Composition for increasing the half-life of a therapeutic drug in cats and method of using the same

Abstract

Compositions and methods of use for increasing the half-life of one or more polypeptides in a cat are provided. The compositions comprise a variant feline IgG Fc region.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 050,535, filed on 10 July 2020, and U.S. Provisional Patent Application No. 63 / 143,720, filed on 29 January 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes an electronically submitted ASCII sequence listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on 23 June 2021, named "47406-0016WO1_SL.txt", and has a size of 25KB.

[0003] This disclosure generally relates to polypeptides (e.g., fusion polypeptides such as polypeptide-Fc domain fusions; or binding molecules such as ligand-binding moieties of antibody or receptor-Fc fusions) whose half-life in cats is increased compared to their wild-type equivalents. [Background technology]

[0004] The Fc region of antibodies plays many, though not limited to, functional roles, including protecting antibodies from degradation via the lysosomal pathway and mediating antibody effector functions. As the use of feline antibodies as therapeutic agents increases, emphasis is being placed not only on selecting the optimal Fab, but also on combining it with the appropriate Fc to achieve the desired half-life and effector function.

[0005] In the art, there are few guidelines regarding increasing the half-life of polypeptide therapeutics (e.g., antibodies) used in cats. This disclosure addresses this lack by providing Fc region variants that improve the serum persistence of polypeptides (e.g., antibodies) in cats. [Overview of the project]

[0006] Provided herein are feline Fc (e.g., Fc region variants of feline IgG) or feline FcRn-binding fragments useful for therapeutic polypeptides. This disclosure features polypeptides exhibiting increased binding to feline FcRn than a control polypeptide (e.g., the feline Fc region of IgG, which is the wild-type equivalent). In some cases, these polypeptides exhibit increased binding to feline FcRn than the control polypeptide at pH 5.5, pH 6.0, and / or pH 6.5. In some cases, these polypeptides can bind to feline FcRn at higher levels at acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5) than at neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some cases, these polypeptides bind to feline FcRn at higher levels at pH 5.5 and / or pH 6.0 than at pH 7.4. This disclosure relates in part to polypeptides with an increased half-life in cats compared to their wild-type equivalent. For example, a binding molecule (e.g., a ligand-binding portion of an antibody or receptor) with an increased half-life is provided compared to a version of the binding molecule not bound to the Fc region or its feline FcRn binding region disclosed herein. Also provided are enzyme-Fc region fusions, ligand-Fc region fusions, nanobody-Fc fusions, and peptide-Fc region fusions, the fusions having an increased half-life compared to their wild-type equivalents. In addition to having one or more substitutions that increase the half-life (compared to the wild-type feline Fc region), the Fc region may also include other substitutions that result in, for example, increased effector function, decreased effector function, increased binding to protein A, and / or decreased polypeptide heterogeneity (e.g., by removing one or more post-translational modifications in the Fc region). The feline Fc region sequence may be derived from any feline antibody. In some cases, the feline Fc region sequence is derived from feline IgG (e.g., IgG1a, IgG1b, IgG2).

[0007] This disclosure comprises a recombinant protein comprising (1) a binding domain or fragment thereof that specifically binds to an epitope of a ligand or protein, wherein the binding domain is (2) bound to a domain comprising an Fc region (CH2+CH3 region) or a feline FcRn binding region as disclosed herein. In some cases, the binding domain comprises (i) six complementarity-determining regions (CDRs), e.g., of a feline or human / humanized antibody; (ii) a nanobody; (iii) a soluble receptor-binding domain or a ligand-binding fragment thereof that binds to a ligand; and (iv) an extracellular domain of a feline receptor protein.

[0008] This disclosure also provides compositions comprising (1) a first polypeptide comprising a first Fc region (e.g., CH2 region, CH3 region, CH2+CH3 region) comprising an Fc region variant of feline IgG as described herein, and (2) a second polypeptide comprising a second Fc region comprising an Fc region variant of feline IgG as described herein. The first and second polypeptides can associate via the first and second Fc regions. In some cases, the amino acid sequences of the first and second Fc regions are the same. In other cases, the amino acid sequences of the first and second Fc regions are different (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25). In some cases, the Fc region variant is a variant of the Fc region of a feline IgG1a antibody. In some cases, the Fc region variant is a variant of the Fc region of the feline IgG1b antibody. In some cases, the Fc region variant is a variant of the Fc region of the feline IgG2 antibody.

[0009] Fusion molecules comprising feline IgG Fc region variants and polypeptides disclosed herein are also disclosed. In some cases, the feline IgG Fc region variant is covalently bound to the polypeptide (e.g., via a hinge region or linker). In some cases, the polypeptide is the ligand-binding domain of a feline receptor protein, the extracellular domain of a feline receptor protein, or the antigen-binding domain. In some cases, the polypeptide is selected from the ligand-binding domain or extracellular domain of feline IL-13Rα1 or IL-13Rα2, feline EPO, feline CTLA4, feline LFA3, feline VEGFR1 / VEGFR3, feline IL-1R, feline GLP-1 receptor agonists, and feline thrombopoietin-binding peptides. In some cases, the polypeptide is an scFv, nanobody, or single-domain antibody. In some cases, the IgG Fc region variant is a variant of the Fc region of a feline IgG1a antibody. In some cases, the Fc region variant of IgG is the same as the Fc region variant of feline IgG1b antibody. In some cases, the Fc region variant of IgG is the same as the Fc region variant of feline IgG2 antibody.

