Anti-IL-5 antibodies and their use

Abstract

The present invention provides novel anti-IL-5 proteins, antibodies, and IL-5 binding fragments thereof that inhibit the binding of IL-5 to the IL-5 receptor and are suitable for administration to human or feline subjects. The present invention also provides novel compositions and methods for treating, alleviating, or preventing lung diseases, cardiovascular diseases, cancer, metabolic diseases, neurological diseases, and infections, comprising administering an effective amount of the anti-IL-5 protein, antibody, or fragment thereof. The methods and compositions are used for the purpose of treating or preventing IL-5-related diseases.

Description

[Technical Field]

[0001] Reference This application claims priority to U.S. Provisional Application No. 63 / 532,157, filed on 11 August 2023, which is incorporated herein by reference in its entirety.

[0002] All documents cited or referenced herein (hereinafter referred to as "Cited Documents"), and all documents cited or referenced in the Cited Documents, together with the manufacturers' instructions, manuals, product specifications, and product sheets relating to any product described herein or any document incorporated herein by reference, are incorporated herein by reference in their entirety and can be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference in the same manner as each individual document is specifically and individually indicated as being incorporated by reference.

[0003] Sequence List The specification of this application, which was initially filed, includes / contains an electronic sequence listing in XML (eXtensible Markup Language) format, created on August 29, 2024, with a file size of 123,249 bytes and filename Y9432-99004.xml. The entirety of this sequence listing is incorporated herein by reference.

[0004] The present invention provides novel anti-IL-5 proteins and antibodies suitable for administration to human or feline subjects. The present invention also provides novel compositions and methods for treating asthma in human or feline subjects, or for inducing anti-asthmatic or anti-allergic effects in these subjects, comprising administering an effective amount of anti-IL-5 protein, antibody, or fragment thereof. These methods and compositions are used for the purpose of treating or preventing IL-5-related diseases. [Background technology]

[0005] Interleukin 5 (IL-5) is an interleukin produced by type 2 T helper cells and mast cells. IL-5 stimulates B cell growth and increases immunoglobulin (mainly IgA) secretion by binding to its receptor. IL-5 is also an important regulator of eosinophil activation.

[0006] IL-5 is a TH2 cytokine consisting of 115 amino acids (in humans; 133 in mice) that is part of the hematopoietic family. Unlike other members of this cytokine family (interleukin-3 and CM-CSF), the active form of this glycoprotein is a homodimer.

[0007] IL-5 has long been associated with the cause of several allergic diseases, including allergic rhinitis and asthma, in which a marked increase in eosinophil counts is observed in peripheral blood, airway tissue, and induced sputum.

[0008] No reference or specification of any document in this application constitutes an acceptance that such document is available as prior art to the present invention. [Overview of the project]

[0009] The present invention provides an antigen-binding protein that specifically binds to interleukin-5 (IL-5). In certain embodiments, the IL-5 binding protein comprises (a) a heavy chain complementarity-determining region 1 (HCDR1) containing X1, X2, X3, X4, X5, X6, X7, and X8 (wherein X1 comprises A, G, I, L, M, W, F, P, or V; X2 comprises A, G, I, L, M, W, F, P, or V; X3 comprises C, S, T, Y, N, or Q; X4 comprises A, G, I, L, M, W, F, P, or V; X5 comprises A, G, I, L, M, W, F, P, or V; X6 comprises H, K, or R; X7 comprises C, S, T, Y, N, or Q; and X8 comprises C, S, T, Y, N, or Q). (b) Heavy chain complementarity determination region 2 (HCDR2) including X1X2X3X4X5X6X7X8 (wherein X1 includes A, G, I, L, M, W, F, P, or V; X2 includes A, G, I, L, M, W, F, P, or V; X3 includes C, S, T, Y, N, or Q; X4 includes A, G, I, L, M, W, F, P, or V; X5 includes A, G, I, L, M, W, F, P, or V; X6 includes H, K, or R; X7 includes C, S, T, Y, N, or Q; X8 includes C, S, T, Y, N, or Q) and (c) X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 Heavy chain complementarity determination region 3 (HCDR3) including (where X1 includes C, S, T, Y, N, or Q; X2 includes H, K, or R; X3 includes E or D; X4 includes C, S, T, Y, N, or Q; X5 includes E or D; X6 includes A, G, I, L, M, W, F, P, or V; X7 includes C, S, T, Y, N, or Q; X8 includes D, E, or G; X9 includes A, G, I, L, M, W, F, P, or V, X 10 This includes A, G, I, L, M, W, F, P, or V, and X 11 This includes D, E, or L, and X 12(d) Light chain complementarity determination region 1 (LCDR1) including X1X2X3X4X5X6 (X1 includes C, S, T, Y, N, or Q, X2 includes C, S, T, Y, N, or Q, X3 includes A, G, I, L, M, W, F, P, or V, X4 includes C, S, T, Y, N, or Q, X5 includes E or D, X6 includes C, S, T, Y, N, or Q), and (e) X1X2X3X4X5X6X7X8X9X 10 Light chain complementarity determination region 2 (LCDR2) including (where X1 includes L or R, X2 includes A, G, I, L, M, W, F, P, or V, X4 includes C, S, T, Y, N, or Q, X5 includes C, S, T, Y, N, or Q, X6 includes D, E, A, G, I, L, M, W, F, P, or V, X7 includes C, S, T, Y, N, or Q, X8 includes C, S, T, Y, N, Q, A, G, I, L, M, W, F, P, or V, X9 includes D, E, C, S, T, Y, N, or Q, X 10 (f) Light chain complementarity determination region 3 (LCDR3) including X1X2X3X4X5X6X7X8X9 (where X1 includes C, S, T, Y, N, or Q; X2 includes C, S, T, Y, N, or Q; X3 includes A, G, I, L, M, W, F, P, or V; X4 includes A, H, I, K X5 includes L, M, P, R, S, V, or Y, X6 includes A, G, I, L, R, S, T, Y, N, or Q, X7 includes A, G, I, L, M, W, F, P, or V, X8 includes C, S, T, F, Y, N, or Q, and X9 includes C, S, T, Y, N, or Q).

[0010] In certain embodiments, the IL-5 binding protein is (a) HCDR1 containing GFTFSNYA (SEQ ID NO: 60) or different at one or two positions, and / or (b) HCDR2 containing IGSGGHYT (SEQ ID NO: 61) or different at one or two positions, and / or (c) TRETDGYX8X9X10 X 11 X 12 HCDR3 comprising (SEQ ID NO: 81), provided that X8 comprises D or G, X9 comprises G or P, X 10 comprises I, L, or M, X 11 comprises D or L, X 12 comprises L, V, or Y), and / or (d) LCDR1 comprising QSISDY (SEQ ID NO: 62) or differing therefrom at one or two positions, and / or (e) LCDR2 comprising X1X2X3X4X5X6SX8X9X 10 , provided that X5 comprises A, F, G, H, P, Q, S, T, V, or Y, and X6 comprises A, D, E, G, L, M, P, S, V, or Y, and / or (f) LCDR3 comprising QX2GX4X5FPX8T (SEQ ID NO: 82), provided that X2 comprises S, N, or Q, X4 comprises A, H, I, L, M, P, S, V, or Y, X5 comprises F, I, L, Q, R, S, or V, and X8 comprises F or Y).

[0011] In certain embodiments, the IL-5 binding protein comprises LCDR2, provided that X1 comprises L or R, X2 comprises I, L, or V, X3 comprises I or F, X4 comprises F, K, N, or Y, X5 comprises A, F, G, H, P, Q, S, T, V, or Y, X6 comprises A, D, E, G, L, M, P, S, V, or Y, X8 comprises A, D, G, L, P, Q, S, V, or Y, X9 comprises E, G, K, L, Q, R, S, T, or Y, and X 10 comprises A, D, I, L, K, N, Q, R, S, or V.

[0012] In certain embodiments, the number of substitutions comprised in the IL-5 binding protein is no more than 2 per CDR compared to HCDR1, HCDR2, and HCDR3 shown in FIG. 3, and LCDR1, LCDR2, and LCDR3 shown in FIG. 4.

[0013] In certain embodiments, the substitutions contained in the IL-5 binding protein are one or less per CDR compared to HCDR1, HCDR2, and HCDR3 shown in Figure 3, and LCDR1, LCDR2, and LCDR3 shown in Figure 4.

[0014] In certain embodiments, the IL-5 binding protein is one or more HCDRs from among SEQ ID NOs: 3, 5, 8, 11, 19, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58, and SEQ ID NOs: 4, 6, 7, Includes one or more LCDRs of sequence numbers 9, 10, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or 59.

[0015] In certain embodiments, the IL-5 binding protein comprises an HCDR of any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58, and an LCDR of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59.

[0016] In certain embodiments, the IL-5 binding protein comprises each HCDR of SEQ ID NO: 20 and each LCDR of SEQ ID NO: 23, each HCDR of SEQ ID NO: 19 and each LCDR of SEQ ID NO: 21, each HCDR of SEQ ID NO: 19 and each LCDR of SEQ ID NO: 22, each HCDR of SEQ ID NO: 19 and each LCDR of SEQ ID NO: 23, each HCDR of SEQ ID NO: 20 and each LCDR of SEQ ID NO: 21, or each HCDR of SEQ ID NO: 20 and each LCDR of SEQ ID NO: 22.

[0017] In a particular embodiment, the IL-5 binding protein is distributed with each HCDR of SEQ ID NO: 24 and each LCDR of SEQ ID NO: 25, each HCDR of SEQ ID NO: 26 and each LCDR of SEQ ID NO: 27, each HCDR of SEQ ID NO: 28 and each LCDR of SEQ ID NO: 29, each HCDR of SEQ ID NO: 30 and each LCDR of SEQ ID NO: 31, each HCDR of SEQ ID NO: 32 and each LCDR of SEQ ID NO: 33, each HCDR of SEQ ID NO: 34 and each LCDR of SEQ ID NO: 35, each HCDR of SEQ ID NO: 36 and each LCDR of SEQ ID NO: 37, each HCDR of SEQ ID NO: 38 and each LCDR of SEQ ID NO: 39, and each HCDR of SEQ ID NO: 40. Includes each LCDR in column number 41, each HCDR in sequence number 42 and each LCDR in sequence number 43, each HCDR in sequence number 44 and each LCDR in sequence number 45, each HCDR in sequence number 46 and each LCDR in sequence number 47, each HCDR in sequence number 48 and each LCDR in sequence number 49, each HCDR in sequence number 50 and each LCDR in sequence number 51, each HCDR in sequence number 52 and each LCDR in sequence number 53, each HCDR in sequence number 54 and each LCDR in sequence number 55, each HCDR in sequence number 56 and each LCDR in sequence number 57, or each HCDR in sequence number 58 and each LCDR in sequence number 59.

[0018] In a particular embodiment, the IL-5 binding protein includes each HCDR of SEQ ID NO: 8 and each LCDR of SEQ ID NO: 7, each HCDR of SEQ ID NO: 11 and each LCDR of SEQ ID NO: 7, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 9, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 10, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 12, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 13, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 14, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 15, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 16, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 17, or each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 18.

[0019] In certain embodiments, the IL-5 binding protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, or at least 95% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58, or comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) identical to any of the above.

[0020] In certain embodiments, the IL-5 binding protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, or at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, or comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) identical to any of the above.

[0021] In certain embodiments, the IL-5 binding protein is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 97% identical to, or contains the same VH domain as, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO: 58.

[0022] In certain embodiments, the IL-5 binding protein is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, or at least 97% identical to, or contains the same VL domain as, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59.

[0023] In certain embodiments, the antigen-binding protein includes a feline framework or a felineized framework. In certain embodiments, the antigen-binding protein includes a canine framework or a canineized framework. In certain embodiments, the antigen-binding protein includes a human framework or a humanized framework.

[0024] In one embodiment, the present invention provides an isolated nucleic acid sequence encoding any one of the above-mentioned anti-IL-5 antibodies or antibody fragments, and a vector that can contain or express any one of the above-mentioned anti-IL-5 antibodies or antibody fragments.

[0025] In another embodiment, the present invention provides recombinant cells comprising a nucleic acid sequence encoding any one of the above-described anti-IL-5 antibodies or antibody fragments, or a vector that contains or can express any one of the above-described anti-IL-5 antibodies or antibody fragments.

[0026] The present invention provides a method for producing one of the above-mentioned anti-IL-5 antibodies or antibody fragments, comprising culturing cells capable of expressing the anti-IL-5 antibody or antibody fragment under conditions that result in the production of the antibody or antibody fragment.

[0027] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one of the above-mentioned anti-IL-5 antibodies or antibody fragments.

[0028] In one embodiment, the present invention provides a method for suppressing IL-5-mediated activation of eosinophils, comprising culturing eosinophils in the presence of one of the above-mentioned anti-IL-5 antibodies or antibody fragments.

[0029] In one embodiment, the present invention provides a method for suppressing an eosinophil-mediated inflammatory response in a subject, comprising administering a therapeutically effective amount of one of the above-mentioned anti-IL-5 antibodies or antibody fragments to the subject.

[0030] In one embodiment, the present invention provides a method for inhibiting the binding of IL-5 to an IL-5 receptor in a subject, comprising administering a therapeutically effective amount of one of the above-mentioned anti-IL-5 antibodies or antibody fragments to the subject.

[0031] In one embodiment, the present invention provides a method for detecting IL-5 in a sample, comprising: incubating the sample with one of the above-mentioned anti-IL-5 antibodies or antibody fragments; and detecting the anti-IL-5 antibody or antibody fragment bound to IL-5 in the sample.

