand) of a canine receptor is an antibody and / or fusion protein that blocks (partially or completely) the binding of a canine receptor to its canine ligand, and vice versa, as determined by standard binding assays (e.g., BIACore®, ELISA or flow cytometry).

[0080] Typically, an antibody or antigen-binding fragment of the invention retains at least 10% of its canine antigen-binding activity (when compared to the parent antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the canine antigen-binding affinity of the parent antibody. It is also contemplated that the antibody or antigen-binding fragment of the invention may contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of an antibody) that do not substantially alter its biological activity.

[0081] "Isolated antibody" refers to a purified state, and in that context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with experimental or therapeutic uses of the binding compounds described herein.

[0082] As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, the first and second antibodies being from different species [U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)]. Typically, the variable domains are obtained from an antibody from a laboratory animal "parent antibody", such as a rodent, and the constant domain sequences are obtained from an animal subject antibody, e.g., human or canine, such that the resulting chimeric antibody is less likely to provoke an adverse immune response in a human or canine subject, respectively, than the parent (e.g., rodent) antibody.

[0083] As used herein, the term "caninized antibody" refers to a form of an antibody that contains sequences from both canine and non-canine (e.g., rat) antibodies. In general, a caninized antibody contains substantially all of at least one or more, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-canine immunoglobulin (e.g., including six CDRs as exemplified below), and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) being of a canine immunoglobulin sequence. As exemplified herein, a caninized antibody contains both three heavy chain CDRs and three light chain CDRs from a rat anti-canine antigen antibody, along with a canine frame or modified canine frame. The modified canine frame contains one or more amino acid changes exemplified herein that further optimize the effectiveness of the caninized antibody, for example, to increase its binding to the canine antigen and / or its ability to block binding of the canine antigen to its natural binding partner. Caninized mouse antibodies or rat anti-dog antibodies that bind to canine IL-31 and IL-31Rα include, but are not limited to, antibodies for use in the invention that contain canine IgGA, IgGB, IgGC or IgGD heavy chains.

[0084] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same.

[0085] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, allowing binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is generally based on the Sequences of Proteins of Immunological Interest [Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 th ed.;NIH Publ.No.91-3242(1991);Kabat,Adv.Prot.Chem.32:1-75(1978);Kabat,et al.,J.Biol.Chem.252:6609-6616(1977);Chothia,et al., J. Mol. Biol. 196:901-917 (1987), or Chothia, et al., Nature 342:878-883 (1989)].

[0086] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region includes amino acid residues from "complementarity determining region" or "CDR" [i.e., CDRL1 (or LCDR1), CDRL2 (or LCDR2) and CDRL3 (or LCDR3) in the light chain variable domain and CDRH1 (or HCDR1), CDRH2 (or HCDR2) and CDRH3 (or HCDR3) in the heavy chain variable domain]. [See Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which defines the CDR region of an antibody by sequence; also see Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which defines the CDR region of an antibody by structure].

[0087] As used herein, the terms "framework" or "FR" residues refer to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0088] As used herein, the term "canine frame" refers to the amino acid sequences of the heavy and light chains of a canine antibody other than the hypervariable region residues defined herein as CDR residues. For caninized antibodies, in most embodiments, the amino acid sequences of the native canine CDRs are replaced in both chains with the corresponding foreign CDRs (e.g., from mouse or rat antibodies). The heavy and / or light chains of the canine antibody may contain some foreign non-CDR residues to preserve the conformation of the foreign CDRs in the canine antibody and / or to modify Fc function, for example, as exemplified below and / or as disclosed in U.S. Pat. No. 10,106,607 B2.

[0089] As used herein, an "anti-pruritic agent" is a compound, polymer, and / or formulation that tends to inhibit, reduce, and / or prevent itch. Anti-pruritic agents are colloquially referred to as anti-itch drugs.

[0090] As used herein, an "anti-pruritus antibody" is an antibody that can act as an anti-pruritus agent in animals, including mammals such as humans, dogs and / or cats, particularly with respect to atopic dermatitis. In a specific embodiment, the anti-pruritus antibody binds to a specific protein in the IL-31 signaling pathway, such as IL-31 or its receptor IL-31Rα. Binding of the anti-pruritus antibody to its corresponding antigen (e.g., IL-31 or IL-31Rα) inhibits, for example, the binding of IL-31 to IL-31Rα, disrupting and / or preventing successful signaling of this pathway, thereby inhibiting, reducing and / or preventing pruritus caused by the IL-31 signaling pathway.

[0091] As used herein, an "anti-inflammatory agent" is a compound, macromolecule, and / or preparation that reduces inflammation by blocking the interaction of certain substances in the body that cause inflammation. The anti-inflammatory agent may be a cFc fusion protein that can act as an anti-inflammatory agent in animals, including mammals such as humans, dogs, and / or cats, particularly with respect to atopic dermatitis. In a specific embodiment, the anti-inflammatory cFc fusion protein binds to a specific protein in the IL-4 / IL-13 signaling pathway, such as IL-4 or IL-13. The binding of the anti-inflammatory cFc fusion protein to its corresponding antigen (e.g., IL-4) inhibits, for example, the binding of IL-4 to IL-4Rα, disrupting and / or preventing signaling of this pathway, thereby disrupting or preventing chronic inflammation associated with atopic dermatitis. The combination of a homodimer of a cIL-4Rα-cFc fusion protein and a homodimer of a cIL-13Rα2-cFc fusion protein acts as an anti-inflammatory agent in the treatment of atopic dermatitis.

[0092] As used herein, a "bispecific fusion protein" is an artificial protein that may be adjacent proteins, e.g., two different biologically active protein domains joined to each other via peptide bonds, e.g., ECD of cIL-4Rα, ECD of cIL-13Rα1, cFc and an optional linker. Alternatively, a bispecific fusion protein may be a heterodimeric fusion protein in which two different biologically active protein domains are individually linked to the fusion partners via peptide bonds, but are linked in the heterodimeric fusion protein by non-peptide bonds, which may be either covalent or non-covalent. For example, a heterodimer formed by combining two monomeric fusion proteins with different ECDs, such as a heterodimer of a cIL-4Rα-cFc fusion protein monomer and a cIL-13Rα2-cFc fusion protein monomer.

[0093] "Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences when they are optimally aligned. If both positions of the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in the two sequences are identical or homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, the comparison is performed when the two sequences are aligned to give the maximum percentage of homology.

[0094] "Isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA or synthetic origin or any combination thereof, where the isolated polynucleotide is not associated with all or a portion of a polynucleotide in which it is found in nature or linked to a polynucleotide with which it is not linked in nature. For purposes of this disclosure, it should be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass an intact chromosome. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences for up to 10 or even up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0095] The term "control sequence" refers to a DNA sequence required for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoters, and may include operator sequences and ribosome binding sites. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0096] A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0097] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and the culture derived therefrom, regardless of the number of transfers. It is also understood that not all progeny have exactly the same DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a clear designation is intended, it will be clear from the context.

[0098] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. As used herein, an amino acid sequence is 100% "identical" to a second amino acid sequence if the amino acid residues of both sequences are identical. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparison is performed over contiguous blocks of amino acid residues contained in a given protein, e.g., a protein or portion of a polypeptide being compared. In particular embodiments, selected deletions or insertions that would otherwise change the correspondence between the two amino acid sequences are taken into account.