[0010] In some embodiments, the present disclosure relates to a polypeptide comprising a feline IgG Fc region variant or its feline FcRn binding region, wherein the polypeptide comprises an amino acid substitution at at least one position selected from the group consisting of: (i) The position corresponding to amino acid position 252 of wild-type cat IgG, where the amino acid substitution is S252W; (ii) A position corresponding to amino acid position 254 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S254R and S254K; (iii) A position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V or L309Y; (iv) A position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of Q311R, Q311V, Q311L, and Q311K; (v) A position corresponding to amino acid position 428 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428M, S428Y, S428H, and S428R; and (vi) One or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type feline IgG, wherein the amino acid positions are based on EU numbering, and the polypeptide provides a polypeptide having an increased binding affinity for feline FcRn when compared to the Fc domain of wild-type feline IgG.

[0011] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 252 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 252 of wild-type feline IgG is S252W.

[0012] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 254 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 254 of wild-type feline IgG is S254R. In some embodiments, the amino acid substitution at position 254 of wild-type feline IgG is S254K.

[0013] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 309 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 309 of wild-type feline IgG is L309V. In some embodiments, the amino acid substitution at position 309 of wild-type feline IgG is L309Y.

[0014] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 311 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311R. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311V. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311K. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311L.

[0015] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 428 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428M.

[0016] In some embodiments, the polypeptide comprises at least the amino acid substitution S428Y. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428Y. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428R. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428H.

[0017] In another embodiment, the polypeptide contains amino acid substitutions at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type cat IgG. In some embodiments, the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293H, E293K, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L, as well as any of the aforementioned conservative amino acid substitutions. In some embodiments, the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293H, E293K, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L.

[0018] In another embodiment, the disclosure relates to a polypeptide comprising a feline IgG Fc region variant or its feline FcRn binding region, wherein the polypeptide comprises two or more amino acid substitutions, the two or more amino acid substitutions comprising the group: (i) An amino acid substitution at the position corresponding to amino acid position 252 of wild-type cat IgG, selected from the group consisting of S252W, S252Y, S252F, and S252R; (ii) An amino acid substitution at the position corresponding to amino acid position 254 of wild-type feline IgG, selected from the group consisting of S254R and S254K; (iii) Amino acid substitutions at the position corresponding to amino acid position 309 of wild-type feline IgG, selected from the group consisting of L309V, L309Y, and L309E; (iv) Amino acid substitutions at the position corresponding to amino acid position 311 of wild-type feline IgG, selected from the group consisting of Q311R, Q311V, Q311L, and Q311K; (v) an amino acid substitution at the position corresponding to amino acid position 428 of wild-type feline IgG, selected from the group consisting of S428L, S428M, S428Y, S428H, and S428R; (vi) amino acid substitutions at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426 and 437 of wild-type cat IgG; and (vii) An amino acid substitution at the position corresponding to amino acid position 434 of wild-type cat IgG, selected from the group consisting of S434F, S434W, S434H, S434R, and S434Y, Here, based on EU numbering, the amino acid positions are such that two or more amino acid substitutions are in different positions, and the polypeptide provides a polypeptide that has increased binding affinity to feline FcRn compared to (a) the Fc domain of wild-type feline IgG and (b) a polypeptide containing only one of the two or more amino acid substitutions.

[0019] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 286 of wild-type cat IgG. In some embodiments, the amino acid substitution is selected from the group consisting of T286E and T286D.

[0020] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 289 of wild-type cat IgG. In some embodiments, the amino acid substitution is selected from the group consisting of T289K and T289H.

[0021] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 301 of wild-type cat IgG. In some embodiments, the amino acid substitution is R301L.

[0022] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 334 of wild-type cat IgG. In some embodiments, the amino acid substitution is R334D.

[0023] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 426 of wild-type cat IgG. In some embodiments, the amino acid substitution is selected from the group consisting of S426L and S426H.

[0024] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 437 of wild-type cat IgG. In some embodiments, the amino acid substitution is T437L.

[0025] In some embodiments, two or more amino acid substitutions are comprised of the following groups: (i) A combination of S252Y and Q311R and / or Q311L; (ii) A combination of S434Y and one or more of S254R, S254K, L262E, T286D, T286E, T289K, E293D, E293K, L309V, L309E, K326D, and Q347L; (iii) The combination of S434F and E380D; (iv) A combination of S428L and one or more of S252R, T286E, Q311V, Q311K, D312T, I377V, I383L, and N389cR; (v) S428L, E380D and S434R; (vi) S428L, E380T and S434R; (vii) The combination of S252R and L262Q; (viii) T260E, L309E and Q355L; (ix) S290V and R344D combination; (x) R301L, E380V and T437L; (xi) T286E and S428H combination; (xii) A combination of R334D and one or more of S428R, T437L, and R301L; (xiii) A combination of S426L and T289H and / or S428H; (xiv) A combination of S428Y and one or more of Q311V, S254R, L309V, T286E, and E380T; and The combination is selected from (xv)S428H and T289H.

[0026] In some embodiments, the polypeptide contains an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

[0027] In some embodiments, the wild-type feline IgG is feline IgG1a containing an Fc domain having an amino acid sequence identical to at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) of SEQ ID NO: 1, feline IgG1b containing an Fc domain having an amino acid sequence identical to at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) of SEQ ID NO: 2, or feline IgG2 containing an Fc domain having an amino acid sequence identical to at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) of SEQ ID NO: 3.