[0032] Therefore, one of the objectives of the present invention is that no prior art products, manufacturing processes for products, or methods of using products are included in the present invention, and the applicant hereby discloses and releases all liability for any prior art products, processes, or methods. Furthermore, the present invention is not intended to include any products, processes, manufacturing processes, or methods of using products that do not meet the description and enablement requirements of the United States Patent and Trademark Office (USPTO) (Section 112, paragraph 1 of the U.S. Patent Act) or the requirements of the European Patent Office (EPO) (Section 83 of the European Patent Convention), and the applicant hereby discloses and releases all liability for any prior art products, manufacturing processes for products, or methods of using products. In practicing the present invention, it may be advantageous to comply with Section 53(c) and Rules 28(b) and (c) of the European Patent Convention (EPC). All rights expressly reserved are reserved to expressly exclude any embodiment that is the subject matter of any patent(s) granted by the applicant in the lineage of this application or any other lineage or any prior application of any third party. Nothing in this specification should be construed as a promise.

[0033] In this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have meanings attributable to them under U.S. patent law, for example, they may mean “include,” “included,” and “including,” and terms such as “consisting essentially of” and “consist essentially of” may have meanings attributable to them under U.S. patent law, for example, they may allow elements not expressly described but should be noted to exclude elements found in the prior art or that affect the fundamental or novel features of the present invention.

[0034] These embodiments and other embodiments are disclosed in the following detailed description or are obvious and incorporated into the detailed description.

[0035] The following detailed description is illustrative and not intended to limit the invention to the specific embodiments described herein. A deeper understanding can be obtained by referring to it in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0036] [Figure 1A] This shows the amino acid sequence of the IL-5 receptor α (IL-5Rα) construct, including the signal sequence, the extracellular domain (ECD) of the feline IL-5 receptor α, the V5 epitope, the TEV protease site, and human IgG1 Fc (SEQ ID NO: 1). [Figure 1B] The amino acid sequence of the IL-5Rα construct, which includes the signal sequence, the extracellular domain (ECD) of the feline IL-5 receptor α, 2×(Gly-Gly-Gly-Ser) (SEQ ID NO: 89), human IgG1, and AviTag (SEQ ID NO: 2), is shown. [Figure 2]The amino acid sequences of the VHCH1 portion (A) (SEQ ID NO: 127), the VLCL portion (B) (SEQ ID NO: 129), and the VLCL portion (C) (SEQ ID NO: 128) of the MtxA and MtxB antibodies described herein are shown. The MtxA and MtxB antibodies share the same VH domain (A) (SEQ ID NO: 127) containing each VHCDR of the mouse anti-feline IL-5 antibody 154. The VL domain (B) (SEQ ID NO: 129) of MtxA and the VL domain (C) (SEQ ID NO: 128) of MtxA share the same VLCDRs as the mouse anti-feline IL-5 antibody 154, but have different FRs. IMGT CDRs are shown in bold. The MtxA and MtxB clones have a potential deamidation site (NG) (within the boxes for B and C) in LCDR3 and a potential oxidation site (M) (within the box for A) in HCDR3. [Figure 3] Sequence numbers (SEQ ID NOs: 3, 5, 8, 11, 19, 20, 24, 26, 28, 30, 32, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58) show the sequence alignment of the VH domains of the binding proteins of the present invention. [Figure 4] Sequence numbers 4, 6, 7, 9, 10, 12-18, 21-23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 90 show the sequence alignment of the VL domains of the binding proteins of the present invention. [Modes for carrying out the invention]

[0037] According to certain exemplary embodiments of the present invention, the IL-5 binding protein is an anti-IL-5 antibody or its antigen-binding fragment. As used herein, the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains of two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as their polymers (e.g., IgM). In typical antibodies, each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interposed by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the antibody (or their antigen-binding moieties) may be identical to the germline sequence of felines, or may be naturally occurring or artificially modified. The amino acid consensus sequence can be defined by analyzing two or more CDRs side by side.

[0038] Antibody residues that significantly influence the affinity and specificity of binding to target antigens are primarily located in the CDR (Cellular Deposition Range). Kabat et al. first proposed a standardized numbering scheme for immunoglobulin variable regions by collecting and aligning the heavy and light chain sequences of immunoglobulins to identify conserved regions, hypervariable regions, and their residues. (Kabat EA et al., 1979, Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain and BP-Microglobulins, Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health). While the Kabat system is a widely adopted standard for numbering antibody residues, the hypervariable regions defined by Kabat do not precisely correspond to the structural aspects of the antigen-binding loop. Chothia and Lesk developed a structure-based numbering scheme by aligning the crystal structures of antibody variable regions and classified CDR loops into a small number of "canonical" classes (Chothia C, et al., 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196:901-17. doi: 10.1016 / 0022-2836(87)90412-8). Advantages of the Chothia numbering scheme include the fact that topologically aligned residues from different antibodies are assigned the same positional number, and that Chothia's definition of CDRs coincides with structural antigen-binding loops in most antibody sequences.Lefranc introduced a novel germline sequence-based system aimed at standardizing the numbering of all proteins in the immunoglobulin superfamily, including T cell receptor chains (Giudicelli V et al., 1997, IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 25:206-11), which was subsequently extended to encompass all variable domains (Lefranc MP et al., 2003, IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. 27:55-77. doi: 10.1016 / S0145-305X(02)00039-3). This system also aligns atypical frameworks (Abhinandan KR et al., 2008, Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol.). Further numbering systems have also been proposed for dividing variable chain sequences into multiple fragments containing a structurally immutable "core" (Gelfand et al., 1998, Algorithmic determination of core positions in the VL and VH domains of immunoglobulin molecules. J Comput Biol. (1998) 5:467-77). In certain embodiments of the present invention, CDR residues are identified according to the standard systems described above. In certain embodiments, the antibody of the present invention is identified by all or a subset of Kabat CDR residues of the antibody sequence described herein.In certain embodiments, the antibody of the present invention is identified by all or a subset of Chothia CDR residues in the antibody sequences described herein. In certain embodiments, the antibody of the present invention is identified by all or a subset of IMGT CDR residues in the antibody sequences described herein. In certain embodiments, the antibody of the present invention is identified by CDR residues defined by two or more systems, including, for example, all or a subset of HCDR1 residues by Kabat, all or a subset of HCDR2 residues by Chothia, all or a subset of HCDR3 residues by Kabat, all or a subset of LCDR1 residues by Kabat, all or a subset of LCDR2 residues by IMGT, and all or a subset of LCDR3 residues by Chothia.

[0039] For reference, the table below shows the relative positions of Kabat, Chothia, and IMGT CDRs mapped to the VH (SEQ ID NO: 3) and VL (SEQ ID NO: 4) of the feline IL-5-binding mouse antibody 154 described herein. In this specification, "XXX" represents X1X2X3X4X5X6X7X8 for HCDR1, X1X2X3X4X5X6X7X8 for HCDR2, and X1X2X3X4X5X6X7X8X9X for HCDR3. 10 X 11 X 12 LCDR1 has X1X2X3X4X5X6, and LCDR2 has X1X2X3X4X5X6X7X8X9X 10 In LCDR3, this is the CDR area represented as X1X2X3X4X5X6X7X8X9. [Table 1]

[0040] In one embodiment, the present invention provides a binding protein suitable for use in mammals, such as felines (but not limited to them). In a particular embodiment, the feline anti-IL-5 binding protein comprises a heavy chain complementarity determination region 1 (HCDR1), a heavy chain complementarity determination region 2 (HCDR2), a heavy chain complementarity determination region 3 (HCDR3), a light chain complementarity determination region 1 (LCDR1), a light chain complementarity region 2 (LCDR2), and a light chain complementarity region 3 (LCDR3).

[0041] In some embodiments, the heavy chain complementarity determination region 1 (HCDR1) has a length of 3 to 25 amino acids. In some embodiments, HCDR1 has a length of 5 to 15 amino acids. In some embodiments, HCDR1 has a length of 6 to 10 amino acids. In some embodiments, HCDR1 has a length of 3 amino acids. In some embodiments, HCDR1 has a length of 4 amino acids. In some embodiments, HCDR1 has a length of 5 amino acids. In some embodiments, HCDR1 has a length of 6 amino acids. In some embodiments, HCDR1 has a length of 7 amino acids. In some embodiments, HCDR1 has a length of 8 amino acids. In some embodiments, HCDR1 has a length of 9 amino acids. In some embodiments, HCDR1 has a length of 10 amino acids. In some embodiments, HCDR1 has a length of 11 amino acids. In some embodiments, HCDR1 has a length of 12 amino acids. In some embodiments, HCDR1 has a length of 13 amino acids. In some embodiments, HCDR1 has a length of 14 amino acids. In some embodiments, HCDR1 is the length of 15 amino acids. In some embodiments, HCDR1 is the length of 16 amino acids. In some embodiments, HCDR1 is the length of 17 amino acids. In some embodiments, HCDR1 is the length of 18 amino acids. In some embodiments, HCDR1 is the length of 19 amino acids. In some embodiments, HCDR1 is the length of 20 amino acids. In some embodiments, HCDR1 is the length of 21 amino acids. In some embodiments, HCDR1 is the length of 22 amino acids. In some embodiments, HCDR1 is the length of 23 amino acids. In some embodiments, HCDR1 is the length of 24 amino acids. In some embodiments, HCDR1 is the length of 25 amino acids.In some embodiments, HCDR1 comprises the amino acid sequence X1X2X3X4X5X6X7X8 (wherein X1 comprises a nonpolar amino acid, X2 comprises a nonpolar amino acid, X3 comprises a polar amino acid, X4 comprises a nonpolar amino acid, X5 comprises a polar amino acid, X6 comprises a polar amino acid, X7 comprises a polar amino acid, and X8 comprises a nonpolar amino acid). In some embodiments, X1 comprises A, G, I, L, M, W, F, P, or V; X2 comprises A, G, I, L, M, W, F, P, or V; X3 comprises C, S, T, Y, N, or Q; X4 comprises A, G, I, L, M, W, F, P, or V; X5 comprises C, S, T, Y, N, or Q; X6 comprises C, S, T, Y, N, or Q; X7 comprises C, S, T, Y, N, or Q; and X8 comprises A, G, I, L, M, W, F, P, or V. In some embodiments, X1 comprises G, X2 comprises F, X3 comprises T, X4 comprises F, X5 comprises S, X6 comprises N, X7 comprises Y, and X8 comprises A. In some embodiments, HCDR1 comprises the amino acid sequence GFTFSNYA (Sequence ID 60).

[0042] In some embodiments, the heavy chain complementarity determination region 2 (HCDR2) has a length of 3 to 25 amino acids. In some embodiments, HCDR2 has a length of 5 to 15 amino acids. In some embodiments, HCDR2 has a length of 6 to 10 amino acids. In some embodiments, HCDR2 has a length of 3 amino acids. In some embodiments, HCDR2 has a length of 4 amino acids. In some embodiments, HCDR2 has a length of 5 amino acids. In some embodiments, HCDR2 has a length of 6 amino acids. In some embodiments, HCDR2 has a length of 7 amino acids. In some embodiments, HCDR2 has a length of 8 amino acids. In some embodiments, HCDR2 has a length of 9 amino acids. In some embodiments, HCDR2 has a length of 10 amino acids. In some embodiments, HCDR2 has a length of 11 amino acids. In some embodiments, HCDR2 has a length of 12 amino acids. In some embodiments, HCDR2 has a length of 13 amino acids. In some embodiments, HCDR2 has a length of 14 amino acids. In some embodiments, HCDR2 is the length of 15 amino acids. In some embodiments, HCDR2 is the length of 16 amino acids. In some embodiments, HCDR2 is the length of 17 amino acids. In some embodiments, HCDR2 is the length of 18 amino acids. In some embodiments, HCDR2 is the length of 19 amino acids. In some embodiments, HCDR2 is the length of 20 amino acids. In some embodiments, HCDR2 is the length of 21 amino acids. In some embodiments, HCDR2 is the length of 22 amino acids. In some embodiments, HCDR2 is the length of 23 amino acids. In some embodiments, HCDR2 is the length of 24 amino acids. In some embodiments, HCDR2 is the length of 25 amino acids.In some embodiments, HCDR2 comprises the amino acid sequence X1X2X3X4X5X6X7X8 (wherein X1 comprises a nonpolar amino acid, X2 comprises a nonpolar amino acid, X3 comprises a polar amino acid, X4 comprises a nonpolar amino acid, X5 comprises a nonpolar amino acid, X6 comprises a basic amino acid, X7 comprises a polar amino acid, and X8 comprises a polar amino acid). In some embodiments, X1 comprises A, G, I, L, M, W, F, P, or V; X2 comprises A, G, I, L, M, W, F, P, or V; X3 comprises C, S, T, Y, N, or Q; X4 comprises A, G, I, L, M, W, F, P, or V; X5 comprises A, G, I, L, M, W, F, P, or V; X6 comprises H, K, or R; X7 comprises C, S, T, Y, N, or Q; and X8 comprises C, S, T, Y, N, or Q. In some embodiments, X1 comprises I, X2 comprises G, X3 comprises S, X4 comprises G, X5 comprises G, X6 comprises H, X7 comprises Y, and X8 comprises T. In some embodiments, HCDR2 comprises the amino acid sequence IGSGGHYT (Sequence ID 61).