[0099] Sequence similarity includes identical residues and non-identical biochemically related amino acids that share similar properties and may be interchangeable.

[0100] "Conservatively modified variants" or "conservative substitutions" refer to the substitution of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), such that changes can occur frequently without changing the biological activity of the protein. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity [e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224(4th Ed.;1987)]. Moreover, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity. Exemplary conservative substitutions are shown directly in Table A below.

[0101] [Table 1]

[0102] Function-conservative variants of the cFc fusion proteins of the present invention are also contemplated by the present invention. As used herein, "function-conservative variants" refers to cFc fusion proteins in which one or more amino acid residues are altered without altering desired properties such as antigen affinity and / or specificity. Such variants include, but are not limited to, replacing amino acids with those having similar properties, such as the conservative amino acid substitutions in Table A above.

[0103] nucleic acid The present invention includes cFc fusion proteins of the invention, and compositions comprising the cFc fusion proteins of the invention in combination with antibodies for use in the invention (see, eg, the Examples below).

[0104] Also included in the present invention are nucleic acids encoding the provided cFc fusion proteins and immunoglobulin polypeptides used in the present invention, including amino acid sequences that are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequence of the caninized antibody provided herein when the comparison is made using a BLAST algorithm, the algorithm parameters being selected to give the greatest match between the respective sequences over the entire length of the respective reference sequences. The present invention further provides nucleic acids encoding fusion proteins and / or immunoglobulin polypeptides, including amino acid sequences that are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when the comparison is made using a BLAST algorithm, the algorithm parameters being selected to give the greatest match between the respective sequences over the entire length of the respective reference sequences.

[0105] As used herein, the percent sequence identity of nucleotides and amino acids can be determined using the C, MacVector (MacVector, Inc., Cary, NC 27519), Vector NTI (Informax, Inc., MD), Oxford Molecular Group PLC (1996) and Clustal W algorithms with alignment default parameters and identity default parameters. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, Advanced Blast search can be used under default filter conditions, for example, using the GCG (Genetics Computer Group, GCG Package Program Manual, Version 7, Madison, WI) pileup program with default parameters.

[0106] The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272 (1993); Madden, T. L., et al., Meth. Enzymol. 266:131-141 (1996); Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J. C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J. Met al.,Comput.Appl.Biosci.10:67-70(1994);ALIGNMENT SCORING SYSTEMS:Dayhoff,MO,et al.,“A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. MODayhoff (ed.), pp. 345-352, (1978); Natl. Structure, vol. 5, suppl. 3.” (1978), MODayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, J. Mol. Biol. al.,Methods 3:66-70(1991);Henikoff,S.,et al.,Proc.Natl.Acad.Sci.USA 89:10915-10919(1992);Altschul,SF,et al.,J.Mol.Evol.36:290-300(1993);ALIGNMENT STATISTICS:Karlin,S.,et al.,Proc.Natl.Acad.Sci.USA 87:2264-2268(1990);Karlin,S.,et al.,Proc.Natl.Acad.Sci.USA 90:5873-5877(1993);Dembo,A.,et al.,Ann.Prob.22:2022-2039(1994);およびAltschul,S.F.“Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research(S.Suhai,ed.),pp.1-14,Plenum,New York(1997)。.

[0107] The cFc fusion proteins of the present invention (and antibodies used in the present invention) can be recombinantly produced by methods known in the art. Mammalian cell lines available as hosts for the expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells. A particularly preferred cell line is selected by determining which cell line has a high expression level. Other cell lines that may be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When a recombinant expression vector encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and / or antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or more preferably, secretion of the antibody into the medium in which the host cell is growing.

[0108] The antibody can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibody of the present invention (or other moieties therefrom) from the production cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, and 0323997, and European Patent Application No. 89303964.4.

[0109] Pharmaceutical Compositions and Administration To prepare pharmaceutical or sterile compositions containing the cFc fusion proteins of the present invention, the proteins may be mixed, alone or together with the antibodies used in the present invention, with a pharma- ceutically acceptable carrier or excipient [see, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0110] Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing with an acceptable carrier, excipient, or stabilizer in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension [see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the pharmaceutical composition containing the cFc fusion protein of the present invention is diluted to an appropriate concentration in a sodium acetate solution at pH 5-6, and NaCl or sucrose is added for isotonicity. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0111] The toxicity and therapeutic efficacy of an antibody composition administered alone or in combination with another agent can be determined, for example, by the LD 50 (a dose lethal to 50% of the population) and ED 50The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. 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. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in dogs. The dosage of such compounds is preferably within the ED range with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form and route of administration used.

[0112] The mode of administration may vary. Suitable routes of administration include oral, rectal, mucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intraarterial. In a specific embodiment, a pharmaceutical composition comprising a cFc-fusion protein of the invention may be administered by an invasive route, such as injection. In a further embodiment of the invention, a pharmaceutical composition comprising a cFc-fusion protein of the invention is administered intravenously, subcutaneously, intramuscularly, intraarterially, or by inhalation (aerosol delivery). Administration by non-invasive routes (e.g., oral; e.g., pill, capsule, or tablet) is also within the scope of the invention.

[0113] Composition can be administered using medical equipment known in the art.For example, pharmaceutical composition of the present invention can be administered by injection with hypodermic needle, including prefilled syringe or autoinjector.Pharmaceutical composition disclosed herein can also be administered using needleless hypodermic injection device, for example, the device disclosed in U.S. Patent No. 6,620,135; No. 6,096,002; No. 5,399,163; No. 5,383,851; No. 5,312,335; No. 5,064,413; No. 4,941,880; No. 4,790,824 or No. 4,596,556.

[0114] The pharmaceutical composition disclosed herein can also be administered by injection. Examples of well-known implants and modules for administering pharmaceutical compositions include U.S. Patent No. 4,487,603 (discloses an implantable microinfusion pump for distributing drugs at a controlled rate); U.S. Patent No. 4,447,233 (discloses a drug infusion pump for delivering drugs at a precise infusion rate); U.S. Patent No. 4,447,224 (discloses a variable flow rate implantable infusion device for continuous drug delivery); U.S. Patent No. 4,439,196 (discloses an osmotic drug delivery system with multi-chamber compartments). Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0115] Alternatively, compositions containing the cFc-fusion proteins of the invention (and optionally the antibodies used in the invention) may be administered locally rather than systemically, often in a depot or sustained release formulation.

[0116] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody and / or cFc fusion protein, the level of symptoms, the immunogenicity of the therapeutic antibody and / or cFc fusion protein, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers enough of the therapeutic antibody and / or cFc fusion protein to bring about improvement of the target disease / condition while simultaneously minimizing undesirable side effects. Thus, the amount of biologic delivered depends, in part, on the particular therapeutic antibody and / or fusion protein and the severity of the condition being treated. Guidance on selecting appropriate doses of therapeutic antibodies is available [e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 344:783-792 (2001); Engl.J.Med.342:613-619(2000);Ghosh et al.New Engl.J.Med.348:24-32(2003);Lipsky et al.New Engl.J.Med.343:1594-1602(2000)].