[0028] In other embodiments, wild-type feline IgG is feline IgG1a containing an Fc domain having the amino acid sequence of SEQ ID NO: 1. In other embodiments, wild-type feline IgG is feline IgG1b containing an Fc domain having the amino acid sequence of SEQ ID NO: 2. In other embodiments, wild-type feline IgG is feline IgG2 containing an Fc domain having the amino acid sequence of SEQ ID NO: 3.

[0029] In some embodiments, the polypeptide further comprises a binding domain. In some embodiments, the binding domain comprises (i) six complementarity-determining regions (CDRs) of an immunoglobulin molecule; (ii) a ligand-binding domain of a feline receptor protein; (iii) a nanobody; or (iv) an extracellular domain of a feline receptor protein. In some embodiments, the binding domain specifically binds to an antigen selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and the complement system complex.

[0030] In some embodiments, the polypeptide further comprises a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonists, and thrombopoietin-binding peptides.

[0031] In some embodiments, the polypeptide binds to feline FcRn at higher levels at acidic pH than at neutral pH. In some embodiments, the polypeptide binds to feline FcRn at higher levels at pH 5.5 than at pH 7.4. In some embodiments, the polypeptide binds to feline FcRn at higher levels at pH 6.0 than at pH 7.4.

[0032] In some embodiments, the polypeptide (1) has an increased half-life in cats compared to one or more control polypeptides, where one or more control polypeptides are identical to one or more polypeptides except that they have the Fc region of the corresponding wild-type cat IgG instead of the Fc region variant of IgG, and / or (2) has increased binding to cat FcRn compared to the control polypeptides, where the amino acid positions are based on EU numbering.

[0033] In some embodiments, the Disclosure provides pharmaceutical compositions comprising (i) a polypeptide described herein and (ii) a pharmaceutically acceptable excipient.

[0034] In some embodiments, this disclosure provides one or more nucleic acids encoding polypeptides described herein.

[0035] In some embodiments, the Disclosure provides one or more expression vectors comprising one or more nucleic acids described herein.

[0036] In some embodiments, the Disclosure provides a host cell comprising one or more nucleic acids or one or more expression vectors as described herein.

[0037] In some embodiments, the Disclosure provides a method for producing one or more polypeptides, the method including: (a) To provide one or more nucleic acids as described herein; (b) Expressing one or more nucleic acids in a host cell culture to produce polypeptides; (c) Recover the polypeptide produced in (b) from the host cell culture.

[0038] In some embodiments, the method further includes formulating a polypeptide as a pharmaceutical formulation.

[0039] In some embodiments, the Disclosure provides a method for treating a feline disease or disorder in a cat that is in need thereof, comprising administering to the cat an effective amount of a composition comprising a pharmaceutical composition described herein.

[0040] In some embodiments, the Disclosure provides a method for preventing a feline disease or disorder in a cat that is in need thereof, comprising administering to the cat an effective amount of a composition comprising a pharmaceutical composition described herein.

[0041] In some embodiments, the disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disorder, gastrointestinal disorder, cardiovascular disease, kidney disease, reproductive dysfunction, infection, or cancer.

[0042] In some embodiments, the disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used to implement or verify the present invention, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, including definitions, this application shall prevail. Materials, methods, and examples are illustrative and not intended to limit the scope of the invention.

[0044] Other features and advantages of the present invention will become apparent from the following embodiments and claims for carrying out the invention. [Brief explanation of the drawing]