[0043] In some embodiments, the heavy chain complementarity determination region 3 (HCDR3) has a length of 3 to 25 amino acids. In some embodiments, HCDR3 has a length of 5 to 15 amino acids. In some embodiments, HCDR3 has a length of 6 to 10 amino acids. In some embodiments, HCDR3 has a length of 3 amino acids. In some embodiments, HCDR3 has a length of 4 amino acids. In some embodiments, HCDR3 has a length of 5 amino acids. In some embodiments, HCDR3 has a length of 6 amino acids. In some embodiments, HCDR3 has a length of 7 amino acids. In some embodiments, HCDR3 has a length of 8 amino acids. In some embodiments, HCDR3 has a length of 9 amino acids. In some embodiments, HCDR3 has a length of 10 amino acids. In some embodiments, HCDR3 has a length of 11 amino acids. In some embodiments, HCDR3 has a length of 12 amino acids. In some embodiments, HCDR3 has a length of 13 amino acids. In some embodiments, HCDR3 has a length of 14 amino acids. In some embodiments, HCDR3 is the length of 15 amino acids. In some embodiments, HCDR3 is the length of 16 amino acids. In some embodiments, HCDR3 is the length of 17 amino acids. In some embodiments, HCDR3 is the length of 18 amino acids. In some embodiments, HCDR3 is the length of 19 amino acids. In some embodiments, HCDR3 is the length of 20 amino acids. In some embodiments, HCDR3 is the length of 21 amino acids. In some embodiments, HCDR3 is the length of 22 amino acids. In some embodiments, HCDR3 is the length of 23 amino acids. In some embodiments, HCDR3 is the length of 24 amino acids. In some embodiments, HCDR3 is the length of 25 amino acids. In some embodiments, HCDR3 is the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12Includes (wherein X1 contains polar amino acids, X2 contains basic amino acids, X3 contains acidic amino acids, X4 contains polar amino acids, X5 contains acidic amino acids, X6 contains nonpolar amino acids, X7 contains polar amino acids, X8 contains acidic amino acids, X9 contains nonpolar amino acids, X 10 It contains nonpolar amino acids, X 11 It contains acidic amino acids, X 12 X contains polar amino acids. In some embodiments, X1 contains C, S, T, Y, N, or Q; X2 contains H, K, or R; X3 contains E or D; X4 contains C, S, T, Y, N, or Q; X5 contains E or D; X6 contains A, G, I, L, M, W, F, P, or V; X7 contains C, S, T, Y, N, or Q; X8 contains E or D; X9 contains A, G, I, L, M, W, F, P, or V; X 10 This includes A, G, I, L, M, W, F, P, or V, and X 11 This includes E or D, and X 12 X includes C, S, T, Y, N, or Q. In some embodiments, X1 includes T, X2 includes R, X3 includes E, X4 includes T, X5 includes D, X6 includes G, X7 includes Y, X8 includes D, X9 includes G, X 10 This includes M or L, X 11 This includes D, X 12 It contains Y. In some embodiments, HCDR3 contains the amino acid sequence TRETDGYDGMDY (SEQ ID NO: 63). In some embodiments, HCDR3 contains the amino acid sequence TRETDGYDGLDY (SEQ ID NO: 64).

[0044] In some embodiments, the light chain complementarity determination region 1 (LCDR1) has a length of 3 to 25 amino acids. In some embodiments, LCDR1 has a length of 5 to 15 amino acids. In some embodiments, LCDR1 has a length of 6 to 10 amino acids. In some embodiments, LCDR1 has a length of 3 amino acids. In some embodiments, LCDR1 has a length of 4 amino acids. In some embodiments, LCDR1 has a length of 5 amino acids. In some embodiments, LCDR1 has a length of 6 amino acids. In some embodiments, LCDR1 has a length of 7 amino acids. In some embodiments, LCDR1 has a length of 8 amino acids. In some embodiments, LCDR1 has a length of 9 amino acids. In some embodiments, LCDR1 has a length of 10 amino acids. In some embodiments, LCDR1 has a length of 11 amino acids. In some embodiments, LCDR1 has a length of 12 amino acids. In some embodiments, LCDR1 has a length of 13 amino acids. In some embodiments, LCDR1 has a length of 14 amino acids. In some embodiments, LCDR1 is the length of 15 amino acids. In some embodiments, LCDR1 is the length of 16 amino acids. In some embodiments, LCDR1 is the length of 17 amino acids. In some embodiments, LCDR1 is the length of 18 amino acids. In some embodiments, LCDR1 is the length of 19 amino acids. In some embodiments, LCDR1 is the length of 20 amino acids. In some embodiments, LCDR1 is the length of 21 amino acids. In some embodiments, LCDR1 is the length of 22 amino acids. In some embodiments, LCDR1 is the length of 23 amino acids. In some embodiments, LCDR1 is the length of 24 amino acids. In some embodiments, LCDR1 is the length of 25 amino acids. In some embodiments, LCDR1 contains the amino acid sequence X1X2X3X4X5X6 (wherein X1 contains polar amino acids, X2 contains polar amino acids, X3 contains nonpolar amino acids, X4 contains polar amino acids, X5 contains acidic amino acids, and X6 contains polar amino acids).In some embodiments, X1 comprises C, S, T, Y, N, or Q; X2 comprises C, S, T, Y, N, or Q; X3 comprises A, G, I, L, M, W, F, P, or V; X4 comprises C, S, T, Y, N, or Q; X5 comprises E or D; and X6 comprises C, S, T, Y, N, or Q. In some embodiments, X1 comprises Q, X2 comprises S, X3 comprises I, X4 comprises S, X5 comprises D, and X6 comprises Y. In some embodiments, LCDR1 comprises the amino acid sequence QSISDY (Sequence ID 62).

[0045] In some embodiments, the light chain complementarity determination region 2 (LCDR2) has a length of 3 to 25 amino acids. In some embodiments, LCDR2 has a length of 5 to 15 amino acids. In some embodiments, LCDR2 has a length of 6 to 10 amino acids. In some embodiments, LCDR2 has a length of 3 amino acids. In some embodiments, LCDR2 has a length of 4 amino acids. In some embodiments, LCDR2 has a length of 5 amino acids. In some embodiments, LCDR2 has a length of 6 amino acids. In some embodiments, LCDR2 has a length of 7 amino acids. In some embodiments, LCDR2 has a length of 8 amino acids. In some embodiments, LCDR2 has a length of 9 amino acids. In some embodiments, LCDR2 has a length of 10 amino acids. In some embodiments, LCDR2 has a length of 11 amino acids. In some embodiments, LCDR2 has a length of 12 amino acids. In some embodiments, LCDR2 has a length of 13 amino acids. In some embodiments, LCDR2 has a length of 14 amino acids. In some embodiments, LCDR2 is 15 amino acids long. In some embodiments, LCDR2 is 16 amino acids long. In some embodiments, LCDR2 is 17 amino acids long. In some embodiments, LCDR2 is 18 amino acids long. In some embodiments, LCDR2 is 19 amino acids long. In some embodiments, LCDR2 is 20 amino acids long. In some embodiments, LCDR2 is 21 amino acids long. In some embodiments, LCDR2 is 22 amino acids long. In some embodiments, LCDR2 is 23 amino acids long. In some embodiments, LCDR2 is 24 amino acids long. In some embodiments, LCDR2 is 25 amino acids long. In some embodiments, LCDR2 contains the amino acid sequence X1X2X3 (where X1 contains polar amino acids, X2 contains nonpolar amino acids, and X3 contains polar amino acids).In some embodiments, X1 comprises C, S, T, Y, N, or Q; X2 comprises A, G, I, L, M, W, F, P, or V; and X3 comprises C, S, T, Y, N, or Q. In some embodiments, X1 comprises Y, X2 comprises A, and X3 comprises S. In some embodiments, LCDR2 comprises the amino acid sequence YAS. In some embodiments, LCDR2 comprises the amino acid sequence X1X2X3X4X5X6X7X8X9 (wherein X1 comprises a nonpolar amino acid, X2 comprises a nonpolar amino acid, X3 comprises a polar amino acid, X4 comprises a nonpolar amino acid, X5 comprises an acidic or nonpolar amino acid, X6 comprises a polar amino acid, X7 comprises a nonpolar or polar amino acid, X8 comprises an acidic or polar amino acid, and X9 comprises a nonpolar amino acid). In some embodiments, X1 includes A, G, I, L, M, W, F, P, or V; X2 includes A, G, I, L, M, W, F, P, or V; X3 includes C, S, T, Y, N, or Q; X4 includes C, S, T, Y, N, or Q; X5 includes D, E, A, G, I, L, M, W, F, P, or V; X6 includes C, S, T, Y, N, or Q; X7 includes C, S, T, Y, N, Q, A, G, I, L, M, W, F, P, or V; X8 includes D, E, C, S, T, Y, N, or Q; and X9 includes A, G, I, L, M, W, F, P, or V. In some embodiments, X1 comprises V or L, X2 comprises F or I, X3 comprises Y, X4 comprises Y or T, X5 comprises D or A, X6 comprises S, X7 comprises Q or A, X8 comprises E or S, and X9 comprises V or I. In some embodiments, LCDR2 comprises the amino acid sequence VFYTDSAEV (SEQ ID NO: 65). In some embodiments, LCDR2 comprises the amino acid sequence LIYYASQSI (SEQ ID NO: 66).

[0046] In some embodiments, the light chain complementarity determination region 3 (LCDR3) has a length of 3 to 25 amino acids. In some embodiments, LCDR3 has a length of 5 to 15 amino acids. In some embodiments, LCDR3 has a length of 6 to 10 amino acids. In some embodiments, LCDR3 has a length of 3 amino acids. In some embodiments, LCDR3 has a length of 4 amino acids. In some embodiments, LCDR3 has a length of 5 amino acids. In some embodiments, LCDR3 has a length of 6 amino acids. In some embodiments, LCDR3 has a length of 7 amino acids. In some embodiments, LCDR3 has a length of 8 amino acids. In some embodiments, LCDR3 has a length of 9 amino acids. In some embodiments, LCDR3 has a length of 10 amino acids. In some embodiments, LCDR3 has a length of 11 amino acids. In some embodiments, LCDR3 has a length of 12 amino acids. In some embodiments, LCDR3 has a length of 13 amino acids. In some embodiments, LCDR3 has a length of 14 amino acids. In some embodiments, LCDR3 is the length of 15 amino acids. In some embodiments, LCDR3 is the length of 16 amino acids. In some embodiments, LCDR3 is the length of 17 amino acids. In some embodiments, LCDR3 is the length of 18 amino acids. In some embodiments, LCDR3 is the length of 19 amino acids. In some embodiments, LCDR3 is the length of 20 amino acids. In some embodiments, LCDR3 is the length of 21 amino acids. In some embodiments, LCDR3 is the length of 22 amino acids. In some embodiments, LCDR3 is the length of 23 amino acids. In some embodiments, LCDR3 is the length of 24 amino acids. In some embodiments, LCDR3 is the length of 25 amino acids.In some embodiments, LCDR3 includes the amino acid sequence X1X2X3X4X5X6X7X8X9 (wherein X1 includes a polar amino acid, X2 includes a polar amino acid, X3 includes a nonpolar amino acid, X4 includes a basic amino acid, X5 includes a polar amino acid, X6 includes a nonpolar amino acid, X7 includes a nonpolar amino acid, X8 includes a polar amino acid, and X9 includes a polar amino acid). In some embodiments, X1 includes C, S, T, Y, N, or Q; X2 includes C, S, T, Y, N, or Q; X3 includes A, G, I, L, M, W, F, P, or V; X4 includes H, K, or R; X5 includes C, S, T, Y, N, or Q; X6 includes A, G, I, L, M, W, F, P, or V; X7 includes A, G, I, L, M, W, F, P, or V; X8 includes C, S, T, Y, N, or Q; and X9 includes C, S, T, Y, N, or Q. In some embodiments, X1 includes Q, X2 includes N, X3 includes G, X4 includes H, X5 includes S, X6 includes F, X7 includes P, X8 includes Y, and X9 includes T. In some embodiments, LCDR3 comprises the amino acid sequence QNGHSFPYT (SEQ ID NO: 67). In some embodiments, LCDR3 comprises the amino acid sequence QQGHSFPYT (SEQ ID NO: 68). In some embodiments, LCDR3 comprises the amino acid sequence QSGHSFPYT (SEQ ID NO: 69).

[0047] In certain embodiments, the anti-IL-5 binding protein comprises one or more CDRs (i.e., one, two, three, four, five, or all six) of the felineized mouse clone 154 disclosed herein. In certain embodiments, the anti-IL-5 binding protein comprises one or more CDRs (i.e., one, two, three, four, five, or all six) of the affinity-matured felineized antibodies disclosed herein. In certain embodiments, the anti-IL-5 binding protein comprises CDRs derived from one or more of the felineized mouse clone 154 and affinity-matured variants provided herein.

[0048] In certain embodiments, the binding protein includes a human antibody or a humanized antibody. In certain embodiments, the binding protein includes a cat antibody or a felineized antibody.