[0117] The determination of the appropriate dose is made by a veterinarian, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, the dose is started at a dose somewhat less than the optimal dose, and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms.

[0118] Compositions comprising the cFc fusion proteins of the invention, alone or together with the antibodies used in the invention, can be provided by continuous infusion or by doses administered, for example, daily, 1 to 7 times per week, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, etc. Doses can be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. The total weekly dose is generally at least 0.05 μg / kg body weight, more generally at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg or more [e.g., Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer [See Immunol. Immunother. 52:133-144 (2003)]. Doses may also be provided to achieve a predetermined target concentration of the cFc-fusion protein of the invention in dog serum, e.g., 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or more. In other embodiments, the cFc-fusion protein of the invention is administered subcutaneously or intravenously at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg / subject weekly, biweekly, "every four weeks," monthly, bimonthly or quarterly.

[0119] As used herein, "inhibit" or "treat" or "treatment" includes postponing the onset of symptoms associated with a disorder or condition and / or reducing the severity of symptoms of such a disorder or condition. The term further includes ameliorating existing uncontrolled or undesirable symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the term refers to the conferring of a beneficial result to a vertebrate subject (e.g., a dog) having a disorder, condition, and / or condition or having a potential for developing such a disorder, disease, or condition.

[0120] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of the cFc-fusion protein of the present invention that is effective when administered alone or in combination with an additional therapeutic agent to a cell, tissue or subject, e.g., a dog, to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such a disease or condition. A therapeutically effective dose further refers to an amount of the antibody and / or fusion protein sufficient to cause at least a partial improvement of a symptom, e.g., treatment, cure, prevention or amelioration of an associated medical condition, or an increase in the rate of treatment, cure, prevention or amelioration of such a condition. When applied to a combination, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will cause an improvement of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% of a diagnostic measure or parameter. An effective amount can also cause an improvement in a subjective measure when a subjective measure is used to assess the severity of the condition.

[0121] Other combination therapies Compositions comprising the cFc fusion proteins of the invention (with or without the antibodies used in the invention) can include one or more additional therapeutic moieties. One such family of therapeutic moieties are Janus kinase (JAK) inhibitors. In a specific embodiment of this type, the JAK inhibitor is [ka] (In the formula, R 1 is optionally substituted with hydroxy 1~4 and pharmaceutically acceptable salts thereof [U.S. Pat. No. 8,133,899; U.S. Pat. No. 8,987,283]. More specifically, the JAK inhibitor is oclacitinib, and even more specifically, oclacitinium maleate.

[0122] An alternative JAK inhibitor that preferentially inhibits JAK1 over JAK3 is 1-[(3R,4S)-4-cyanotetrahydropyran-3-yl]-3-[(2-fluoro-6-methoxy-4-pyridyl)amino]pyrazole-4-carboxamide, [ka] and pharma- ceutically acceptable salts thereof [see WO 2018 / 108969].

[0123] Yet another alternative JAK inhibitor is 3-azetidineacetonitrile, also known as 1-(cyclopropylsulfonyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-(supplied by CAS) {1-(cyclopropanesulfonyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile (supplied by USAN Program chemical consultant); [ka] and pharma- ceutically acceptable salts thereof [see U.S. Patent Application Publication No. 2020 / 0339585].

[0124] Another therapeutic component that can be added to the composition of the present invention can be a spleen tyrosine kinase (SYK) inhibitor.One such SYK inhibitor is (1S,4R)-4-hydroxy-2,2-dimethyl-4-{5-[3-methyl-5-(4-methyl-pyrimidin-2-ylamino)-phenyl]-1,3-thiazol-2-yl}-cyclohexanecarboxylic acid or its pharma- ceutically acceptable salt [see, for example, U.S. Patent No. 8,759,366].

[0125] Further therapeutic ingredients that can be added to the compositions of the present invention include: [ka] and pharmaceutically acceptable salts thereof (see also U.S. Pat. No. 7,696,222, U.S. Pat. No. 8,546,422, U.S. Pat. No. 8,637,541, WO 2010 / 099039, WO 2010 / 031183, and U.S. Pat. No. 8,546,422).

[0126] These additional therapeutic components may be administered to the canine subject prior to, in conjunction with, or following administration of a composition comprising an antibody and / or fusion protein of the invention.

[0127] The magnitude of the prophylactic or therapeutic dose of the above JAK inhibitor, SYK inhibitor, or chemoattractant receptor homologous molecule will of course vary depending on the nature and severity of the condition being treated, as well as the particular inhibitor and its route of administration. This will also vary according to a variety of factors, including the age, weight, general health, sex, diet, time of administration, rate of excretion, drug combinations and response of the individual dog. In general, the daily dose is about 0.001 mg to about 100 mg per kg of dog body weight, preferably 0.01 mg to about 10 mg per kg of dog body weight. In another embodiment, the daily dose is about 0.2 mg to about 1.0 mg per kg of dog body weight. In another embodiment, the daily dose is about 0.1 mg to about 3.0 mg per kg of dog body weight. However, in some cases, it may be necessary to use dosages outside these limits. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. For example, a formulation intended for oral administration may contain from 0.05 mg to 5 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 99.95 percent of the total composition. Dosage unit forms generally contain from about 0.1 mg to about 0.4 g of active ingredient, typically 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg or 400 mg.

[0128] [Example] [Example 1] Homodimeric Fc fusion proteins Generation of recombinant fusion proteins: The recombinant fusion proteins listed in Tables 2B and 2C below were obtained from commercial manufacturers after providing them with the correct amino acid sequence for the selected fusion protein. The amino acid sequence can be obtained from publicly available protein databases such as GenBank, for example, the accession numbers of the full-length amino acid sequences include: Canis lupus familiaris interleukin-4 receptor subunit alpha isoform X1 accession number XP_022275636.1, Canis lupus familiaris interleukin-13 receptor subunit alpha-1 isoform X2 accession number XP_038306633.1, and Canis lupus familiaris interleukin-13 receptor subunit alpha-2 precursor accession number NP_001003075.1. Typically, to recombinantly produce these fusion proteins, DNA encoding the canine fusion protein is chemically synthesized and then cloned into an appropriate expression vector (e.g., pcDNA3.4 expression vector) to produce the protein in cells such as CHO or HEK-293 cells. Thus, commercial manufacturers select the optimal nucleotide sequence that encodes the amino acid sequence of the fusion protein, chemically synthesize the nucleic acid, insert the nucleic acid into an expression vector that produces the corresponding recombinant fusion protein, and then purify the expressed fusion protein. The nucleic acid sequences are typically manufactured at commercial suppliers in a process that requires the following steps: 1. Based on the target gene sequence, design and synthesize several oligonucleotides with a length of about 100 nucleotides (the synthesized overlapping oligonucleotides include ECD, hinge region, and cFc); 2. Using the polymerase chain reaction (PCR) to assemble oligonucleotides together to obtain the full-length gene sequence; and 3. The PCR product is purified using a DNA gel extraction kit and used as the insert in the subsequent cloning step. In this case, the recombinant fusion protein was produced in CHO cells and purified using Protein A column chromatography.