[0045] [Figure 1]The Biacore sensorgrams of feline IgG1a Fc variants S428L, S428M, S428Y, S434F, S434W, and S434H from the NNK Library are shown. In each figure, thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 2] This shows the Biacore sensorgrams of feline IgG1a Fc variants S252W, S252Y, and S252F from the NNK library. The Biacore sensorgram for wild-type (WT) is also shown. In each figure, thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 3] The amino acid sequence alignment of the wild-type feline IgG1a Fc region (SEQ ID NO: 1), the wild-type feline IgG1b Fc region (SEQ ID NO: 2), and the presumed wild-type feline IgG2 Fc region (SEQ ID NO: 3) is shown. The hinge region is located between the triangles. The arrows indicate cysteine ​​residues within the hinge region that are thought to be involved in the disulfide bridge between the two heavy chains (Strietzel et al., 2014, Vet.Immunol.Immunopathol., 158:214-223). [Figure 4] The amino acid sequence alignment of the wild-type cat IgG1a Fc region (SEQ ID NO: 1) and the human IgG1 Fc region is shown based on EU numbering. The 55 amino acid positions used in the generation of the phage library described in Example 2 are highlighted and underlined. [Figure 5A] This Carterra LSA sensorgram shows the interaction between the S252Y feline IgG1a Fc variant and feline FcRN from a phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 5B]This Carterra LSA sensorgram shows the interaction between the S252Y feline IgG1a Fc variant and feline FcRN from a phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 6A] This Carterra LSA sensorgram shows the interaction between the S252Y+Q311R feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 6B] This Carterra LSA sensorgram shows the interaction between the S252Y+Q311R feline IgG1a Fc variant and feline FcRN at pH 7.4 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 7A] This Carterra LSA sensorgram shows the interaction between the S252Y+Q311K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 7B] This Carterra LSA sensorgram shows the interaction between the S252Y+Q311K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 8A]This Carterra LSA sensorgram shows the interaction between the S252Y+Q311V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 8B] This Carterra LSA sensorgram shows the interaction between the S252Y+Q311V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 9A] This Carterra LSA sensorgram shows the interaction between the S252Y+Q311L feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 9B] This Carterra LSA sensorgram shows the interaction between the S252Y+Q311L feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 10A] This Carterra LSA sensorgram shows the interaction between the S434Y feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 10B]This Carterra LSA sensorgram shows the interaction between the S434Y feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 11A] This Carterra LSA sensorgram shows the interaction between the S434Y+S254R feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 11B] This Carterra LSA sensorgram shows the interaction between the S434Y+S254R feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 12A] This Carterra LSA sensorgram shows the interaction between the S434Y+S254K feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 12B] This Carterra LSA sensorgram shows the interaction between the S434Y+S254K feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 13A]This Carterra LSA sensorgram shows the interaction between the S434Y+L262E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 13B] This Carterra LSA sensorgram shows the interaction between the S434Y+L262E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 14A] This Carterra LSA sensorgram shows the interaction between the S434Y+T286D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 14B] This Carterra LSA sensorgram shows the interaction between the S434Y+T286D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 15A] This Carterra LSA sensorgram shows the interaction between the S434Y+T286E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 15B]This Carterra LSA sensorgram shows the interaction between the S434Y+T286E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 16A] This Carterra LSA sensorgram shows the interaction between the S434Y+T289K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 16B] This Carterra LSA sensorgram shows the interaction between the S434Y+T289K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 17A] This Carterra LSA sensorgram shows the interaction between the S434Y+E293D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 17B] This Carterra LSA sensorgram shows the interaction between the S434Y+E293D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 18A]This Carterra LSA sensorgram shows the interaction between the S434Y+E293K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 18B] This Carterra LSA sensorgram shows the interaction between the S434Y+E293K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 19A] This Carterra LSA sensorgram shows the interaction between the S434Y+L309V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 19B] This Carterra LSA sensorgram shows the interaction between the S434Y+L309V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 20A] This Carterra LSA sensorgram shows the interaction between the S434Y+L309E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 20B]This Carterra LSA sensorgram shows the interaction between the S434Y+L309E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 21A] This Carterra LSA sensorgram shows the interaction between the S434Y+K326D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 21B] This Carterra LSA sensorgram shows the interaction between the S434Y+K326D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 22A] This Carterra LSA sensorgram shows the interaction between the S434Y+Q347L feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 22B] This Carterra LSA sensorgram shows the interaction between the S434Y+Q347L feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 23A]This Carterra LSA sensorgram shows the interaction between the S434Y+S426L feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 23B] This Carterra LSA sensorgram shows the interaction between the S434Y+S426L feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 24A] This Carterra LSA sensorgram shows the interaction between the S434F feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 24B] This Carterra LSA sensorgram shows the interaction between the S434F feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 25A] This Carterra LSA sensorgram shows the interaction between the S434F+E380D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 25B]This Carterra LSA sensorgram shows the interaction between the S434F+E380D feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 26A] This Carterra LSA sensorgram shows the interaction between the S428L+T286E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 26B] This Carterra LSA sensorgram shows the interaction between the S428L+T286E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 27A] This Carterra LSA sensorgram shows the interaction between the S428L+Q311V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 27B] This image shows a Carterra LSA sensorgram of the interaction between the S428L+Q311V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 28A]This Carterra LSA sensorgram shows the interaction between the S428L+Q311K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 28B] This Carterra LSA sensorgram shows the interaction between the S428L+Q311K feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 29A] This Carterra LSA sensorgram shows the interaction between the S428L+D312T feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 29B] This Carterra LSA sensorgram shows the interaction between the S428L+D312T feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 30A] This Carterra LSA sensorgram shows the interaction between the S428L+I377V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 30B]This Carterra LSA sensorgram shows the interaction between the S428L+I377V feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 31A] This Carterra LSA sensorgram shows the interaction between the S428L+I383L feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 31B] This image shows a Carterra LSA sensorgram of the interaction between the S428L+I383L feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 32A] This Carterra LSA sensorgram shows the interaction between the S428L+N389cR feline IgG1a Fc variant and feline FcRN at pH 6.0 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 32B] This Carterra LSA sensorgram shows the interaction between the S428L+N389cR feline IgG1a Fc variant and feline FcRN at pH 7.4 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 33A]This Carterra LSA sensorgram shows the interaction between the S428L+E380D+S434R feline IgG1a Fc variant and feline FcRN at pH 6.0 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 33B] This Carterra LSA sensorgram shows the interaction between the S428L+E380D+S434R feline IgG1a Fc variant and feline FcRN at pH 7.4 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 34A] This Carterra LSA sensorgram shows the interaction between the S428L+E380T+S434R feline IgG1a Fc variant and feline FcRN at pH 6.0 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 34B] This Carterra LSA sensorgram shows the interaction between the S428L+E380T+S434R feline IgG1a Fc variant and feline FcRN at pH 7.4 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 35A] This Carterra LSA sensorgram shows the interaction between the Q311R feline IgG1a Fc variant and feline FcRN from a phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 35B]This Carterra LSA sensorgram shows the interaction between the Q311R feline IgG1a Fc variant and feline FcRN from a phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 36A] This Carterra LSA sensorgram shows the interaction between the R392E feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 36B] This Carterra LSA sensorgram shows the interaction between the R392E feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 37A] This Carterra LSA sensorgram shows the interaction between the S252R+L262Q feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 37B] This image shows a Carterra LSA sensorgram of the interaction between the S252R+L262Q feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 38A]This Carterra LSA sensorgram shows the interaction between the S252R+A378E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 38B] This Carterra LSA sensorgram shows the interaction between the S252R+A378E feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 39A] This Carterra LSA sensorgram shows the interaction between the T260E+L309E+Q355L feline IgG1a Fc variant and feline FcRN at pH 6.0 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 39B] This Carterra LSA sensorgram shows the interaction between the T260E+L309E+Q355L feline IgG1a Fc variant and feline FcRN at pH 7.4 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 40A] This Carterra LSA sensorgram shows the interaction between the T286E+S428R feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 40B]This Carterra LSA sensorgram shows the interaction between the T286E+S428R feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 41A] This Carterra LSA sensorgram shows the interaction between the S290V+R334D feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 41B] This Carterra LSA sensorgram shows the interaction between the S290V+R334D feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 42A] This Carterra LSA sensorgram shows the interaction between the R301L+E380V+T437L feline IgG1a Fc variant and feline FcRN at pH 6.0 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 42B] This Carterra LSA sensorgram shows the interaction between the R301L+E380V+T437L feline IgG1a Fc variant and feline FcRN at pH 7.4 in the phage display library. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 43A]This Carterra LSA sensorgram shows the interaction between the S428L feline IgG1a Fc variant from the phage display library and feline FcRN at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 43B] This Carterra LSA sensorgram shows the interaction between the S428L feline IgG1a Fc variant from the phage display library and feline FcRN at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 44A] This Carterra LSA sensorgram shows the interaction between the S428L+S252R feline IgG1a Fc variant and feline FcRN from the phage display library at pH 6.0. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 44B] This Carterra LSA sensorgram shows the interaction between the S428L+S252R feline IgG1a Fc variant and feline FcRN from the phage display library at pH 7.4. Irregular lines represent measured data, and smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 45A] This Carterra LSA sensorgram shows the interaction between wild-type feline IgG1a Fc variant (SEQ ID NO: 1) and feline FcRN at pH 6.0. Irregular lines represent measured data, while smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 45B]This Carterra LSA sensorgram shows the interaction between wild-type feline IgG1a Fc variant (SEQ ID NO: 1) and feline FcRN at pH 7.4. Irregular lines represent measured data, while smooth lines are approximation curves using a 1:1 interaction model. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 46] A flowchart of a two-compartment pharmacokinetic (PK) model with linear clearance, using a nonlinear mixed-effects (NLME) model, is shown, which was used to describe the serum concentrations of anti-NGF monoclonal antibody (mAb) variants. [Figure 47] The serum concentrations observed for wild-type (WT) antibodies and antibody variants S252W, S428Y, S428Y+L309V, S428Y+Q311V, and S428Y+S254R are shown individually (results from two animals per antibody / variant). [Figure 48] The predicted serum concentration profiles for wild-type (WT) antibody and antibody variants S252W, S428Y, S428Y+L309V, S428Y+Q311V, and S428Y+S254R in a typical 2kg cat receiving a single IV dose of 2mg / kg antibody / variant are shown. [Figure 49A] This figure shows Biacore sensorgrams illustrating the binding of wild-type feline IgG1a. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49B] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49C] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49D]This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49E] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49F] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49G] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49H] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49I] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49J] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49K] This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 49L]This figure shows Biacore sensorgrams illustrating the binding of feline IgG1a variants. In each figure, thin lines represent measurement data, and thick lines represent approximation curves. Y-axis: Resonance units (RU); X-axis: Time (seconds). [Figure 50] The serum concentrations of the wild-type and Fc variants, observed individually in two animals for each variant, are shown. [Figure 51] This shows the predicted serum concentration profile of monoclonal antibodies that hold wild-type (WT) IgG1a Fc or IgG1a Fc variants. [Modes for carrying out the invention]