[0049] In certain embodiments, amino acid residues are mutated to residues that preserve the properties of the amino acid side chain. Examples of amino acid side chain properties include polar amino acids (C, S, T, Y, N, Q), nonpolar amino acids (A, G, I, L, M, W, F, P, V), basic amino acids (H, K, R), acidic amino acids (E, D), hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids containing the following side chains: aliphatic side chains (G, A, V, L, I, P), hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains (C, M), carboxylic acid and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), and aromatic-containing side chains (H, F, Y, W). The letters in parentheses represent single-letter amino acid codes. Amino acid substitutions within each group are called conservative substitutions. It is well known that polypeptides having modified amino acid sequences in which one or more amino acid residues are deleted, added, and / or substituted can retain their original biological activity (Mark DF et al., Proc. Natl. Acad. Sci. USA 81:5662-5666 (1984); Zoller MJ and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. et al., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. USA 79: 6409-6413 (1982)). The number of amino acids that are mutated is not limited, but it is usually within 40%, preferably within 35%, and more preferably within 30% (e.g., within 25%) of the amino acids in each CDR. The identity of the amino acid sequence can be determined as described herein.

[0050] This invention provides recombinant antibodies designed or modified to minimize antigenicity in felines and humans. In certain embodiments, the antibodies are further modified to eliminate T cell epitopes.

[0051] As used herein, the term “cat” refers to all members of the Felidae family. Domestic cats, purebred and / or mixed-breed pet cats, and wild or feral cats are all animals of the Felidae family.

[0052] As used herein, the terms “human framework” or “feline framework” refer to the amino acid sequences of the heavy and light chains of a feline antibody, excluding the hypervariable region residues defined herein as CDR residues. With respect to humanized antibodies, in certain embodiments, feline CDRs that closely match those of IL-5-binding antibodies from other species are identified within the variable region sequences of the heavy and light chains of the human antibody. In certain embodiments, the natural human CDRs are replaced in both chains with the corresponding exogenous CDRs (e.g., those derived from rat or mouse antibodies). With respect to feline antibodies, in certain embodiments, feline CDRs that closely match those of IL-5-binding antibodies from other species are identified within the variable region sequences of the heavy and light chains of the feline antibody. In certain embodiments, the natural feline CDRs are replaced in both chains with the corresponding exogenous CDRs (e.g., those derived from rat or mouse antibodies). If necessary, the heavy and / or light chains of humanized or felineized antibodies may contain specific mutated or exogenous non-CDR residues, such as framework amino acid residues that differ between germline antibody sequences, or mutations that preserve the conformation of exogenous CDRs within the antibody.

[0053] In dogs, there are five major isotypes (IgA, IgG, IgM, IgD, IgE) and two forms of the light chain (κ and λ). In dogs, there are four subtypes of IgG: IgGA, IgGB, IgGC, and IgGD (Bergeron et al, 2014, Comparative functional characterization of canine IgG subclasses. Veterinary Immunology and Immunopathology. 157:31-41). In cats, there are three subtypes of IgG: IgG1a, IgG1b, and IgG2 (Streitzel et al. 2014, In vitro functional characterization of feline IgGs. Vet Immunol Immunopathol 158, 214-223, doi.org / 10.1016 / j.vetimm.2014.01.012).

[0054] The present invention provides canine and feline antibodies engineered to modulate one or more effector functions or circulating half-lives. The hinge domain and constant domain of the antibodies bind to host receptors or complement proteins to mediate effector functions and regulate antibody circulation. In certain embodiments, one or more effector functions are enhanced. In certain embodiments, one or more effector functions are reduced or eliminated. In certain embodiments, the antibodies of the present invention include modifications to modulate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC). A non-limiting example is the manipulation of residues Met242 and / or Leu243 (EU number) in the constant region of canine IgGB to reduce effector function (e.g., Lund et al., Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG. J Immunol., 1991, 147:2657-62). In certain embodiments, the IgGB constant domain of the present invention includes substitutions M242A and L243A. In certain embodiments, the second constant domain (CH2) and / or the third constant domain (CH3) include mutations and combinations of mutations from the wild type designed to modulate binding to the FcRn (neonatal Fc) receptor. In the canine constant domain, such mutations include substitutions of Ala426 (e.g., A426Y or A426H), substitutions of Thr286 (e.g., T286L or T286Y), substitutions of Tyr436 (e.g., Y436H), and combinations of such mutations (including, but not limited to, A426Y+T286L, A426Y+Y436H, A426H+T286L, and A426H+T286Y). In certain embodiments, the chimeric or canine antibody of the present invention includes, but is not limited to, substitutions of the amino acid Asn434, such as N434H.In the feline constant region, such mutations include, but are not limited to, substitutions of Ser428 including S428Y or S428L, substitutions of Gln311 including Q311V, substitutions of Leu309 including L309V, substitutions of Thr286 including T286E, substitutions of Glu380 including E380T, and combinations of such mutations including, but are not limited to, S428Y+Q311V, S428Y+L309V, S428Y+Q311V+T286E, S428Y+Q311V+E380T, and S428Y+L309V+E380T. In certain embodiments, the chimeric antibody or feline antibody of the present invention includes, but is not limited to, substitutions of the amino acids Ser428 and / or Ser434, including S428L and / or S434H.

[0055] As used herein, the term “antibody” includes the antigen-binding fragment of a complete antibody molecule. As used herein, the “antigen-binding portion” of an antibody, the “antigen-binding fragment” of an antibody, and similar terms include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. As used herein, the terms “specifically bind” or “specifically bind” mean that the IL-5 binding proteins of the present invention react with or bind to IL-5 more frequently, more rapidly, for longer periods, and / or with higher affinity than to other antigens. For example, the IL-5 binding proteins bind to IL-5 with significantly higher affinity (e.g., at least 2x, 5x, 10x, 20x, 50x, 100x, 500x, 1000x, 10,000x or more) than to other proteins or peptides. In certain embodiments, the IL-5 binding protein binds to IL-5 with an equilibrium dissociation constant (KD) (with respect to the epitope or target to which it binds) of, for example, 10⁻⁴M or less, e.g., 10⁻⁵M, 10⁻⁶M, 10⁷M, 10⁸M, 10⁹M, 10⁻¹¹M, or 10⁻¹²M. It will be understood by those skilled in the art that an antibody that specifically binds to a target of one species (e.g., IL-5) may also specifically bind to an orthologue of IL-5.

[0056] Antibody antigen-binding fragments can be obtained from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques for manipulating and expressing DNA encoding the antibody's variable and optionally constant domains. Such DNA is known and / or readily available, for example, from commercial suppliers, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated using chemical or molecular biological techniques to, for example, position one or more variable and / or constant domains in a suitable configuration, introduce codons, form cysteine ​​residues, or modify, add, or delete amino acids.

[0057] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv(scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide, or constrained FR3-CDR3-FR4 peptides). As used herein, the term “antigen-binding fragment” also includes domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (monovalent nanobodies, divalent nanobodies, etc.), small module immunotherapies (SMIPs), and other manipulated molecules such as shark variable IgNAR domains.

[0058] In certain embodiments, the antigen-binding fragment of the antibody includes at least one variable domain. The variable domain may be of any size or amino acid composition and generally includes at least one CDR adjacent to or in-frame to one or more framework sequences. In an antigen-binding fragment having a VH domain conjugated to a VL domain, the VH domain and the VL domain can be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer and may include VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of the antibody may include a monomeric VH or VL domain.

[0059] In certain embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragment of the antibody of the present invention include (i) VH-CH1, (ii) VH-CH2, (iii) VH-CH3, (iv) VH-CH1-CH2, (V) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3, (vii) VH-CL, (viii) VL-CH1, (ix) VL-CH2, (x) VL-CH3, (xi) VL-CH1-CH2, (xii) VL-CH1-CH2-CH3, (xiii) VL-CH2-CH3, and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations described above, the variable and constant domains may be directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region consists of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and provides a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibody of the present invention may include homodimers or heterodimers (or other polymers) of any of the variable and constant domain configurations described above, non-covalently linked to each other and / or to one or more monomer VH or VL domains (e.g., by disulfide bonds).

[0060] The term "diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (e.g., P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO 93 / 11161). Generally, a diabody is a dimer of two polypeptide chains. In each polypeptide chain, the light chain variable region (VL) and heavy chain variable region (VH) on the same chain are connected by a short linker (e.g., a linker of about 5 residues) that prevents them from binding to each other. Because the linker between them is too short, the VL and VH within the same polypeptide chain cannot form a single-stranded V-region fragment, but instead form a dimer. Therefore, a diabody has two antigen-binding domains. By combining the VL and VH regions for two types of antigens (a and b), VLa-VHb and VLb-VHa are formed via a linker of approximately 5 residues. When these are co-expressed, they are secreted as bispecific Db. The antibody of the present invention may be such a Db.

[0061] Single-chain antibodies (also known as "scFv") can be prepared by ligating the heavy chain V region and the light chain V region of an antibody (see Pluckthun, “The Pharmacology of Monoclonal Antibodies” Vol. 113, eds. Rosenburg and Moore, Springer Verlag, NY, pp. 269-315 (1994) for a review of scFv). Methods for preparing single-chain antibodies are well known in the art (see, for example, U.S. Patents 4,946,778, 5,260,203, 5,091,513, and 5,455,030). In such scFvs, the heavy chain V region and the light chain V region are linked via a linker, preferably a polypeptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883). The heavy chain V region and the light chain V region of the scFv may originate from the same antibody or from different antibodies. The peptide linker used to link the V regions can be any single-chain peptide consisting of 12 to 19 residues. The DNA encoding the scFv can be amplified by PCR using a primer pair defining both ends, with the entire DNA selected from the DNA encoding the heavy chain or the V region of the heavy chain of the antibody, and the DNA encoding the light chain or the V region of the light chain of the antibody, or a partial DNA encoding a desired amino acid sequence, as a template. Further amplification can then be performed using a combination of the DNA encoding the peptide linker portion and primer pairs defining both ends of the DNA linked to the heavy chain and light chain, respectively. After constructing the DNA encoding scFv, an expression vector containing this DNA and a host transformed by this expression vector can be obtained using conventional methods. Furthermore, scFv can be obtained using the obtained host according to conventional methods. These antibody fragments can be produced in the host by obtaining the gene encoding the antibody fragment and expressing it as described above.Antibodies conjugated to various types of molecules, such as polyethylene glycol (PEG), can be used as modified antibodies. Methods for modifying antibodies have already been established in the relevant art. The term "antibody" in this invention includes the antibodies described above.

[0062] As used herein, the term "Kd" refers to the dissociation constant of antibody-antigen interactions. The dissociation constant Kd and the binding constant Ka are quantitative measures of affinity. In equilibrium, free antigen (Ag) and free antibody (Ab) are in equilibrium with antigen-antibody complexes (Ag-Ab), and the rates of individual reactions are quantified by the rate constants ka and kd. In equilibrium, ka[Ab][Ag] = kd[Ag-Ab]. The dissociation constant Kd is given by Kd = kd / ka = [Ag][Ab] / [Ag-Ab]. Kd usually has units of concentration such as M, mM, nM, or pM. When comparing the affinity of antibodies expressed in terms of Kd, a lower value indicates a higher affinity for IL-5. The binding constant Ka is given by Ka = ka / kd = [Ag-Ab] / [Ag][Ab]. Ka typically has units that are reciprocals of concentration, such as M-1, mM-1, nM-1, and pM-1. As used herein, the term "binding strength" refers to the strength of antigen-antibody binding, taking valency into account.

[0063] The obtained antibodies can be purified until homogeneous. Antibodies can be isolated and purified by methods routinely used for the isolation and purification of proteins. Antibodies can be isolated and purified by using a combination of one or more methods appropriately selected from, for example, column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Such methods are not limited to those listed above. Examples of chromatographic methods include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography. These chromatographic methods can be carried out using liquid-phase chromatography such as HPLC and FPLC. Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, examples of protein A columns include HyperD, POROS, and Sepharose FF (Pharmacia). Antibodies can also be purified by utilizing antigen binding using a carrier on which the antigen is immobilized.

[0064] As used herein, the term “therapeutic agent” refers to any drug or substance that produces a beneficial effect on a mammalian recipient. Therefore, “therapeutic agents” include both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0065] As used herein, “treatment” means reducing, curing, and / or preventing the onset of at least one symptom of a particular disease or condition.

[0066] The anti-IL-5 proteins (including antibodies or fragments thereof) described herein are useful in improving or alleviating symptoms of IL-5-related diseases and disorders, or in treating or preventing them. The anti-IL-5 proteins or fragments thereof, and combinations with other agents, are administered in therapeutically effective doses to subjects in need of treatment for IL-5-related diseases and disorders in the form of the pharmaceutical compositions described herein.

[0067] In certain embodiments, the method includes improving or alleviating the symptoms of a disease of interest, or treating or preventing a disease of interest. In certain embodiments, an anti-IL-5 protein, antibody, or fragment thereof inhibits the binding of IL-5 to IL-5 receptors and / or eosinophil receptors and is administered, for example, alone or in combination with a second agent, for the treatment, improvement, symptom reduction, or prevention of lung diseases, cardiovascular diseases, cancer, infections, neurological disorders, allergic / inflammatory diseases, or metabolic diseases.

[0068] Non-limiting examples of cardiovascular diseases for which antibody compositions and methods are used to improve, alleviate, treat, or prevent symptoms include hypertension, cardiotoxicity of anticancer drugs, cardiotoxicity of anthracyclines, cardiotoxicity of quinolones, heart failure of any cause, ischemia, heart attack, stroke, atherosclerosis, ventricular fibrillation, hypertension, thrombosis, and embolism.