[0129] The nucleic acid encoding the cFc fusion protein of the present invention comprises the coding sequence of the extracellular domain (ECD) or fragment thereof of the selected canine interleukin receptor, i.e., cIL-4Rα, cIL-13Rα1 or cIL-13Rα2, and the coding sequence of the canine IgG hinge region together with canine IgG (cFc). The resulting fusion protein comprises, in order from N-terminus to C-terminus, the ECD, the hinge region (bold), and the cFc. The cFc and hinge region can be derived from canine IgGA, IgGB, IgGC or IgGD. The cFc fusion protein may have amino acid substitutions to allow for an extended half-life in vivo or to eliminate some effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC) [see, e.g., U.S. Pat. No. 10,106,607 B2].

[0130] The two monomers of the homodimer are held together by disulfide bonds formed by cysteine ​​residues in the hinge region of each monomer. The homodimeric proteins can be made in separate host cells (such as CHO cells) and then combined after purification from their respective producing cells. The homodimeric proteins can be administered to dogs via various routes such as IV, SC, IP or IM. The homodimeric proteins can be administered at doses ranging from 0.1ug / kg to 20mg / kg or more. Typically, the homodimeric proteins can be administered at doses ranging from 0.1mg / kg to 10mg / kg.

[0131] An example of a homodimeric Fc fusion protein of the present invention is as follows: cIL-4Rα-cIgGB-Fc [SEQ ID NO: 5]

number

[0132] cIL-13Rα1-cIgGB-Fc [SEQ ID NO: 6]

number

[0133] cIL-13Rα2-cIgGB-Fc [SEQ ID NO: 7]

number

[0134] [Example 2] Homodimeric cFc fusion protein with extended half-life Understanding the structure and characteristics of the interaction between the so-called neonatal Fc receptor (FcRn) and IgG antibodies has provided the basis for antibody or Fc engineering research to improve the serum half-life of IgG antibodies and Fc fusion proteins. The mechanism behind the serum half-life extension of proteins and approaches to extend the serum half-life of such proteins has been described by several researchers [see, for example, Ko et al., BioDrugs 35:147-157 (2021)]. Homologous proteins with extended half-life are synthesized and recombinantly produced from nucleotide sequences encoding the desired amino acid sequences, as described in Example 1 above.

[0135] Examples of recombinant cFc fusion proteins with extended in vivo half-lives are provided below. In these examples, the canine cFc is IgGB, however, the use of alternative cFcs, i.e. IgGA, IgGC and IgGD in the cFc fusion proteins of the invention are also part of the invention.

[0136] Canine IgG-B Fc was originally defined by Tang et al. [Vet Immunology & Immunopathology, 80:259-270 (2001)] as comprising the amino acid sequence of SEQ ID NO:51, provided below.

[0137]

number

[0138] The amino acid sequences of the cFc portion of the recombinant fusion proteins contain amino acid substitutions (bold and underlined) that confer higher affinity binding to FcRn than wild-type cFc at mildly acidic pH (e.g., pH 6.0) while at the same time having a binding affinity similar to that exhibited by wild-type cFc at neutral pH (e.g., pH 7.0-7.2). The hinge region of each sequence is in bold but not underlined.

[0139] cIL-4Rα-cIgGB-Fc-YTE [SEQ ID NO: 8] CH2 numbering (L18Y / A20T / T22E)

number

[0140] cIL-13Rα1-cIgGB-Fc-YTE [SEQ ID NO: 9] CH2 numbering (L18Y / A20T / T22E)

number

[0141] cIL-13Rα2-cIgGB-Fc-YTE [SEQ ID NO: 10] CH2 numbering (L18Y / A20T / T22E)

number

[0142] cIL-4Rα-cIgGB-Fc-H [SEQ ID NO: 11] CH2 numbering (N202H)

number

[0143] cIL-4Rα-cIgGB-Fc-YD [SEQ ID NO: 12] CH2 numbering (L18Y / T22D)

number

[0144] cIL-13Rα2-cIgGB-Fc-YD [SEQ ID NO: 13] CH2 numbering (L18Y / T22D)

number

[0145] The bolded amino acid residues are the hinge region, and the bolded and underlined amino acid residues are substitutions to increase the in vivo half-life of the fusion protein.

[0146] [Example 3] Bispecific Fc fusion proteins Bispecific cFc fusion proteins are generally considered a better alternative to homodimeric cFc fusion proteins because each of the two monomers of the bispecific cFc fusion protein binds to a different target protein. In theory, this can significantly reduce the overall production cost. Thus, one such bispecific cFc fusion protein that was produced contained a heterodimer consisting of a first monomer containing, in N- to C-terminal order, the ECD of IL-13Rα1, the hinge region of IgGB, and the cFc of IgGB, whereas the second monomer contains, in N- to C-terminal order, the ECD of cIL-4Rα, the hinge region of IgGB, and the cFc of IgGB. Another bispecific cFc fusion protein comprised a heterodimer consisting of a first monomer containing, from N- to C-terminus, the ECD of IL-13Rα2, the hinge region of IgGB and the cFc of IgGB, while the second monomer contained, from N- to C-terminus, the ECD of cIL-4Rα, the hinge region of IgGB and the cFc of IgGB.

[0147] The heterodimeric protein is synthesized and recombinantly produced from a nucleotide sequence encoding the desired amino acid sequence similar to that described in Example 1 above. The heterodimeric protein is administered to the dog via various routes such as IV, SC, IP or IM. The heterodimeric protein may be administered at a dose ranging from 0.1 ug / kg to 20 mg / kg or more. Typically, the heterodimeric protein may be administered at a dose ranging from 0.1 mg / kg to 10 mg / kg.

[0148] To form a bispecific fusion protein, it is important to make amino acid substitutions in the Fc portion of the fusion protein of each binding partner to promote the formation of a heterodimer rather than a homodimer of the two Fc fusion proteins. Several possible methods or combinations of specific amino acid substitutions in the Fc portion of the canine Fc fusion protein that can be used to promote heterodimer formation are provided in Table 1 below. These substitutions favor either the knob-into-hole method for Fc heterodimerization or the electrostatic attraction between different Fc chains to enable heterodimerization [see Moore et al., Methods, 154:38-50 (2019) and Brinkmann & Kontermann, MABS, 9:182-212 (2017) for a comprehensive discussion of these amino acid substitutions].

[0149] One bispecific fusion protein, cIL-4Rα-13Rα1_ZW1-cFc, is a heterodimer of cIL-4Rα-cIgGB-Fc-ZW-A and cIL-13Rα1-cIgGB-Fc-ZW-B. Another bispecific fusion protein, cIL-4Rα-13Rα2_ZW1-cFc, is a heterodimer of cIL-4Rα-cIgGB-Fc-ZW-A and It is a heterodimer with cIL-13Rα2-cIgGB-Fc-ZW-B.