[0046] The use of polypeptides (e.g., antibodies, ligand-binding domains of receptors, enzymes, ligands, peptides) is increasing as therapeutic agents for the prevention and treatment of a wide variety of feline diseases. In particular, for the prevention or treatment of chronic diseases that require repeated polypeptide administration, it is important to develop polypeptides with long half-lives.

[0047] Accordingly, this disclosure features feline immunoglobulin Fc regions or feline FcRn binding regions containing mutations that enhance the half-life of one or more polypeptides comprising these sequences. Polypeptides comprising these domains and methods of use thereof are also disclosed. These peptides can be used for a variety of therapeutic and diagnostic applications.

[0048] Where values ​​are given as a range, it should be understood that the description includes disclosure of all possible subranges within that range and specific numerical values ​​that fall within that range, regardless of whether a specific numerical value or subrange is explicitly stated. All numerical notations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations and may vary as appropriate in increments of 1.0 or 0.1 (+) or (-), or by + / - 15%, 10%, 5%, or 2%. It should be understood that all numerical notations are preceded by the word "approximately," although this is not always explicitly stated. It should also be understood that the reagents described herein are merely illustrative, and their equivalents are known in the art, although this is not always explicitly stated.

[0049] When the term "approximately" is used herein to refer to a measurable value such as a quantity or concentration, it means that the specified quantity may vary by 20%, 10%, 5%, 1%, 0.5%, or 0.1%.

[0050] Cat antibodies Cats typically possess three IgG heavy chains, known as IgG1a, IgG1b, and IgG2. These heavy chains represent three distinct subclasses of feline IgG. The amino acid and DNA sequences of these heavy chains are available from Strietzel et al., 2014, Vet.Immunol.Immunopathol., 158:214-223 and the GENBANK database. For example, the amino acid sequence of the feline IgG1a heavy chain is GENBANK accession number BAA32229.1, the feline IgG1b heavy chain is GENBANK accession number BAA32230.1, and the feline IgG2 heavy chain is GENBANK accession number KF811175.1. Feline antibodies also contain two types of light chains: kappa and lambda. The DNA and amino acid sequences of these light chains are also available from the GENBANK database. For example, the light chain amino acid sequence of Nekokappa has accession number AF198257.1, and the light chain of Nekoramuda has accession number E07339.1.