[0069] Non-limiting examples of infectious diseases for which antibody compositions and methods are used to improve, alleviate, treat, or prevent symptoms include AIDS, pleurodromicosis (AHD, echinococcosis), amoebic infection (amebic dysentery), schistosomiasis, anisakiasis, anthrax, babesiosis (babesiosis), balanthidiasis (balanthidiasis), baylissalis infection (raccoon roundworm), schistosomiasis of Bilharz, blastosmycosis (blastomycosis), borreliosis, botulism, Brainard's diarrhea, brucellosis, bovine spongiform encephalopathy (BSE), and Dysdiiasis, capillary disease (capillary infection), chronic fatigue syndrome (CFS), Chagas disease (trypanosomiasis), varicella (varicella-zoster virus), chlamydia pneumonia, cholera, Creutzfeldt-Jakob disease (CJD), liver fluke infection, cutaneous larval migrans (CLM) (hookworm infection), coccidioidomycosis, conjunctivitis, coxsackievirus A16 (hand, foot, and mouth disease), cryptococcosis, cryptosporidiosis, house mosquito (West Nile virus vector), cyclosporiasis (cyclosporiasis), cysticercosis Neurocysticercosis, cytomegalovirus infection, dengue fever, dipyridinium infection (canine and feline tapeworms), Ebola hemorrhagic fever, encephalitis, amoebic dysentery, amoebic dysentery disper infection, amoebic dysentery Hartmann's infection, amoebic dysentery (amebiasis), amoebic pinworm infection, enterovirus infection (other than polio), Epstein-Barr virus infection, Escherichia coli infection, food poisoning, foot-and-mouth disease, fungal dermatitis, gastroenteritis, Group A streptococcal infection, Group B streptococcal infection, leprosy, hantavirus pulmonary syndrome, head lice infestation, Helicobacter - Helicobacter pylori infection, blood disorders, Hendra virus infection, hepatitis (HCV, HBV), shingles, HIV infection, human ehrlichiosis, human parainfluenza virus infection, influenza, isosporiasis (isospora infection), Lassa fever, leishmaniasis, kala-azar (leishmania infection), lice (body lice, head lice, genital lice), Lyme disease, malaria, Marburg hemorrhagic fever, measles, meningitis, mosquito-borne diseases, Mycobacterium avium complex (MAC) infection, Naegleria infection,Examples of diseases that can cause illness include hospital-acquired infections, nonpathogenic enteroamebic infections, onchocerciasis (river blindness), opistolariasis (opistolus infection), parvovirus infections, plague, Pneumocystis pneumoniae (PCP), polio, Q fever, rabies, respiratory syncytial virus (RSV) infection, rheumatic fever, Rift Valley fever, river blindness (onchocerciasis), rotavirus infection, roundworm infections, salmonellosis, Salmonella enteritis, scabies, bacterial dysentery, herpes zoster, sleeping sickness, smallpox, streptococcal infections, tapeworm infections, tetanus, toxic shock syndrome, tuberculosis, ulcers (peptic ulcerative disease), valley fever, Vibrio parahaemolyticus infection, Vibrio vulnificus infection, viral hemorrhagic fever, warts, waterborne infections, West Nile virus infection (West Nile encephalitis), pertussis, and yellow fever. ,

[0070] Non-limiting examples of allergic / inflammatory diseases for which antibody compositions and methods are used to improve, alleviate, treat, or prevent symptoms include asthma, bronchial asthma, rheumatoid arthritis, inflammatory bowel disease, type 2 diabetes, diabetes mellitus and hearing loss (DAD), Barringer-Wallace syndrome, inflammatory diseases, rheumatic fever, pulmonary arterial hypertension, innate immune response, chronic obstructive pulmonary disease, pulmonary embolism, pericarditis, aortic coarctation, Tetralogy of Fallot, aortic stenosis, mitral stenosis, aortic regurgitation, mitral regurgitation, pneumoconiosis, bronchiectasis, cardiomyopathy, and endothelial nitroglycerin resistance.

[0071] Non-limiting examples of lung diseases for which antibody compositions and methods are used to improve, alleviate, treat, or prevent symptoms include acute pneumonia, pulmonary fibrosis, interstitial pneumonia, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, asthma, refractory asthma, systemic inflammatory response syndrome (SIRS), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), sarcoidosis, chronic idiopathic pulmonary thromboembolism, diffuse panbronchiolitis, cystic fibrosis, allergic alveolitis, lung cancer, obesity-related hypoventilation syndrome, alveolar hypoventilation syndrome, and chronic transplant rejection lung. Particularly important diseases are pulmonary fibrosis, interstitial pneumonia, pulmonary hypertension, asthma, COPD, and SIRS.

[0072] Non-limiting examples of cancers for which antibody compositions and methods are used to improve, alleviate, treat, or prevent symptoms include bladder cancer, hematological cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gingival cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, cervical cancer, ovarian cancer, prostate cancer, skin cancer, gastric cancer, testicular cancer, tongue cancer, or uterine cancer, or malignant neoplasms, undifferentiated carcinoma, giant cell carcinoma and spindle cell carcinoma, small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilosa cell carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; and mixed hepatocellular carcinoma and cholangiocarcinoma. Synthetic carcinoma; trabecular carcinoma; adenoid cystic carcinoma; adenocarcinoma in an adenomatous polyp; adenocarcinoma of familial polyposis coli; solid carcinoma; malignant carcinoid tumor; branchial adenocarcinoma; papillary adenocarcinoma; achromatic carcinoma; eosinophilic carcinoma; Adrenocortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; earwax; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; Adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant cysticoma; malignant granulosa cell tumor; malignant erythroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; Gromangios sarcoma; malignant melanoma; achromatic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevi; epithelioid sarcoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müllerian mixed tumor; Nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated germ cell tumor; fetal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; malignant angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic gingivoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pineal glandoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma;Astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; gangliblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granulocyte tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis Examples include sarcoma fungoides; other certain non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0073] Non-limiting examples of neurological diseases in which antibody compositions and methods are used to improve, alleviate, treat, or prevent symptoms include Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, Khuf's disease, Lewy body dementia, neurofibrillary tangles, Rosenthal fibers, Mallory vitreous humor, senile dementia, myasthenia gravis, Gilles de la Tourette syndrome, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), epilepsy, Creutzfeldt-Jakob disease, hearing loss / dystonia syndrome, Leigh syndrome, Leber hereditary optic neuropathy (LHON), Parkinson's syndrome, dystonia, motor neuron disease, neuropathy / ataxia / retinitis pigmentosa (N). Examples include ARP, maternal Leigh syndrome (MILS), Friedreich's ataxia, hereditary spastic paraplegia, Möll-Tranebyag syndrome, Wilson's disease, sporadic Alzheimer's disease, sporadic amyotrophic lateral sclerosis, sporadic Parkinson's disease, autonomic dysfunction, hypertension, sleep disorders, neuropsychiatric disorders, depression, schizophrenia, schizoaffective disorder, Korsakoff psychosis, mania, anxiety disorders, phobias, learning disabilities or memory impairment, amnesia or age-related memory loss, attention deficit disorder, dysthymia, major depressive disorder, obsessive-compulsive disorder, psychoactive substance use disorder, panic disorder, bipolar affective disorder, severe bipolar affective (mood) disorder (BP-1), migraines, hyperactivity and motor disorders.

[0074] Non-limiting examples of metabolic diseases for which antibody compositions and methods are used to improve, alleviate, treat, or prevent symptoms include metabolic syndrome, diabetes (type 1 diabetes, type 2 diabetes, gestational diabetes, etc.), impaired glucose tolerance, obesity, diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, dyslipidemia (hypertriglyceridemia, hypercholesterolemia, low HDL cholesterol, postprandial hyperlipidemia, etc.), hypertension, hypertriglyceridemia, severe hypertriglyceridemia, hypercholesterolemia, familial or genetic hypercholesterolemia, fatty liver disease, non-alcoholic fatty liver disease (NFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, mixed dyslipidemia, atherosclerosis, and coronary heart disease.

[0075] Anti-IL-5 proteins, antibodies, or antibody fragments may be administered in combination with one or more active agents, including other analgesics. Such active ingredients include analgesics, antihistamines, antipyretics, anti-inflammatory agents, antibiotics, antivirals, and anticytokine agents. Active ingredients include TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, hepatocyte growth factor (HGF), and hepcidin agonists, antagonists, and modulators (including reactive antibodies to any of the above and reactive antibodies to any of their receptors).The active ingredients are not limited to these, but include 2-arylpropionic acid, aceclofenac, acemetacin, acetylsalicylic acid (aspirin), alclofenac, aluminoprofen, amoxiprine, ampylon, arylalkanoate, azapropazon, benolilate / benolilate, benoxaprofen, bromfenac, carprofen, celecoxib, choline magnesium salicylate, clofezon, COX-2 inhibitors, dexibprofen, dexketoprofen, diclofenac, diflunisal, droxicam, ethenzamide, etodolac, etoricoxib, faislamin, fenamic acid, fenbufen, fenoprofen, flufenamic acid, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indomethacin, and fluenzamic acid. Other examples include doprofen, kebzon, ketoprofen, ketrolac, romoxicam, loxoprofen, lumiracoxib, magnesium salicylate, meclofenamic acid, mefenamic acid, meloxicam, metamisole, methyl salicylate, mofebutazone, nabumetone, naproxen, n-arylanthranilic acid, nerve growth factor (NGF), oxamethacin, oxaprozin, oxicam, oxyfenbutazone, parecoxib, phenazone, phenylbutazone, phenylbutazone, piroxicam, pirprofen, profen, proglummetacin, pyrazolidine derivatives, lofecoxib, salicylsalicylic acid, salicylamide, salicylate, sulfinpyrazone, sulindac, suprofentenoxicam, tiaprofenic acid, tolfenamic acid, tolmetine, and valdecoxib.

[0076] Antihistamines are any compounds that antagonize the action of histamine or the release of histamine from cells (such as mast cells). Examples of antihistamines, though not limited to these, include acribastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclidine, cetirizine, cetirizine analogs, chlorpheniramine, clemastine, CS560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR609, hydroxyzine, levocabastine, loratidine, methoscopolamine, mizolastine, nolastemizole, phenindamine, promethazine, pyriramine, terfenadine, and tranilast.

[0077] Antibiotics are not limited to these, but include amikacin, aminoglycosides, amoxicillin, ampicillin, ansamycin, arsphenamine, azithromycin, azurocillin, aztreonam, bacitracin, carbasephalosporins, carbapenems, carbenicillin, cefaclor, cefadroxil, cephalexin, cefalothin, cefalotin, cephamandol, cefazolin, cefdinir, cefditoren, cefepime, cefixime, cefoperazone, and cefotaxy. Cefoxitin, cefpodoxime, cefprodil, ceftazidime, ceftibuten, ceftizoxime, ceftoviprole, ceftriaxone, cefraximin, cephalosporins, chloramphenicol, cilastatin, ciprofloxacin, clarithromycin, clindamycin, cloxacillin, colistin, cotrimoxazole, dalfopristin, demeclocycline, dicloxacillin, zilithromycin, doripenem, doxycycline, enoxacin, ertapenem, erythromycin, ethambutol, flucloxacillin, ho Sufomycin, furazolidone, fusidic acid, gatifloxacin, geldanamycin, gentamicin, glycopeptides, herbimycin, imipenem, isoniazid, kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin, loracalbef, macrolides, mafenide, meropenem, methicillin, metronidazole, mezlocillin, minocycline, monobactams, moxifloxacin, mupirocin, nafcillin, neomycin, netylmycin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin Oxytetracycline, paromomycin, penicillin, penicillin derivatives, piperacillin, platensimycin, polymyxin B, polypeptide derivatives, prontodil, pyrazinamide, quinolone derivatives, quinupristin, rifampicin, rifampin, roxithromycin, spectinomycin, streptomycin, sulfacetamide, sulfamethizol, sulfanilimide, sulfasalazine, sulfisoxazole, sulfonamide derivatives, teicoplanin, telithromycin, tetracycline, tetracycline derivatives, ticarcillin, tinidazole,Examples include tobramycin, trimethoprim, trimethoprim-sulfamethoxazole, troleandmycin, trovafloxacin, and vancomycin.

[0078] Examples of active ingredients include aldosterone, beclomethasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids, and triamcinolone. Any suitable combination of these active ingredients can also be conceived.

[0079] Formulation and method of administration For in vivo use, the therapeutic agents described herein are generally added to a pharmaceutical composition prior to administration. Such a composition contains one or more of the therapeutic compounds described herein as active ingredients (i.e., in levels sufficient to produce a statistically significant effect on the symptoms of cystic fibrosis when measured using a representative assay). The pharmaceutical composition comprises one or more such compounds in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for a particular dosage form. Furthermore, other pharmaceutically active ingredients (including other therapeutic agents) may (but may not) be present in the composition.

[0080] The antibodies of the present invention can be formulated according to standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) and may include pharmaceutically acceptable carriers and / or additives. The present invention relates to compositions (including reagents and pharmaceuticals) comprising the antibodies of the present invention and pharmaceutically acceptable carriers and / or additives. Exemplary carriers include surfactants (e.g., PEG and Tween), excipients, antioxidants (e.g., ascorbic acid), colorants, flavorings, preservatives, stabilizers, buffers (e.g., phosphoric acid, citrate, and other organic acids), chelating agents (e.g., EDTA), suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, and straighteners. However, the carriers that can be used in the present invention are not limited to this list. In fact, other commonly used carriers such as light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts may also be used as appropriate. The composition may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; and amino acids such as glycine, glutamine, asparagine, arginine, and lysine. When this composition is prepared as an aqueous solution for injection, the aqueous solution for injection may include, for example, physiological saline, dextrose, and other auxiliary agents (suitable solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol and PEG), and nonionic surfactants (polysorbate 80 and HCO-50), such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride).