[0150] [Table 2]

[0151] Examples of monomers of bispecific Fc fusion proteins of the invention are listed below with amino acid substitutions shown in bold and underlined:

[0152] cIL-4Rα-cIgGB-Fc-ZW-A [SEQ ID NO: 18]

number

[0153] cIL-13Rα1-cIgGB-Fc-ZW-B [SEQ ID NO: 19]

number

[0154] cIL-13Rα2-cIgGB-Fc-ZW-B [SEQ ID NO: 20]

number

[0155] Prior art amino acid sequences of four canine IgGs: cIgGA [SEQ ID NO: 1] Prior Art

number

[0156] cIgGB [SEQ ID NO: 2] Prior Art

number

[0157] cIgGC [SEQ ID NO: 3] Prior Art

number

[0158] cIgGD [SEQ ID NO: 4] Prior Art

number

[0159] Prior art amino acid sequences of the canine IgG hinge region of four canine IgGs: cIgGA hinge region [SEQ ID NO: 21] Prior art

number

[0160] cIgGB hinge region [SEQ ID NO: 22] Prior art

number

[0161] cIgGC hinge region [SEQ ID NO: 23] Prior art

number

[0162] cIgGD hinge region [SEQ ID NO: 24] Prior art

number

[0163] Prior art amino acid sequences of the ECDs of cIL-4Rα, cIL-13Rα1 and cIL-13Rα2 cIL-4Rα [SEQ ID NO: 48] Prior Art

number

[0164] cIL-13Rα1 [SEQ ID NO: 49] Prior Art

number

[0165] cIL-13Rα2 [SEQ ID NO: 50] Prior Art

number

[0166] [Table 3]

[0167] [Table 4]

[0168] [Table 5]

[0169] [Example 4] Binding of cFc fusion proteins to canine IL-4 and IL-13 method: The binding constants of the cFc fusion proteins provided in Tables 3 and 4 below were determined using OCTET® HTX. All kinetic measurements were performed with OCTET® HTX using the SA® biosensor and DATA ACQUISITION® 12.0 software. 10 μg / mL of biotin-labeled antigen, either canine IL-4 (cIL-4) or canine IL-13 (cIL-13), was loaded onto the SA® biosensor for 120 seconds. The biosensor was then placed into 1× pH 7.0 TBS / casein buffer for 60 seconds for the blocking phase. For the association phase, the antigen-loaded biosensor was placed into 2-fold serial dilutions of wild-type receptor Fc fusions, bispecific receptor Fc fusions, or FcRn mutant receptor Fc fusions recognizing cIL-4 or cIL-13 antigens from 1 μM to 15.6 nM in 1× pH 7.0 TBS / casein buffer for 30 seconds. The last well was buffer only and the sensor was used for subtraction of the reference sensor. Finally, the biosensor was placed in 1x pH 7.0 TBS / casein buffer for 120 seconds for the dissociation phase. The results were then analyzed using Data Analysis 12.0 software and the curves were fitted using a 1:1 binding model.

[0170] result: The association rate constants (ka), dissociation rate constants (kdis) and dissociation constants (KD) of the cIL-4Rα-cFc and cIL-13Rα1-cFc, as well as the cIL-13Rα2-cFc homodimer and heterodimer fusion proteins are shown below in Tables 3 and 4. As can be seen from Table 3 below, the binding constant (KD) of the unmodified cIL-4Rα-cFc homodimer with cIL-4 is approximately 1×10 -12 In sharp contrast, the KD of the heterodimeric bispecific cIL4Rα-IL13Rα1_ZW1-cFc with cIL-4 was approximately 10,000-fold higher (approximately 1 × 10 -8In other words, the homodimeric cIL-4Rα-cFc binds cIL-4 approximately four orders of magnitude stronger than the heterodimeric bispecific cIL4Rα-IL13Rα1_ZW1-cFc. Of note, the modified homodimeric cIL-4Rα-cFc-H binds canine IL-4 (1 × 10 -10 ) is approximately two orders of magnitude higher than the KD of the unmodified cIL-4Rα-Fc homodimer, and the KD of the modified homodimer cIL-4Rα-cFc-YTE for binding to cIL-4 (1 × 10 -11 ) was approximately one order of magnitude higher than that of the engineered homodimer. Furthermore, binding of the heterodimeric bispecific cIL4Rα-IL13Rα2_ZW1-cFc to cIL-4 was undetectable by this assay. Thus, engineering the homodimeric cIL-4Rα-cFc to increase its half-life reduced the affinity for cIL-4 by approximately 10- to 100-fold for the homodimeric cIL-4Rα-cFc-NH and cIL-4Rα-cFc-YTE, respectively, whereas the cIL4Rα-IL13Rα1_ZW1-cFc heterodimer showed an approximately four order of magnitude reduction in affinity for cIL-4 compared to the unengineered cIL-4Rα-cFc homodimer, indicating that the affinity of the dimeric cIL4Rα-IL13Rα2_ZW1-cFc for cIL-4 was very low and undetectable by this experimental procedure.

[0171] [Table 6]

[0172] The binding constant (KD) of unmodified cIL-13Rα1-cFc to cIL-13 is approximately 5×10 -9 M (see Table 4 below). Engineering cIL-13Rα1-cFc to further increase its half-life, i.e., generating cIL-13Rα1-cFc-YTE, reduced its affinity for cIL-13 by approximately 4-fold, i.e., the KD was reduced to 2×10 -8 In particular, the heterodimeric bispecific cIL4Rα-IL13Rα1_ZW1-cFc increased cIL-13 (approximately 4 × 10 ) more potently than unmodified IL-13Rα1-cFc.-10 M, see Table 4 below). This is in direct contrast to the binding of the exact same heterodimer to cIL-4, which, as noted above, was reduced by nearly four orders of magnitude relative to the corresponding cIL-4Rα-cFc homodimer (see Table 3 above). These results indicate that heterodimer formation can result in dramatic differences in the binding affinities of the two individual monomers of the heterodimer for their respective binding partners.

[0173] From Table 4 below, unmodified cIL-13Rα2-cFc had a titer of approximately 7.5×10 -13 It is clear that the cIL-13Rα2-cFc homodimer binds extremely strongly to cIL-13 with a KD of 1.0 M. In fact, the binding of the cIL-13Rα2-cFc homodimer to cIL-13 is 3-4 orders of magnitude stronger than that of the cIL-13Rα1-cFc homodimer to cIL-13. When cIL-13Rα2-cFc was modified to either cIL-13Rα2-cFc-YTE or cIL-13Rα2-cFc-YD to extend its half-life, the binding affinity for cIL-13 was slightly increased, i.e., the KD was reduced by half (approximately 4.0 × 10 -13 Strikingly, both homodimers of cIL-13Rα2-cFc-YTE or cIL-13Rα2-cFc-YD were reduced to approximately 4 × 10 -9 M (see Table 4 below).

[0174] [Table 7]

[0175] In summary, binding affinity for IL-4 is significantly reduced when the cIL-4Rα-cFc homodimer is replaced by a heterodimer of cIL-4Rα-cFc-ZW-A with either cIL-13Rα1-cFc-ZW-B or cIL-13Rα2-cFc-ZW-B to form cIL4Rα-IL13Rα1_ZW1-cFc and cIL-4Rα-IL13Rα2_ZW1-cFc, respectively, but the reduction in affinity is substantially greater for the cIL-4Rα-IL13Rα2_ZW1-cFc heterodimer. On the other hand, the corresponding binding affinity of IL-13 is increased when the cIL-13Rα1-cFc homodimer is replaced by the cIL4Rα-IL13Rα1_ZW1-cFc heterodimer, whereas the binding affinity of IL-13 is substantially decreased when the cIL-13Rα2-cFc homodimer is replaced by the cIL-4Rα-IL13Rα2_ZW1-cFc heterodimer.