[0051] The CH2 region of the feline Fc domain: The CH2 region of feline antibodies contains or consists of amino acids 231–340 (according to EU numbering) of feline IgG antibodies. It is understood that the CH2 region may contain 1–6 (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0052] The amino acid sequence of the CH2 region of feline IgG1a is provided below:PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAK (SEQ ID NO: 4)

[0053] The amino acid sequence of the CH2 domain of feline IgG1b is provided below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDK(Sequence ID 5)

[0054] The amino acid sequence of the CH2 domain of feline IgG2 is provided below: VPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAK(Sequence ID 6)

[0055] The CH3 region of the feline Fc domain: The CH3 region of feline antibodies contains or consists of amino acids 341–447 (according to EU numbering) of feline IgG antibodies. It is understood that the CH3 region may contain 1–6 (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0056] The amino acid sequence of the CH3 domain of feline IgG1a is provided below: GQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (Sequence ID 7)

[0057] The amino acid sequence of the CH3 domain of feline IgG1b is provided below: GQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (Sequence ID 8)

[0058] The amino acid sequence of the CH3 domain of feline IgG2 is provided below: GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (Sequence ID 9)

[0059] Fc region of the feline Fc domain: The Fc region of feline IgG antibodies contains or consists of amino acids 231-447 (according to EU numbering).

[0060] The amino acid sequence of the Fc domain of feline IgG1a is provided below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKA KGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (Sequence ID 1)

[0061] The amino acid sequence of the Fc domain of feline IgG1b is provided below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKD KGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (Sequence 2)

[0062] The amino acid sequence of the Fc domain of feline IgG2 is provided below: VPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (Sequence ID 3)

[0063] Table 1 below compares the amino acid sequences of the CH2 and CH3 domains of human IgG1, feline IgG1a, feline IgG1b, and feline IgG2 based on EU numbering. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0064] Feline IgG Fc substitution improves half-life Increased serum persistence is a beneficial property for therapeutic polypeptides. This disclosure features substitutions of the Fc regions of wild-type feline IgG1a, IgG1b, and IgG2, the substitutions improving the half-life in cats of one or more polypeptides containing these Fc regions compared to one or more control polypeptides, where one or more control polypeptides are identical to one or more polypeptides except that they have the Fc region of the corresponding wild-type feline IgG instead of the Fc region variant of IgG. Substitutions that increase half-life may be made in one or more of the feline CH2 region, feline CH3 region, or in association with the feline Fc (e.g., CH2+CH3) region.

[0065] This disclosure relates to a polypeptide comprising a feline IgG Fc region variant or its feline FcRn binding region, wherein the polypeptide has an amino acid substitution at at least one position selected from the group consisting of: (i) The position corresponding to amino acid position 252 of wild-type cat IgG, where the amino acid substitution is S252W; (ii) A position corresponding to amino acid position 254 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S254R and S254K; (iii) A position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V or L309Y; (iv) A position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of Q311R, Q311V, Q311L, and Q311K; (v) A position corresponding to amino acid position 428 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428M, S428Y, S428H, and S428R; and (vi) One or more amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type cat IgG, Here, based on EU numbering, the amino acid positions provide a polypeptide that exhibits increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0066] In some embodiments, the polypeptide exhibits increased binding affinity to feline FcRn at a pH of approximately 5.0 to approximately 6.5 (e.g., approximately 5.5 or approximately 6.0) compared to the Fc domain of wild-type feline IgG.

[0067] In some embodiments, the polypeptide includes an amino acid substitution at the position corresponding to amino acid position 252 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 252 of wild-type feline IgG is S252W.

[0068] In some embodiments, the polypeptide includes an amino acid substitution at the position corresponding to amino acid position 254 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 254 of wild-type feline IgG is S254R. In some embodiments, the amino acid substitution at position 254 of wild-type feline IgG is S254K.

[0069] In some embodiments, the polypeptide comprises amino acid substitutions L309V or L309Y.

[0070] In some embodiments, the polypeptide contains an amino acid substitution at the position corresponding to amino acid position 311 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311R. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311V. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311K. In some embodiments, the amino acid substitution at position 311 of wild-type feline IgG is Q311L.

[0071] In some embodiments, the polypeptide includes an amino acid substitution at the position corresponding to amino acid position 428 of wild-type feline IgG. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428M.

[0072] In some embodiments, the polypeptide contains at least the amino acid substitution S428Y. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428Y. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428R. In some embodiments, the amino acid substitution at position 428 of wild-type feline IgG is S428H.

[0073] In another embodiment, the polypeptide contains amino acid substitutions at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type cat IgG. In some embodiments, the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293H, E293K, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L, as well as any of the aforementioned conservative amino acid substitutions. In some embodiments, the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293H, E293K, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L.