[0081] The antibodies of the present invention can be encapsulated in microcapsules (microcapsules made of hydroxycellulose, gelatin, polymethyl methacrylate, etc.) and prepared as components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, “Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980)). Furthermore, methods for producing sustained-release drugs are well known and can be applied to the antibodies of the present invention (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech. 12: 98-105 (1982); U.S. Patent No. 3,773,919; European Patent Application No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP: 133,988).

[0082] With regard to the treatment of a disease state / condition, the term "therapeutic dose" refers to the amount of a compound, either alone or in a pharmaceutical composition, that, in a single or multiple dose, is capable of producing some detectable positive effect on any symptom, aspect, or characteristic of the disease state / symptom. Such an effect does not necessarily have to be absolute to be beneficial.

[0083] As used herein, the terms “treat,” “treating,” and “treatment” include administering a compound before the clinical onset of a disease state / symptom to prevent any symptom, and administering a compound after the clinical onset to reduce or eliminate any symptom, aspect, or characteristic of a disease state / symptom. Such treatment does not necessarily have to be absolute to be beneficial.

[0084] In certain embodiments, the therapeutic agent of the present invention can be administered systemically, for example, orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an absorbable edible carrier. The therapeutic agent of the present invention may be encapsulated in hard or soft-shell gelatin capsules, compressed into tablets, or added directly to the patient's diet. For oral therapeutic administration, the active compound can be used in combination with one or more excipients in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and formulations must contain at least 0.1% of the active compound. The percentage (%) of the compositions and formulations may, of course, vary and can be about 2 to about 60% of the weight of a given unit dosage form. The amount of the active compound in such therapeutically useful compositions is such that an effective dose level is obtained.

[0085] Tablets, lozenges, pills, capsules, etc., may contain binders such as tragacanth gum, acacia, corn starch, or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch, potato starch, or alginic acid, and may also contain lubricants such as magnesium stearate, sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, the composition and formulation may also contain a liquid carrier such as vegetable oil or polyethylene glycol in addition to the above types of materials. Various other materials may be present to coat or otherwise modify the physical shape of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar. Syrups or elixirs may contain an active ingredient, sucrose or fructose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavoring. Naturally, all materials used in the preparation of the unit dosage form must be pharmaceutically acceptable and substantially non-toxic in the amounts used. Furthermore, the active compound can be incorporated into sustained-release formulations and devices.

[0086] The active compound may also be administered intravenously or intraperitoneally by injection or infusion. Solutions of the active compound or its salts may be prepared in water and optionally mixed with a non-toxic surfactant. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as in oil. These preparations contain preservatives to inhibit microbial growth under normal storage and use conditions.

[0087] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are adapted for immediate preparation of sterile injection or infusion solutions or dispersions, sometimes encapsulated in liposomes. In all cases, the final dosage form must be sterile, fluid, and stable under manufacturing and storage conditions. The liquid carrier or medium may be a solvent or liquid dispersion medium, for example, containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by liposome formation, maintaining the required particle size in the case of dispersions, or by the use of surfactants. Inhibition of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. Long-term absorption of the injectable composition can be achieved by using absorption-delaying substances, such as aluminum monostearate and gelatin, in the composition.

[0088] Sterile injection solutions can be prepared by adding the required amount of active compound along with the various other components mentioned above to a suitable solvent, and then, if necessary, by filtration sterilization. For sterile powders used in the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield powders of the active ingredients present in a pre-sterile filtered solution and any desired additional components.

[0089] Useful doses of the compounds of the present invention can be determined by comparing their in vitro activity with their in vivo activity in animal models. In certain embodiments, useful doses are approximately 0.1 mg / kg to approximately 5 mg / kg, or approximately 0.5 mg / kg to approximately 2 mg / kg. Methods for extrapolating effective doses in humans and animals of different sizes are well known in the art (see, for example, U.S. Patent No. 4,938,949).

[0090] The amount of compound, or its activated salt or derivative, required for treatment varies not only depending on the specific salt selected, but also on the route of administration, the nature of the symptoms being treated, the patient's age, and the symptoms, and is ultimately left to the discretion of the attending physician or clinician.

[0091] However, generally speaking, an appropriate dose is in the range of about 0.5 to about 100 mg per kg of recipient's body weight per day, for example, about 10 to about 75 mg, for example, 3 to about 50 mg, preferably in the range of 6 to 90 mg / kg / day, and most preferably in the range of 15 to 60 mg / kg / day.

[0092] The compound is conveniently administered in unit dosage forms, for example, each unit dosage form contains 5 to 1000 mg of the active ingredient, conveniently 10 to 750 mg, and most conveniently 50 to 500 mg.

[0093] Ideally, the active ingredient should be administered so that the peak plasma concentration of the active compound is approximately 0.5–75 μM, preferably 1–50 μM, and most preferably 2–30 μM. This can be achieved, for example, by intravenous injection of a 0.05–5% solution of the active ingredient (sometimes in saline) or by oral administration as a bolus containing approximately 1–100 mg of the active ingredient. The desired blood concentration can be maintained by continuous infusion to approximately 0.01–5.0 mg / kg / hour, or by intermittent infusion containing approximately 0.4–15 mg / kg of the active ingredient(s).

[0094] The desired dose can be conveniently administered as a single dose or as divided doses, for example, two, three, four, or more times per day, at appropriate intervals. The divided dose itself can also be further divided into a predetermined number of doses, for example, at roughly spaced intervals.

[0095] Exemplary IL-5 receptor α (IL-5Rα) constructs useful for screening, identifying, and evaluating anti-IL-5 antibodies that inhibit receptor binding include: [Table 2]

[0096] Although the present invention and its advantages are described in detail, it should be understood that various changes, substitutions, and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

[0097] The present invention is further illustrated in the following examples, which are provided for illustrative purposes only and are not intended to limit the invention in any way. [Examples]

[0098] Example 1. Preparation and characterization of mouse antibody 154 that binds to feline IL-5. Five Balb / C mice were immunized with feline IL-5 (NP_001009845) fused to mouse IgG2a Fc (BAC44883.1) using AbCellera's proprietary method. Titer was measured by flow cytometry assay using beads coated with feline IL-5. Serum of different dilutions was incubated with feline IL-5 (Kingfisher Biotech, RP1152F) coated beads at 37°C for 30 minutes. The beads were washed, and binding of serum antibody to the beads was detected using a fluorescently labeled anti-mouse IgG secondary antibody (Jackson Immuno Research). Fluorescence was measured using high-throughput plate-based flow cytometry with an INTELLICYT® IQUE® ScreenerPlus. Lymph nodes, spleens, and bone marrow were collected from three mice that showed significant IL-5 titers. Cells were isolated from all tissues, and plasma cells were enriched using flow cytometry. A concentrated plasma cell suspension was injected into an AbCellera microfluidic screening device. A total of 590,000 single cells were screened using the microfluidic device. Single cells secreting feline IL-5 specific antibody were identified and isolated using a bead-based assay. Beads coated with anti-mouse IgG antibody (Jackson Immuno Research) were flowed into the microfluidic screening device and incubated with single antibody-secreting cells. IgG secreted from plasma cells was captured on the beads by a constant region. Binding to the secreted IgG immobilized on the beads was then evaluated using fluorescently labeled feline IL-5 antigen. Positive hits were identified using machine vision and collected using an automated robot-based protocol.

[0099] NGS sequencing libraries (MiSeq, Illumina) were generated using automated workstations (Bravo, Agilent) via single-cell polymerase chain reaction (PCR) and custom molecular biology protocols. Sequencing data was analyzed using a custom bioinformatics pipeline to obtain heavy-chain and light-chain sequence pairs for each recovered antibody-secreting cell. 576 binders were recovered from screening devices and sequenced, yielding 456 high-confidence sequences. 252 unique antibodies were identified and annotated with their closest germline (V[D]J) genes and degree of somatic hypermutation. Antibodies were considered members of the same clonal family if they shared the same putative heavy-chain and light-chain V and J genes and had the same CDR3 length. This process identified 93 families. Based on diversity and the lack of cysteine-induced risk, 48 antibodies were selected for expression and purification. The variable (V[D]J) region of each antibody chain was synthesized and inserted into a mammalian expression vector along with either the constant domain of mouse IgG2a or the kappa constant domain using a custom automated high-throughput cloning pipeline. The heavy and light chain expression vectors were transiently transfected into HD-293F cells, and the antibodies were purified using protein A chromatography. These antibodies were prepared in phosphate-buffered saline (PBS) at pH 7.2.

[0100] The purified mAbs were quantified using a UV absorption spectrophotometer at 280 nm.

[0101] These mouse antibodies were characterized by evaluating their purity, thermal stability, binding to feline IL-5, and inhibition of feline IL-5 binding to IL-5 receptor α (IL-5Rα).

[0102] The purity of the expressed and purified mAbs was analyzed by denatured capillary decyl sulfate sodium gel electrophoresis (SDS-PAGE).

[0103] The melting point (Tm) of the antibody was evaluated by differential scanning fluorescence (DSF) using a SYPRO® Orange fluorescent probe (Thermo Fisher Scientific, S6653). 6 μL of a 350 μg / mL mAb solution in phosphate-buffered saline (pH 7.4) (PBS) was mixed with 6 μL of a 19-fold concentrated SYPRO® Orange solution diluted in PBS. Thermal denaturation (thermal unfolding), evaluated by the change in fluorescence, was measured using a Bio-Rad C1000 Touch thermal cycler (Bio-Rad Laboratories) with a CFX96 real-time system readerhead (Bio-Rad Laboratories). The excitation and emission wavelengths were 450–490 nm and 560–580 nm, respectively. The fluorescence signal was measured at a starting temperature of 25°C and increased to 95°C at a rate of 0.5°C / min. Data were analyzed using Bio-Rad CFX Maestro software (v1.1), and the melting curve was integrated. Tm was defined as the local minimum obtained from the differential of the melting curve.

[0104] To measure the binding affinity of antibodies to feline IL-5, high-throughput SPR experiments using a 384 ligand array format were performed on a Carterra LSA instrument equipped with an HC-30M chip type (Carterra-bio). First, the chip surface was activated by flowing a freshly prepared 1:1:1 activation mix of 100 mM MES (pH 5.5), 100 mM NHS, and 400 mM EDC for 7 minutes. Antibodies diluted to 10 μg / mL with 10 mM sodium acetate (pH 4.25) buffer and 0.01% Tween were bound to the chip surface for 10 minutes using the instrument's 96 multi-flow channel printhead. The chip surface was quenched by flowing 1 M ethanolamine for 7 minutes, followed by two washes in 25 mM MES (pH 5.5) buffer for 15 seconds each. Feline IL-5 (Kingfisher Biotech, RP1152F) was used at the following concentrations: 1000, 333.3, 111.1, 37.0, 12.3, 4.1, 1.4, 0.45, and 0.15 nM.

[0105] To investigate the inhibitory ability of a mouse anti-feline IL-5 antibody on the binding of feline IL-5 to the feline IL-5 receptor α (IL-5Rα), an IL-5 receptor inhibition assay was constructed. For this assay, a fusion protein was created by ligating the extracellular domain of feline IL-5Rα (XP_011278466.1) with the V5 epitope, the TEV protease site, and human IgG1 Fc. The V5 epitope is derived from a small epitope (Pk) found in the P and V proteins of paramyxoviruses of the Simianvirus 5 family. The sequence of this construct is shown in Figure 1A (SEQ ID NO: 1). The signal sequence (METDTLLLWVLLLWVPGSTG: SEQ ID NO: 70) was added to the N-terminus, and the corresponding DNA construct was subcloned into pcDNA3.4 (Life Technologies). This construct was expressed using the Expi293 method (ThermoFisher Scientific), and the IL-5Rα-Fc construct was purified from the adapted Expi293 medium using HiTrap Protein A chromatography. The IL-5Rα-Fc construct was further purified by size exclusion chromatography using a TOSOH TSKgel G3000Swxl column to isolate the dimer form.

[0106] The receptor inhibition assay was completed using a Biacore T200 instrument. The Biacore assay format involved conjugating an anti-human Fc antibody (Cytiva, catalog number BR100839) to a CM5 sensor chip via amine coupling to reach approximately 1,000 resonance units (RUs), binding to the IL-5 receptor α-Fc, and then flowing either feline IL-5 (Kingfisher Biotech, catalog number RP1152F) or an IL-5 / anti-IL-5 antibody mixture (mixed in a 1:1 ratio). The affinity of feline IL-5 (Kingfisher Biotech, catalog number RP1152F-100) to feline IL-5RA-Fc was 31 nM (ka=1.9E+5, kd=6.0E-3). The antibody and IL-5 were mixed in a 1:1 molar ratio, and antibody-IL-5 complexes at concentrations of 12.5 nM, 25 nM, and 50 nM were flowed onto the receptor. The observed RU values ​​when IL-5 was flowed alone were 60 RU at 50 nM, 30 RU at 25 nM, and 15 RU at 12.5 nM. The inhibition rate (%) was determined by the following formula: 100 - [(RU of the mixture / RU of IL-5) x 100].