[0176] [Example 5] Inhibition of STAT-6 phosphorylation by cFc fusion proteins The ability of cFc fusion proteins to block IL-4 and IL-13 mediated signaling, as measured by inhibition of STAT-6 phosphorylation in DH82 cells, was determined as follows:

[0177] material: 1. Actively growing DH82 cells: Merck Animal Health Lot:628-011, 24Oct14 2. HBSS, 1X: Corning, Catalog 21-022-CM 3. AlphaLISA p-STAT6(Tyr641) Assay Kit: Perkin Elmer, catalog ALSU-PST6-A10K 4. Recombinant canine IL-4: R&D Systems, Catalog: 752-CL / CF 5a. cFc fusion protein samples for IL-4 research (i) cIL4Rα-cFc (ii) cIL4Rα-IL13Rα1_ZW1-cFc (iii) cIL4Rα-IL13Rα2_ZW1-cFc 5b. cFc fusion protein samples for IL-13 studies: (i) cIL13Rα1-cFc (ii) cIL13Rα2-cFc (iii) cIL4R-IL13Rα1_ZW1-cFc (iv) cIL4R-IL13Rα2_ZW1-cFc 6. About Perkin Elmer® Envision

[0178] method: 1. 1x10 cells per well in tissue culture plates 5 DH82 cells (2.5 × 10 5 Cells were seeded in 40 μL at a density of 100 cells / mL and incubated at 37°C for 2 h. 2. cFc fusion proteins were pre-diluted to 2000 nM (final concentration in well 500 nM) and then serially diluted 3-fold in Hank's Balanced Salt Solution (HBSS). Protein was added by transferring 20 μL / well to each location on a tissue culture plate containing DH82 cells. 3. (a) Canine IL-4 was diluted to 10 ng / mL in HBSS (2.5 ng / mL in well) and 20 μL was added to each well of the plate. The plate was incubated at 37° C. for 15 minutes, or alternatively, (b) Canine IL-13 was diluted to 20 ng / mL in HBSS (5 ng / mL in well) and 20 μL was added to each well of the plate. The plate was incubated at 37° C. for 15 minutes. 4. The plate was removed from the incubator and 20 μL of 4× Lysis Buffer from the AlphaLISA® p-STAT-6 Assay Kit was added to each well of the plate. The plate was agitated at 350 rpm on a plate shaker at room temperature for 10 minutes. 5. Acceptor mix was prepared from the AlphaLISA® p-STAT6 Assay Kit and 15 μL per well was added to 30 μL of cell lysate in a 96-well ½ area plate. The plate was sealed and agitated at 350 rpm for 2 minutes, then incubated at room temperature for 1 hour. 6. Donor mix was prepared in a subdued lab from the AlphaLISA® p-STAT6 Assay Kit and 15 μL / well was added to each plate. Plates were sealed, covered with foil, agitated at 350 rpm for 2 minutes, then incubated at room temperature for 1 hour. 7. Plates were read using the AlphaScreen setting on a Perkin Elmer® EnVison.

[0179] result: (a) The IC50 for inhibition of cIL-4-mediated STAT6 phosphorylation by cIL4Rα-cFc, cIL4Rα-IL13Rα1_ZW1-cFc, and cIL4Rα-IL13Rα2_ZW1-cFc in DH82 is provided in Table 5A below. As can be seen, both the homodimeric cIL-4Rα-cFc and the heterodimeric bispecific cIL-4Rα-IL-13Rα1_ZW1-cFc inhibit 50% of IL-4-mediated STAT6 phosphorylation at concentrations of ∼80 pM and ∼50 pM, respectively, whereas cIL-4Rα-IL-13Rα2_ZW1-cFc inhibits 50% of cIL-4-mediated STAT6 phosphorylation at a concentration more than three orders of magnitude higher (i.e., ∼0.2 μM) than either cIL4Rα-cFc or cIL4Rα-IL13Rα1_ZW1-cFc. Interestingly, in direct contrast to the heterodimeric cIL-4Rα-IL13R2α construct, the heterodimeric cIL-4Rα-IL13R1α construct bound at least as well, if not more strongly, to cIL-4 (see Table 4) than the homodimeric cIL-4Rα-cFc, a relationship that is consistent with the IC values ​​in Table 5A. 50 Matches the data. (b) IC for inhibition of cIL-13-mediated STAT6 phosphorylation by cIL13Rα1-cFc, cIL13Rα2-cFc, cIL4Rα-IL13Rα1_ZW1-cFc, and cIL4Rα-IL13Rα2_ZW1-cFc in DH82 cells. 50 are provided below in Table 5B. As can be seen, cIL-13Rα2-cFc inhibits 50% of cIL-13-mediated STAT6 phosphorylation at a concentration of approximately 165 pM, while cIL13Rα1-cFc, cIL4Rα-IL13Rα1_ZW1-cFc, and cIL4Rα-IL13Rα2_ZW1-cFc all inhibit 50% of cIL-13-mediated STAT6 phosphorylation well above nanomolar concentrations. Thus, the concentration of cIL-4Rα-IL-13Rα1_ZW1-cFc that inhibits 50% of cIL-13-mediated STAT6 phosphorylation is 6-fold lower than that of cIL-13Rα1-cFc but still approximately 20-fold higher than that of cIL-13Rα2-cFc, whereas the concentration of cIL-4Rα-IL-13Rα2_ZW1-cFc that inhibits 50% of cIL-13-mediated STAT6 phosphorylation is approximately 200-fold higher than that of cIL-13Rα2-cFc. Thus, the unmodified cIL-13Rα2-cFc homodimer surprisingly not only binds cIL-13 more strongly than cIL-4Rα-IL-13Rα2_ZW1-cFc (see Table 4 above), but also consistently inhibits cIL-13-mediated STAT6 phosphorylation at concentrations substantially lower than those found for cIL-4Rα-IL-13Rα2_ZW1-cFc (see Table 5B below).

[0180] [Table 8]

[0181] [Table 9]

[0182] Table 6 below summarizes the results obtained and compares the dissociation constants of the various binding partners with their respective IC 50As can be seen, the optimal cytokine trap is a homodimer of cIL-4Rα-cFc containing a homodimer of cIL-13Rα2-cFc.

[0183] [Table 10]

[0184] [Example 6] Prior art caninized antibodies against canine IL-31 Antibodies that may be useful in the present invention are those described in US Patent No. 9,206,253 B2 and US Patent No. 10,150,810 B2. Preferably, these antibodies have the following light and heavy chain sequences:

[0185] Caninized heavy chain sequences from mouse antibody clone M14 and canine IgG-B: [SEQ ID NO: 14] Prior art

number

[0186] Caninized light chain sequence derived from mouse antibody clone M14 and canine light chain constant region: [SEQ ID NO: 15] Prior Art

number

[0187] Z-HC: caninized heavy chain sequence: [SEQ ID NO: 16] Prior art

number

[0188] Z-LC: caninized light chain sequence: [SEQ ID NO: 17] Prior art

number

[0189] [Table 11]

[0190] [Example 7] Caninized antibody against IL-31Rα Rat monoclonal antibody against canine IL-31Rα: Monoclonal antibodies against canine IL-31Rα were produced by immunizing rats multiple times over a period of 3-4 weeks with the extracellular domain (ECD) of canine IL-31Rα (using 25 μg of antigen / animal each time). After immunization, serum was collected from each animal and tested against canine IL-31Rα ECD by ELISA. Lymph node cells from animals with the highest IL-31Rα ECD reactivity were fused with the myeloma SP2 / 0 cell line to produce hybridomas. Approximately 10 days after fusion, supernatants from growing hybridomas were screened by ELISA on IL-31Rα ECD protein-coated plates using the protocol described below. Three rat monoclonal antibodies, 44E3, 10A12 and 28F12, were selected for caninization. These caninized antibodies bind tightly to canine IL-31Rα.