[0074] In another embodiment, the disclosure relates to a polypeptide comprising a feline IgG Fc region variant or its feline FcRn binding region, wherein the polypeptide comprises two or more amino acid substitutions, the two or more amino acid substitutions comprising the group: (i) An amino acid substitution at the position corresponding to amino acid position 252 of wild-type cat IgG, selected from the group consisting of S252W, S252Y, S252F, and S252R; (ii) An amino acid substitution at the position corresponding to amino acid position 254 of wild-type feline IgG, selected from the group consisting of S254R and S254K; (iii) Amino acid substitutions at the position corresponding to amino acid position 309 of wild-type feline IgG, selected from the group consisting of L309V, L309Y, and L309E; (iv) Amino acid substitutions at the position corresponding to amino acid position 311 of wild-type feline IgG, selected from the group consisting of Q311R, Q311V, Q311L, and Q311K; (v) an amino acid substitution at the position corresponding to amino acid position 428 of wild-type feline IgG, selected from the group consisting of S428L, S428M, S428Y, S428H, and S428R; (vi) amino acid substitutions at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426 and 437 of wild-type cat IgG; and (vii) An amino acid substitution at the position corresponding to amino acid position 434 of wild-type cat IgG, selected from the group consisting of S434F, S434W, S434H, S434R, and S434Y, Here, based on EU numbering, the amino acid positions are such that two or more amino acid substitutions are in different positions, and the polypeptide provides a polypeptide that has increased binding affinity to feline FcRn compared to (a) the Fc domain of wild-type feline IgG and (b) a polypeptide containing only one of the two or more amino acid substitutions.

[0075] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 286 of wild-type cat IgG, and the amino acid substitution is selected from the group consisting of T286E and T286D.

[0076] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 289 of wild-type cat IgG, and the amino acid substitution is selected from the group consisting of T289K and T289H.

[0077] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 301 of wild-type cat IgG, and the amino acid substitution is R301L.

[0078] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 334 of wild-type cat IgG, and the amino acid substitution is R334D.

[0079] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 426 of wild-type cat IgG, and the amino acid substitution is selected from the group consisting of S426L and S426H.

[0080] In some embodiments, two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 437 of wild-type cat IgG, and the amino acid substitution is T437L.

[0081] In some embodiments, two or more amino acid substitutions are comprised of the following groups: (i) A combination of S252Y and Q311R and / or Q311L; (ii) A combination of S434Y and one or more of S254R, S254K, L262E, T286D, T286E, T289K, E293D, E293K, L309V, L309E, K326D, and Q347L; (iii) S434F and E380D; (iv) A combination of S428L and one or more of the following: S252R, T286E, Q311V, Q311K, D312T, I377V, I383L, N389cR; (v) S428L, E380D and S434R; (vi) S428L, E380T and S434R; (vii) The combination of S252R and L262Q; (viii) T260E, L309E and Q355L; (ix)S290V and R344D; and (x) Select from R301L, E380V and T437L.

[0082] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

[0083] In some cases, the Disclosure provides CH2 region variants of feline IgG that include an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 4-6. The Disclosure also provides CH2 region variants of feline IgG that include an amino acid sequence that differs by 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) from any one of SEQ ID NOs: 4-6.

[0084] In other cases, the Disclosure features CH3 region variants of feline IgG that include an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs. The Disclosure also features CH3 region variants of feline IgG that include an amino acid sequence that differs by 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) from any one of SEQ ID NOs.

[0085] In certain cases, the Disclosure features Fc region variants of feline IgG that include an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 1-3. Disclosed are Fc region variants of feline IgG that include an amino acid sequence which differs from any one of SEQ ID NOs: 1-3 by 1 to 20 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids).

[0086] In some embodiments, one or more polypeptides comprising a feline IgG Fc CH2 region variant are provided, wherein the CH2 region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 4-6.

[0087] In some embodiments, the features include one or more polypeptides comprising a feline IgG Fc CH3 region variant, wherein the CH3 region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 7-9.

[0088] In some embodiments, the polypeptide comprises one or more polypeptides containing an Fc region variant of feline IgG, wherein the Fc region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 1 to 3.

[0089] As noted elsewhere, in some embodiments, the polypeptide further includes at least one additional amino acid substitution in the region corresponding to amino acid positions 250-256, 285-288, 307-315, 376-380, 383-392, or 428-437 of wild-type feline IgG, where the amino acid positions are based on EU numbering, and the polypeptide exhibits increased binding to feline FcRn compared to the Fc domain of wild-type feline IgG.

[0090] In some embodiments, the polypeptide includes at least one additional amino acid substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) selected from those disclosed in Table 2 below. [Table 2]

[0091] Substitutions may be made on one or both strands of the CH2 domain, CH3 domain, or Fc domain. In some cases, substitutions on both strands of the CH2 domain, CH3 domain, or Fc domain are identical. In some cases, substitutions on both strands of the CH2 domain, CH3 domain, or Fc domain are not identical. In some cases, the Fc region contains one or more additional substitutions that result in an increase or decrease in effector function and / or improvement of product heterogeneity.

[0092] Other substitutions that can be combined with substitutions that improve half-life The development of therapeutic polypeptides / proteins (e.g., monoclonal antibodies) is a complex process that requires the coordination of a series of intricate operations to produce the desired polypeptide / protein. These operations include optimizing specificity, affinity, functional activity, expression levels in artificial cell lines, long-term stability, removal or enhancement of effector functions, and the development of commercially viable manufacturing and purification methods. This disclosure encompasses any additional substitutions that facilitate one or more of the above objectives.

[0093] In some embodiments, substitutions are introduced to reduce the effector function of the feline Fc region. Such substitutions are known to those skilled in the art and may be at one or more positions (e.g., 1, 2, 3, 4, 5, 6, or 7) of the feline IgG. An exemplary example is WO2019 / 035010A1.