[0107] Mouse antibody clone 154 was selected for felineization, and its variable domain sequences for the heavy chain variable domain (SEQ ID NO: 3) and light chain variable domain (SEQ ID NO: 4) are shown in Figures 3 and 4, respectively. Each CDR region is shown according to the definition of IMGT. For clone 154, the purity (%) of the purified antibody, its thermal stability, affinity for feline IL-5, and the inhibition rate (%) of IL-5 receptor α are listed in Table 1. [Table 3]

[0108] Example 2. Feline transformation of clone 154 and characterization of the clone To felineize clone 154, we compared the framework of clone 154 with our own expression antibody database to search for the feline framework with the highest degree of identity. The proprietary antibody expression database was constructed using next-generation sequencing of feline PBMCs (peripheral blood mononuclear cells) and contains approximately 600,000 unique sequences of the VH (heavy chain variable), VL (lambda chain variable), and VK (kappa chain variable) domains. Twelve kappa cat frameworks were transplanted into the kappa CDR of clone 154 using Kabat definitions, and two feline heavy chain frameworks were transplanted into the heavy chain CDR of clone 154 using Kabat definitions. The twelve feline kappa chain clones were reformatted together with clone 154 using the feline kappa chain constant domain (GenBank: ATI97438.1). The two feline heavy chain clones were reformatted together with clone 154 using the feline IgG1a constant domain (GenBank: BAA32229.1). The constructs of the twelve feline kappa chain clones and the two feline heavy chains were subcloned into the pcDNA3.4 expression vector (Thermo Fisher). The heavy-chain and light-chain chimeric clone 154 was also subcloned into a mammalian expression vector. All possible combinations of the heavy-chain and light-chain felineized clones were co-transfected into HEK293 cells along with the chimeric clone 154, and IgG in the prepared medium was purified using MabSelect SuRe Protein A resin. The purified different felineized IgGs were referred to as matrix clones (abbreviated as Mtx). These antibodies were buffer-exchanged with 20 mM acetate and 136 mM NaCl (pH 5.5). Size exclusion chromatography (SEC) using an Agilent AdvanceBio SEC300A column showed no antibody aggregates exceeding 1% for any of the antibodies.

[0109] MtxA and MtxB antibodies showed the highest affinity for feline IL-5 when measured by surface plasmon resonance (SPR) using a Biacore T200 instrument. A 30 μg / ml goat anti-feline IgG-Fc specific fragment (Jackson Immuno Research, 102-005-008) was immobilized on a CM5 sensor chip (Cytiva; 29104988) at 10 μL / min for 420 seconds in 10 mM acetate (pH 5.0) by NHS coupling, and the remaining sites were blocked with ethanolamine at 10 μL / min for 420 seconds. Anti-feline IL-5 antibody at 2 μg / ml was captured on the anti-feline IgG CM5 chip at 10 μL / min for 60 seconds. Feline IL-5 (Kingfisher Biotech, RP1152F) was used as the test substance. Five serial dilutions (100, 50, 25, 12.5, 6.25 nM) were applied to a sensor chip at 30 μl / min for 120 seconds, followed by dissociation in PBS-P+ (Cytiva; 28995084) for 600 seconds. The binding kinetics of feline IL-5 to the antibody are shown in Figure 2 below. The IL-5 receptor inhibition assay was further completed by capturing biotinylated receptors on the Series S CAP chip and Biotin CAPture reagent (Cytiva). To perform this assay, a fusion protein was constructed by linking the extracellular domain of feline IL-5Rα (XP_011278466.1) with a 2X-Gly-Gly-Gly-Ser (SEQ ID NO: 89) linker, human IgG1 Fc, and an AviTag sequence (Figure 1B, SEQ ID NO: 2). This fusion protein was then subcloned into a pcDNA3.4 mammalian expression vector. This construct was expressed in CHO-S cells, purified by protein A chromatography, and then polished using SEC chromatography. The purified recombinant protein was biotinylated in vitro using BirA biotin ligase. Antibodies were serially diluted with running buffer (1×PBSP+, Cytiva) and pre-incubated with 50 nM IL-5 (Kingfisher) in multiple molar ratios. Binding was evaluated by injecting these samples onto the captured receptor for 180 seconds.The inhibition rate (%) was calculated by dividing the Rmax of the pre-mixed samples by the average Rmax of the IL-5-only samples collected throughout the entire assay. The binding inhibitory activity of each antibody against feline IL-5 was measured (Table 2). [Table 4]

[0110] The sequences of the variable domains and the first constant domain of the MtxA and MtxB antibodies are shown in Figures 2A-C (SEQ ID NOs: 127-129). Both antibodies have the same heavy chain variable domain (SEQ ID NO: 5). The MtxA light chain variable domain (SEQ ID NO: 6) and the MtxB light chain variable domain (SEQ ID NO: 7) are compatible with the common heavy chain variable domain. The individual frameworks and CDRs of the MtxA-derived light chain variable domain are interchangeable with the corresponding frameworks and CDRs of the MtxB-derived light chain variable domain.

[0111] Example 3. Elimination of potential sequence-related risks in LCDR3 and HCDR3. Asparagine deamidation at asparagine residues and oxidation of methionine residues can adversely affect antibody efficacy and stability (Xu et al., 2019. MABS, 11:239-264). These post-translational modifications can be particularly problematic when present within the CDR. In MtxA and MtxB clones, a potential deamidation site (NG) exists in LCDR3 (within the frame in Figure 2B (SEQ ID NO: 129) and 2C (SEQ ID NO: 128)), and a potential oxidation site (M) exists in HCDR3 (within the frame in Figure 2A (SEQ ID NO: 127)). To eliminate potential sequence-related risks, a DNA construct encoding the MtxB clone with substitutions was synthesized (GeneArt). For the NG site in LCDR3, Asn (position 106) was substituted with either Gln or Ser, and for Met (position 115) in HCDR3, Leu was substituted. DNA constructs containing light chain variants were fused to the constant domain of feline kappa strands. These DNA constructs were subcloned into pcDNA3.4 expression vectors (ThermoFisher) and expressed using the ExpiCHO system (ThermoFisher). Antibodies were purified by mAbSelect SuRe chromatography (Cytiva) and buffered with 20 mM sodium acetate and 136 mM sodium chloride (pH 5.5). These variants were tested in SPR experiments using a Biacore T200 instrument to measure their affinity for feline IL-5 (Kingfisher Biotech; RP1152F). Goat anti-feline IgG was immobilized on a Series S CM5 sensor chip (Cytiva), and the original MtxB clone and variants containing amino acid substitutions were captured on the chip at 5 μg / mL. Feline IL-5 (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 0nM) was flowed at a contact time of 120 seconds, a dissociation time of 600 seconds, and a flow rate of 30 μl / min. The kinetic parameters of the mutant MatrixB antibodies are shown in Table 3. [Table 5]

[0112] Example 4. Affinity maturation of felineized MtxB clones To improve the affinity of the MtxB clone to feline IL-5, an affinity maturation project was conducted. The following is an overview of the STEM (Stage-Enhanced Maturation) platform used in the affinity maturation strategy. A highly diverse range of CDR sequences was evaluated through iterative library construction and selection / screening processes. In Stage A1, six individual CDR phage libraries were designed with maximum diversity, while restricting the use of amino acids at each position to those predicted to be acceptable based on bioinformatics and structural prediction analysis. These individual libraries were selected on antigens to create a pool of functional CDR variants. After screening, the CDR pool from the library showing the best performance was PCR amplified and paired by overlap PCR to create a composite library for Stage B. Subsequently, this library was selected under stringent conditions to obtain candidates with improved affinity.

[0113] To create six separate libraries for each CDR, the light chain of MtxB (SEQ ID NO: 7) was subcloned into a phagemide vector. For the heavy chain, the variable region, CH1 region, and partial hinge region (SEQ ID NO: 5) were fused to cleaved pIII and subcloned into a phagemide vector. Primers were designed to individually introduce mutations targeting specific residues adjacent to each CDR (Table 4). [Table 6]

[0114] Mutagenic primers included NNK codons to randomize the target site to all 20 amino acids. Individual PCR fragments were paired by overlap PCR to construct a complete gene library. Overlap PCR was performed on a large scale, and the resulting gels were purified and subcloned into phagemide vectors. Each library was transformed into E. coli to grow and propagate Fab phages. After phage production overnight, the phages were precipitated from the culture supernatant. The amplified input phages were coated with 10 μg / ml feline IL-5 (Kingfisher Biotech, catalog no. RP1152F) and incubated for 1 hour in wells of an Immulon4 HBX high-binding plate blocked with 1% bovine serum albumin-containing phosphate-buffered saline (PBS, pH 7.4). The plates were then washed with PBS (pH 7.4) and acid eluted. The output phages were used to infect E. coli and amplified for the next round of panning. Washing was performed under either low-stringency or high-stringency conditions. Under low stringency conditions, three 3-minute washes were performed in each of the four rounds. Under high stringency conditions, five 5-minute washes were performed in the first three rounds, and ten 5-minute washes were performed in round 4. The number of phage inputs and outputs from each panning selection was measured by phage titer. After the final selection round, phages were seeded onto E. coli lawns, and 252 colonies obtained from each selection strategy were cultured in 96-well plates for growth and Fab production. For binding tests, Fab supernatant was pre-incubated with goat anti-feline IgG F(ab')2 to dimerize Fab, and this complex was coated with 10 μg / ml feline IL-5 (Kingfisher Biotech; Cat#RP1152F) and added to an Immulon 4 HBX high-binding plate blocked with 1% bovine serum albumin-containing PBS (pH 7.4). To detect the binding of Fab to feline IL-5, donkey anti-goat antibody labeled with horseradish peroxidase was added to each well. Clones that showed higher binding affinity than the original MtxB Fab were sequenced to identify unique clones.The majority of the positive clones were derived from the LCDR2 library, and one positive clone was derived from the HCDR3 library. After normalizing the concentration of the positive Fab variant using quantitative ELISA, they were tested with feline IL-5 ELISA as described above. The sequences of each LCDR2-positive and HCDR3-positive clone, along with their ELISA values ​​from the feline IL-5 assay, are shown in Table 5. [Table 7]

[0115] Example 5. Elimination of potential sequence-related risks by combining affinity-mature variants. Antibodies containing variants to remove potential deamidation and oxidation sites were combined with affinity-matured variants obtained from Stage A1 above, and tested for their binding affinity to feline IL-5 and their ability to inhibit the binding of feline IL-5 to feline IL-5RA. For this experiment, the variable domain framework was derived from MtxA (VH: SEQ ID NO: 5, VL: SEQ ID NO: 6), and the modified CDRs (HC1, HC2, LC1, LC2, LC3) of each variant antibody chain were as shown in Table 6 below. [Table 8]

[0116] DNA constructs (GeneArt) were created for two heavy chain variants containing the feline IgG1a constant domain and three light chain variants containing the feline kappa chain constant domain, and all of these were subcloned into pcDNA3.4 (ThermoFisher Scientific). Each combination of these heavy and light chain variants was introduced into ExpiCHO cells to prepare conditioned media. Feline IgG was purified by MabSelect SuRe chromatography and buffered with 20 mM sodium acetate and 136 mM sodium chloride (pH 5.5). The binding affinity of different feline IgGs to feline IL-5 was determined using the same SPR method described in the section on eliminating potential sequence-induced risks of LCDR3 and HCDR3, and is shown in Table 7 below. [Table 9]

[0117] Example 6. Further affinity maturation of feline MtxB clones An additional round of affinity maturation was performed using the STEM (Stage-Enhanced Maturation) platform. In this round, both Stage A1 and Stage B were performed in the same manner as described above. The individual CDR libraries created in Stage A1 were panned again to increase their titer and enhance the diversity of the CDR pool before library construction in Stage B. In this panning, 96 multiwells were coated with a higher concentration of 20 μg / ml to capture more diverse clones. Furthermore, a transient heat treatment at 58°C was performed to remove unstable clones.

[0118] For Stage A1, 10 μg of the CDR library was transformed into E. coli to grow and propagate Fab phages. After phage production overnight, the phages were precipitated from the culture supernatant and resuspended in blocking buffer (1% BSA / PBS). Before selection, unstable clones were removed from the library using a transient heat treatment at 58°C for 10 minutes. Next, the input phages were incubated for 1 hour in a 96-multiwell plate coated with 20 μg / ml feline IL-5. The ELISA plate was then washed with PBS (pH 7.4) and eluted with acid. The output phages were used to infect E. coli and amplified for selection in the next round. Each wash was 5 minutes long, with 3, 5, 5, and 5 washes performed in the panning rounds from rounds 1 to 4, respectively. ELISA screening using feline IL-5 was performed on the output from round 4, and ELISA-positive clones were sequenced. Good diversity was obtained from the LCDR2, LCDR3, and HCDR3 libraries.

[0119] Based on the screening results of Stage A1 panning, Stage B libraries were created by combining libraries LCDR2, LCDR3, and HCDR3. Fragments containing pre-selected CDR pools from these libraries were randomly paired by overlap PCR to create new libraries incorporating mutations across all target CDRs. PCR amplification of LCDR2, LCDR3, and HCDR3 was performed using phages amplified after selection in the fourth round. PCR amplification fragments containing light and heavy chains were ligated into phagemid vectors and transformed into E. coli. The number of transformants was approximately 2E+10. The plasmid DNA from the libraries was purified using the midi-prep method. 10 μg of plasmid DNA from the libraries was used to transform E. coli for growth and propagation of Fab phages. After phage production overnight, the phages were precipitated from the culture supernatant and resuspended in blocking buffer (1% BSA / PBS). Before selection, the phages were treated at 58°C for 10 minutes to remove unstable clones. The phages were incubated for 30 minutes in an ELISA plate coated with feline IL-5 and blocked. After washing the ELISA plate with PBS (pH 7.4), the bound phages were eluted with acid. The output phages were then used to infect E. coli and amplified for selection in the next round. For rounds 1, 2, 3, and 4, the ELISA plates were coated with 10 μg / ml, 8 μg / ml, 5 μg / ml, and 1 μg / ml, respectively. The number of washes after each round was 3, 5, 10, and 10 for rounds 1, 2, 3, and 4, respectively. 93 clones obtained from the output of round 4 were cultured overnight to produce Fab, and their binding to feline IL-5 was tested in ELISA format. To perform this assay, the supernatant from the overnight culture was coated with 10 μg / ml feline IL-5 (Kingfisher Biotech, catalog number RP1152F) and incubated on an Immulon 4 HBX high-binding plate blocked with 1% bovine serum albumin-containing phosphate-buffered saline (PBS, pH 7.4).As additional controls, we included the MtxB clone and 1H3-C6, the clone with the highest affinity in the first stage A1 step. Fab bound to feline IL-5 was detected using an anti-feline Fab'2-HRP conjugate.