[0191] ELISA procedure: 1. Coat a 96-well half-area plate with 25 μL / well of IL-31Rα (1 μg / mL in PBS buffer). Incubate the plate at 4° C. overnight. 2. Wash the plate three times with PBST (PBS + 0.05% Tween 20). 3. Block plates with blocking buffer (PBS with 5% FBS) at 25ul / well for 30 minutes at room temperature. 4. Transfer 25ul / well hybridoma supernatant to a 96-well plate and incubate at room temperature for 60 minutes. 5. Wash the plate 3 times with PBST. 6. Add 25ul / well of Anti-Rat IgG-HRP conjugate, 1:4000 dilution in blocking buffer to the plate and incubate at room temperature for 60 minutes. 7. Wash the plate 5 times with PBST. 8. Add TMB-based reagent to the plate for a colorimetric reaction for 20-30 minutes. 9. Stop the reaction with 0.16M sulfuric acid. 10. Read the plate with a plate reader. Using this procedure, several hybridomas secreting antibodies reactive with canine IL-31Rα were identified.

[0192] Generation of caninized antibodies and binding of caninized antibodies to canine IL-31Rα: The nucleotide and deduced amino acid sequences of the HC and LC of selected rat antibodies reactive with canine IL-31Rα were determined. The amino acid sequences representing the three HC CDRs and the three LC CDRs for each antibody were also determined. These CDRs were used to develop caninized antibodies that bind to the canine IL-31Rα ECD. Binding of the caninized antibodies to IL-31Rα was determined by ELISA as follows:

[0193] material: 1. Anti-dog IgG (cFc specific)-peroxidase (Sigma-Aldrich SAB3700109-1.5MG) 2. TMB-ELISA Substrate (Thermo-Fisher Cat. No. 34028) 3. PBS pH 7.4 (Thermo-Fisher catalog number 10010001) 4. Tris-buffered saline with Tween 20 (TBST) (Sigma-Aldrich T9039-10PAK)

[0194] method: 1. Coat immunoplates with cIL-31Rα at 1 μg / mL, 100 μL / well in PBS buffer. Incubate plates at 37°C for 1-2 hours or at 4°C overnight. 2. Wash the plate 3 times with TBST buffer. 3. Block the plate with blocking buffer (0.5% BSA in TBST) for 45-60 minutes at room temperature. 4. Dilute the anti-cIL31-Rα antibody three-fold in blocking buffer in the dilution plate and transfer the diluted antibody to the cIL-31Rα-coated plate and incubate at room temperature for 45-60 minutes. 5. Wash the plate 3 times with TBST. 6. Add 1:2000 diluted HRP-conjugated anti-dog IgG Fc to the plate and incubate at room temperature for 45-60 minutes. 7. Wash the plate 3 times with TBST. 8. Add TMB-ELISA substrate to the plate for colorimetric reaction for 10-15 minutes. 9. 1M H 3 PO 4 The reaction is stopped by. 10. Read the plate on a plate reader at 450 nM.

[0195] result: FIG. 1 shows the binding activity of related chimeric and caninized anti-canine IL-31Rα antibodies as assessed by ELISA. Different designs of rat antibody 44E2 were evaluated by ELISA. The ELISA results show that all caninized antibodies bind to canine IL-31Rα with an EC50 similar to that of the chimeric 44E2 antibody, but c44E2 H5k1 binds to canine IL-31Rα with an EC50 most similar to that of the corresponding chimeric 44E2 antibody.

[0196] Figure 2 shows the binding activity of related chimeric and caninized anti-canine IL-31Rα antibodies as assessed by ELISA. Different designs of rat antibody 10A12 were evaluated by ELISA. The ELISA results show that one of the caninized antibodies (c10A12 H2L6) binds to canine IL-31Rα with an EC50 that is even lower than that of the corresponding chimeric 10A12 antibody.

[0197] Figure 3 shows the binding activity of related chimeric and caninized anti-canine IL-31Rα antibodies as assessed by ELISA. Different designs of rat antibody 28F12 were evaluated by ELISA. The ELISA results show that the caninized antibodies bind to canine IL-31Rα with an EC50 that is lower than that of the chimeric 28F12 antibody.

[0198] Below are examples of amino acid sequences of chimeric (rat-dog) and caninized antibodies that may be used in the present invention. The amino acids representing the CDRs are underlined.

[0199] r10A12VH-cIgGBm [SEQ ID NO: 25]

number

[0200] c10A12VH1-cIgGBm [SEQ ID NO: 26]

number

[0201] c10A12VH2-cIgGBm [SEQ ID NO: 27]

number

[0202] r10A12VL-cCl [SEQ ID NO: 28]

number

[0203] c10A12VL4-cCl [SEQ ID NO: 29]

number

[0204] c10A12VL5-cCl [SEQ ID NO: 30]

number

[0205] c10A12VL6-cCl [SEQ ID NO: 31]

number

[0206] r28F12VH-cIgGBm [SEQ ID NO: 32]

number

[0207] c28F12VH1-cIgGBm [SEQ ID NO: 33]

number

[0208] c28F12VH2-cIgGBm [SEQ ID NO: 34]

number

[0209] r28F12VL-cCk [SEQ ID NO: 35]

number

[0210] c28F12VL1-cCk [SEQ ID NO: 36]

number

[0211] c28F12VL2-cCk [SEQ ID NO: 37]

number

[0212] c28F12VL3-cCk [SEQ ID NO: 38]

number

[0213] c28F12VL4-cCk [SEQ ID NO: 39]

number

[0214] r44E2VH-cIgGBm [SEQ ID NO: 40]

number

[0215] c44E2VH1-cIgGBm [SEQ ID NO: 41]

number

[0216] c44E2VH4-cIgGBm [SEQ ID NO: 42]

number

[0217] c44E2VH5-cIgGBm [SEQ ID NO: 43]

number

[0218] r44E2VL-cCk [SEQ ID NO: 44]

number

[0219] c44E2VL1-cCk [SEQ ID NO: 45]

number

[0220] c44E2VL2-cCk [SEQ ID NO: 46]

number

[0221] c44E2VL4-cCk [SEQ ID NO: 47]

number

[0222] [Table 12]

[0223] [Table 13]

[0224] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

Claims

1. 1. A composition comprising a homodimer comprising a pair of canine interleukin-4 receptor alpha-canine fragment crystallizable domain fusion proteins (cIL-4Rα-cFc fusion proteins), or a homodimer comprising a pair of canine interleukin-13 receptor alpha 2-canine fragment crystallizable domain fusion proteins (cIL-13Rα2-cFc fusion proteins), or both a homodimer comprising said pair of cIL-4Rα-cFc fusion proteins and a homodimer comprising said pair of cIL-13Rα2-cFc fusion proteins, each of the pair of cIL-4Rα-cFc fusion proteins comprises the extracellular domain (ECD) or fragment thereof of canine interleukin-4 receptor alpha (cIL-4Rα) that binds canine interleukin-4 (cIL-4) and a first canine fragment crystallizable region (cFc); and each of the pair of cIL-13Rα2-cFc fusion proteins comprises the extracellular domain (ECD) of canine interleukin-13 receptor alpha 2 (cIL-13Rα2) or a fragment thereof that binds canine interleukin-13 (cIL-13), and a second cFc; A composition wherein said first cFc and said second cFc are either the same or different.