[0120] The results are shown in Table 8A, comparing the relative Fab binding affinity of each Fab disclosed herein to feline IL-5. Binding to IL-5 was detected using the anti-feline Fab'2-HRP conjugate. Differences in the amino acid composition of CDR compared to MtxB are underlined. The data in the last column represent the absorbance after substrate addition for detecting the HRP conjugate. [Table 10] [Table 11]

[0121] While preferred embodiments of the present invention have been described in detail above, it should be understood that many obvious modifications of the present invention are possible without departing from the spirit or scope of the invention, and the invention as defined in the above paragraphs is not limited to the specific details described above.

Claims

1. An antigen-binding protein that specifically binds to interleukin 5 (IL-5), (a) X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 heavy chain complementarity determining region 1 (HCDR1) comprising (provided that X 1 comprises A, G, I, L, M, W, F, P, or V, and X 2 comprises A, G, I, L, M, W, F, P, or V, and X 3 comprises C, S, T, Y, N, or Q, and X 4 comprises A, G, I, L, M, W, F, P, or V, and X 5 comprises A, G, I, L, M, W, F, P, or V, and X 6 comprises H, K, or R, and X 7 comprises C, S, T, Y, N, or Q, and X 8 comprises C, S, T, Y, N, or Q), and (b) X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 Heavy chain complementarity determination region 2 (HCDR2) including (where X 1 This includes A, G, I, L, M, W, F, P, or V, and X 2 This includes A, G, I, L, M, W, F, P, or V, and X 3 This includes C, S, T, Y, N, or Q, and X 4 This includes A, G, I, L, M, W, F, P, or V, and X 5 This includes A, G, I, L, M, W, F, P, or V, and X 6 is H, K, or R, X 7 This includes C, S, T, Y, N, or Q, and X 8 (including C, S, T, Y, N, or Q) (c) X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 Heavy chain complementarity determination region 3 (HCDR3) including (where X 1 This includes C, S, T, Y, N, or Q, and X 2 is H, K, or R, X 3 This includes E or D, and X 4 This includes C, S, T, Y, N, or Q, and X 5 This includes E or D, and X 6 This includes A, G, I, L, M, W, F, P, or V, and X 7 This includes C, S, T, Y, N, or Q, and X 8 This includes D, E, or G, and X 9 This includes A, G, I, L, M, W, F, P, or V, and X 10 This includes A, G, I, L, M, W, F, P, or V, and X 11 This includes D, E, or L, and X 12 (including C, L, S, T, V, Y, N, or Q) (d) X 1 X 2 X 3 X 4 X 5 X 6 Light chain complementarity determination region 1 (LCDR1) (X 1 This includes C, S, T, Y, N, or Q, and X 2 This includes C, S, T, Y, N, or Q, and X 3 This includes A, G, I, L, M, W, F, P, or V, and X 4 This includes C, S, T, Y, N, or Q, and X 5 This includes E or D, and X 6 (including C, S, T, Y, N, or Q) (e) X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 Light chain complementarity determination region 2 (LCDR2) including (where X 1 This includes L or R, and X 2 This includes A, G, I, L, M, W, F, P, or V, and X 2 This includes A, G, I, L, M, W, F, P, or V, and X 4 This includes C, S, T, Y, N, or Q, and X 5 This includes C, S, T, Y, N, or Q, and X 6 This includes D, E, A, G, I, L, M, W, F, P, or V, and X 7 This includes C, S, T, Y, N, or Q, and X 8 This includes C, S, T, Y, N, Q, A, G, I, L, M, W, F, P, or V, X 9 This includes D, E, C, S, T, Y, N, or Q, and X 10 (including A, G, I, L, M, W, F, P, Y or V) (f) X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 light chain complementarity determining region 3 (LCDR3) comprising (provided that X 1 comprises C, S, T, Y, N or Q, and X 2 comprises C, S, T, Y, N or Q, and X 3 comprises A, G, I, L, M, W, F, P or V, and X 4 comprises A, H, I, K, L, M, P, R, S, V or Y, and X 5 comprises C, F, I, L, R, S, T, Y, N or Q, and X 6 comprises A, G, I, L, M, W, F, P or V, and X 7 comprises A, G, I, L, M, W, F, P or V, and X 8 comprises C, S, T, F, Y, N or Q, and X 9 comprises C, S, T, Y, N or Q), the antigen-binding protein.

2. (a) HCDR1 contains GFTFSNYA (SEQ ID NO: 60) or differs in one or two positions, and / or (b) HCDR2 contains IGSGGHYT (SEQ ID NO: 61) or differs in one or two positions, and / or (c) HCDR3 is TRETDGYX 8 X 9 X 10 X 11 X 12 (Sequence No. 81) includes (however, X 8 This includes D or G, and X 9 This includes G or P, and X 10 This includes I, L, or M, and X 11 This includes D or L, and X 12 (including L, V, or Y), and / or (d) LCDR1 contains QSISDY (Sequence ID 62) or differs in one or two positions, and / or (e) LCDR2 is X 1 X 2 X 3 X 4 X 5 X 6 SX 8 X 9 X 10 Includes (however, X 5 This includes A, F, G, H, P, Q, S, T, V, or Y, and X 6 (including A, D, E, G, L, M, P, S, V, or Y), and / or (f) LCDR3 is QX 2 GX 4 X 5 FPX 8 Includes T (Sequence No. 82) (however, X 2 is S, N, or Q, X 4 This includes A, H, I, L, M, P, S, V, or Y, and X 5 This includes F, I, L, Q, R, S, or V, and X 8 The antigen-binding protein according to claim 1, wherein the antigen-binding protein includes F or Y.

3. In LCDR2, X 1 This includes L or R, and X 2 This includes I, L, or V, and X 3 This includes I or F, and X 4 This includes F, K, N, or Y, and X 5 This includes A, F, G, H, P, Q, S, T, V, or Y, and X 6 This includes A, D, E, G, L, M, P, S, V, or Y, and X 8 This includes A, D, G, L, P, Q, S, V, or Y, and X 9 This includes E, G, K, L, Q, R, S, T, or Y, and X 10 The antigen-binding protein according to claim 1, comprising A, D, I, L, K, N, Q, R, S, or V.

4. The antigen-binding protein according to any one of claims 1 to 3, comprising two or fewer substitutions per CDR compared to HCDR1, HCDR2, and HCDR3 shown in Figure 3, and LCDR1, LCDR2, and LCDR3 shown in Figure 4.

5. The antigen-binding protein according to any one of claims 1 to 3, comprising one or fewer substitutions per CDR compared to HCDR1, HCDR2, and HCDR3 shown in Figure 3, and LCDR1, LCDR2, and LCDR3 shown in Figure 4.

6. One or more HCDRs from among SEQ ID NOs: 3, 5, 8, 11, 19, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58, and SEQ ID NOs: 4, 6, 7, 9, 10, 12, 1 3. An antigen-binding protein according to any one of claims 1 to 5, comprising one or more LCDRs from among SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO:

59.

7. HCDR of any one of the following: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58, and SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: The antigen-binding protein according to any one of claims 1 to 5, comprising: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59 LCDR.

8. The antigen-binding protein according to claim 1, comprising each HCDR of SEQ ID NO: 20 and each LCDR of SEQ ID NO: 23, each HCDR of SEQ ID NO: 19 and each LCDR of SEQ ID NO: 21, each HCDR of SEQ ID NO: 19 and each LCDR of SEQ ID NO: 22, each HCDR of SEQ ID NO: 19 and each LCDR of SEQ ID NO: 23, each HCDR of SEQ ID NO: 20 and each LCDR of SEQ ID NO: 21, or each HCDR of SEQ ID NO: 20 and each LCDR of SEQ ID NO:

22.

9. Each HCDR of SEQ ID NO: 24 and each LCDR of SEQ ID NO: 25, each HCDR of SEQ ID NO: 26 and each LCDR of SEQ ID NO: 27, each HCDR of SEQ ID NO: 28 and each LCDR of SEQ ID NO: 29, each HCDR of SEQ ID NO: 30 and each LCDR of SEQ ID NO: 31, each HCDR of SEQ ID NO: 32 and each LCDR of SEQ ID NO: 33, each HCDR of SEQ ID NO: 34 and each LCDR of SEQ ID NO: 35, each HCDR of SEQ ID NO: 36 and each LCDR of SEQ ID NO: 37, each HCDR of SEQ ID NO: 38 and each LCDR of SEQ ID NO: 39, each HCDR of SEQ ID NO: 40 and each LCDR of SEQ ID NO: 41, each H of SEQ ID NO: 42 The antigen-binding protein according to claim 1, comprising the CDR and the LCDR of SEQ ID NO: 43, the HCDR of SEQ ID NO: 44 and the LCDR of SEQ ID NO: 45, the HCDR of SEQ ID NO: 46 and the LCDR of SEQ ID NO: 47, the HCDR of SEQ ID NO: 48 and the LCDR of SEQ ID NO: 49, the HCDR of SEQ ID NO: 50 and the LCDR of SEQ ID NO: 51, the HCDR of SEQ ID NO: 52 and the LCDR of SEQ ID NO: 53, the HCDR of SEQ ID NO: 54 and the LCDR of SEQ ID NO: 55, the HCDR of SEQ ID NO: 56 and the LCDR of SEQ ID NO: 57, or the HCDR of SEQ ID NO: 58 and the LCDR of SEQ ID NO:

59.

10. The antigen-binding protein according to claim 1, comprising each HCDR of SEQ ID NO: 8 and each LCDR of SEQ ID NO: 7, each HCDR of SEQ ID NO: 11 and each LCDR of SEQ ID NO: 7, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 9, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 10, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 12, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 13, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 14, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 15, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 16, each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO: 17, or each HCDR of SEQ ID NO: 5 and each LCDR of SEQ ID NO:

18.

11. An antigen-binding protein according to any one of claims 1 to 10, comprising a heavy chain framework (FR1H + FR2H + FR3H + FR4H) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to, or identical to, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO:

58.

12. An antigen-binding protein according to any one of claims 1 to 11, comprising a light chain framework (FR1L + FR2L + FR3L + FR4L) that is at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, or at least 95% identical to, identical to, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO:

59.

13. Sequence ID 3, Sequence ID 5, Sequence ID 8, Sequence ID 11, Sequence ID 19, Sequence ID 20, Sequence ID 24, Sequence ID 26, Sequence ID 28, Sequence ID 30, Sequence ID 32, Sequence ID 34, Sequence ID 36, Sequence ID 38, Sequence ID 40, Sequence ID 42, Sequence ID 44, Sequence ID 46, Sequence ID 48, Sequence ID 50, Sequence ID 52, Sequence ID 54, Sequence ID 56 or Sequence ID 58 are at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95%, or at least 97%, identical to these, or are identical to these V H An antigen-binding protein according to any one of claims 1 to 12, comprising a domain.

14. Sequence IDs 4, 6, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or 59 are at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95%, or at least 97%, identical to or the same as these. L An antigen-binding protein according to any one of claims 1 to 13, comprising a domain.

15. An isolated nucleic acid sequence encoding an anti-IL-5 antibody or antibody fragment according to any one of claims 1 to 14.

16. A vector comprising the nucleic acid described in claim 15.

17. Recombinant cells comprising the nucleic acid according to any one of claims 15 or 16.

18. A cell expressing an antigen-binding protein according to any one of claims 1 to 14, or a nucleic acid according to claim 15 or 16.

19. A method for producing an antigen-binding protein according to any one of claims 1 to 14, comprising culturing the cells according to claim 18 under conditions that result in the production of the antigen-binding protein.

20. A pharmaceutical composition comprising a therapeutically effective amount of the anti-IL-5 binding protein according to any one of claims 1 to 14.

21. A method for suppressing IL-5-mediated activation of eosinophils, comprising culturing the eosinophils in the presence of the anti-IL-5 protein described in any one of claims 1 to 14.

22. A method for suppressing a target eosinophil-mediated inflammatory response, comprising administering a therapeutically effective amount of the anti-IL-5 protein described in any one of claims 1 to 14 to the target.

23. A method for inhibiting the binding of IL-5 to an IL-5 receptor in a subject, comprising administering a therapeutically effective amount of the anti-IL-5 protein described in any one of claims 1 to 14 to the subject.

24. The method according to claim 22 or 23, wherein the subject includes cats.

25. A method for detecting IL-5 in a sample, comprising: incubating the sample with an anti-IL-5 protein according to any one of claims 1 to 14; and detecting the anti-IL-5 protein bound to the IL-5 in the sample.

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