2. A composition comprising a canine interleukin-4 receptor alpha-canine fragment crystallizable domain fusion protein (cIL-4Rα-cFc fusion protein), or a canine interleukin-13 receptor alpha 2-canine fragment crystallizable domain fusion protein (cIL-13Rα2-cFc fusion protein), or both the cIL-4Rα-cFc fusion protein and the cIL-13Rα2-cFc fusion protein, the cIL-4Rα-cFc fusion protein comprises the extracellular domain (ECD) of canine interleukin-4 receptor alpha (cIL-4Rα) or a fragment thereof that binds canine interleukin-4 (cIL-4), and a first canine fragment crystallizable region (cFc); the cIL-13Rα2-cFc fusion protein comprises the extracellular domain (ECD) of canine interleukin-13 receptor alpha 2 (cIL-13Rα2) or a fragment thereof that binds canine interleukin-13 (cIL-13), and a second cFc; A composition wherein said first cFc and said second cFc are either the same or different.

3. 3. The composition of claim 1 or 2, wherein the first cFc and the second cFc each comprise an amino acid sequence independently selected from the group consisting of: SEQ ID NO: 1, an amino acid sequence having at least 90%, 95%, or 99% identity to SEQ ID NO: 1; SEQ ID NO: 2, an amino acid sequence having at least 90%, 95%, or 99% identity to SEQ ID NO: 2; SEQ ID NO: 3, an amino acid sequence having at least 90%, 95%, or 99% identity to SEQ ID NO: 3; SEQ ID NO: 4, an amino acid sequence having at least 90%, 95%, or 99% identity to SEQ ID NO: 4; and SEQ ID NO: 51, or an amino acid sequence having at least 90%, 95%, and 99% identity to SEQ ID NO:

51.

4. each of the cIL-4Rα-cFc fusion proteins further comprises a first canine hinge region, the first canine hinge region being positioned between the ECD or fragment thereof of the cIL-4Rα and the first cFc; and 3. The composition of claim 1, wherein each of the cIL-13Rα2-cFc fusion proteins further comprises a second canine hinge region, wherein the second canine hinge region is located between the ECD or fragment thereof of the cIL-13Rα2 and the second cFc, and the first canine hinge region and the second canine hinge region are either the same or different.

5. 5. The composition of claim 4, wherein the first canine hinge region and the second canine hinge region each comprise an amino acid sequence independently selected from the group consisting of SEQ ID NO:21, an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:21, SEQ ID NO:22, an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:22, SEQ ID NO:23, an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:23, SEQ ID NO:24, and an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:

24.

6. the ECD of cIL-4Rα is comprising SEQ ID NO: 48 or an amino acid sequence having at least 85%, 90%, 95% identity to SEQ ID NO: 48; or the ECD of cIL-13Rα2 comprises SEQ ID NO:50 or an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:50; The composition according to claim 1 or 2.

7. 5. The composition of claim 4, wherein the first canine hinge region comprises a first hinge region sequence that is identical to an amino acid sequence in a protein naturally found in a canine (including naturally occurring variants thereof), and the first hinge region sequence is the only sequence located between the ECD or fragment thereof of the cIL-4Rα and the first cFc.

8. 5. The composition of claim 4, wherein the second canine hinge region comprises a second hinge region sequence that is identical to an amino acid sequence in a protein naturally found in a canine (including naturally occurring variants thereof), and the second hinge region sequence is the only sequence located between the ECD or fragment thereof of the cIL-13Rα2 and the second cFc.

9. 3. The composition of claim 1, wherein each of the cIL-4Rα-cFc fusion proteins consists solely of an amino acid sequence identical to the amino acid sequence of a protein naturally found in a dog (including naturally occurring variants thereof), and / or wherein each of the cIL-13Rα2-cFc fusion proteins consists solely of an amino acid sequence identical to the amino acid sequence of a protein naturally found in a dog (including naturally occurring variants thereof).

10. 3. The composition of claim 1 or 2, wherein each of the cIL-4Rα-cFc fusion proteins comprises an amino acid sequence having at least 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:5, or wherein each of the cIL-4Rα-cFc fusion proteins comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:

12.

11. 3. The composition of claim 1, wherein each of the cIL-13Rα2-cFc fusion proteins comprises an amino acid sequence having at least 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 7, or wherein each of the cIL-13Rα2-cFc fusion proteins comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO:

13.

12. The composition of claim 1 or 2, further comprising a canine anti-pruritic antibody or a caninized anti-pruritic antibody.

13. 13. The composition of claim 12, wherein the canine anti-pruritic antibody or caninized anti-pruritic antibody is selected from the group consisting of a caninized antibody capable of binding to canine interleukin-31 (cIL-31), a canine antibody capable of binding to cIL-31, a caninized antibody capable of binding to canine interleukin-31R (cIL-31R), and a canine antibody capable of binding to cIL-31R.

14. the caninized antibody capable of binding to cIL-31, (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 17; The composition of claim 13 comprising:

15. the caninized antibody capable of binding to cIL-31R, (i) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26 and SEQ ID NO:27, and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; (ii) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33 and SEQ ID NO: 34, and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39; and (iii) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; 15. The composition of claim 14, selected from the group consisting of:

16. The composition of claim 1 or 2, further comprising one or more additional components selected from the group consisting of a Janus kinase (JAK) inhibitor, a spleen tyrosine kinase (SYK) inhibitor, or an antagonist of a chemoattractant receptor-homologous molecule expressed on TH2 cells.

17. The JAK inhibitor (a) 【Chemical 1】 A compound represented by the formula (wherein R １ is C optionally substituted by hydroxy １～４ alkyl) or a pharmaceutically acceptable salt thereof; (b) 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, or (c) 【Chemistry 3】 or a pharmaceutically acceptable salt thereof 17. The composition of claim 16, wherein: (a) a cIL-13Rα2-cFc fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13; or (b) an antibody comprising: (b-1) A heavy chain of a caninized antibody capable of binding to canine IL-31, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO:

16. and (b-2) A light chain of a caninized antibody capable of binding to canine IL-31, wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO:

17. A nucleic acid encoding

19. A vector comprising the nucleic acid described in claim 18.

20. A cell containing the vector described in claim 19.

21. A method for treating atopic dermatitis, comprising administering the composition of claim 1 or 2 to a dog having atopic dermatitis.

22. A composition as described in claim 1 or 2 for use in the treatment of atopic dermatitis in canine subjects.

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