[0094] In some embodiments, substitutions are introduced into the Fc region of wild-type feline IgG to enhance binding to protein A, thereby facilitating purification by protein A chromatography. Such substitutions are known to those skilled in the art and can be at one or more positions (e.g., 1, 2, 3, 4, 5, 6, or 7) of feline IgG. An exemplary example is WO2019 / 035010A1.

[0095] In some embodiments, additional amino acid substitutions are made to alter the binding affinity to FcRn compared to the parent polypeptide or wild-type polypeptide (e.g., to increase or decrease the binding affinity to FcRn).

[0096] In some embodiments, the polypeptide includes the hinge region of the feline antibody. In some embodiments, modifications can be made to the hinge region of the feline antibody to increase its half-life.

[0097] Polypeptide containing feline IgG Fc variant This disclosure encompasses any polypeptides that can benefit from increasing their half-life in cats. To increase the half-life, these polypeptides are designed to include Fc region variants (e.g., CH2 region, CH3 region, CH2+CH3 region) as disclosed above.

[0098] Exemplary polypeptides include, but are not limited to, whole antibodies, scFv, nanobodies, ligand-binding moieties of receptors, cytokines, growth factors, enzymes, and peptides. For example, the CH3 domain variant disclosed above may be bound to scFv nanobodies, ligand-binding moieties of receptors (e.g., the ligand-binding moieties of feline IL-13Rα1 or IL-13Rα2), cytokines, growth factors, enzymes, or peptides. As used herein, the terms “nanobody,” “VHH,” “VHH antibody fragment,” and “single-domain antibody” are used interchangeably herein to refer to the variable domain of a single heavy chain of antibodies of the type found in camelids, which are typically naturally occurring and lack a light chain. Preferred nanobodies are well known to those skilled in the art, and examples include nanobodies from camels, dromedaries, llamas, and alpacas. Alternatively, the Fc region variant disclosed above may be bound to these polypeptides. In another embodiment, a feline or feline antibody is modified to include the Fc region variant disclosed herein.

[0099] In some embodiments, the polypeptides of this disclosure include an antibody hinge region. The hinge region may be positioned between the ligand-binding domain of the antigen or polypeptide and an Fc region variant. In some cases, the hinge region is bound to the C-terminus of a cytokine, growth factor, enzyme, or peptide, and the hinge region is bound to the N-terminus of an Fc region variant. Exemplary hinge region sequences are provided below. IgG1a:KTDHPPGPKPCDCPKCP(SEQ ID NO: 10); IgG1b:KTDHPPGPKPCDCPKCP(SEQ ID NO: 11); IgG2:KTASTIESKTGEGPKCP(SEQ ID NO: 12);

[0100] The hinge region in the recombinant protein of this disclosure, when used, may contain 0 to 6 amino acid substitutions (i.e., 0, 1, 2, 3, 4, 5, or 6) compared to the amino acid sequence described in any one of SEQ ID NOs. 10–12. In some cases, the hinge region used in the recombinant protein of this disclosure is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in any one of SEQ ID NOs. 10–12.

[0101] One or more polypeptides of this disclosure may include a binding domain. The binding domain can specifically bind to a protein, subunit, domain, motif, and / or epitope of a select target as described herein. In some embodiments, one or more polypeptides (e.g., fusion polypeptides) may include a protein, where the protein is a therapeutic protein as described herein. In some embodiments, the target (e.g., the target of the binding domain) or the therapeutic protein (e.g., fusion polypeptide) is selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, adresin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1 antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, IgE, angiotensin type 1 (AT1) receptor, Angiotensin type 2 (AT2) receptor, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte-stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CC1, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CC L7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, C D10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33(p6 7 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 , CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-related Antigen, DAN, DCC, DcR3, DC-SIGN, Degradation Promoter, des(1-3)-IGF-I (brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, Endothelin Receptor, Enkephalinase, eNOS, Eot, Eotaxin 1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-Selecti N, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast-Activating Protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-Stimulating Hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, G CSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-Alpha 1, GFR-Alpha 2, GFR-Alpha 3, GITR, GLP1, GLP2, Glucagon, Glut4, Glycoprotein IIb / IIIa (GP IIb / IIIa), GM-CSF, gp130, gp72, GRO, GnRH, Growth hormone-releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, cardiac myosin, cytomegalovirus (CMV), growth hormone (GH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL- 9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL-1R, IL-2 R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R IL-33R), interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha A6, Integrin Beta 1, Integrin Beta 2, Interferon Gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP(TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis Y antigen, Lewis Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian duct inhibitor, Mug, MuSK, NAIP, NAP, NAV1.7, NCAD, N-C Doherin, NCA90, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neuroturin, Nerve Growth Factor (NGF), NGFR, NGF-Beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PD1, PDL1, PDGF, PDG F, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, Rheumatoid Factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, Serum Albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (Tumor-Associated Glycoprotein-72), TARC, TCA-3, T Cell Receptor (e.g., T Cell Receptor Alpha / Beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, Testicular PLAP-like Alkaline Phosphatase, TfR, TGF, TGF-Alpha, TGF-Beta, TGF-Beta Pan TGF-link R1(ALK-5), TGF-link R11, TGF-link RIIb, TGF-link RIII, TG F-link 1, TGF-link 2, TGF-link 3, TGF- Regulation of factor 4, TGF-factor 5, activator Ck-1 Recombinant Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-protein TNF-Link 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1Apo-2、DR4)、TNFRSF10B(TRAIL R2DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF). R1CD120a, p55–60, TNFRSF1B(TNF RII CD120b, p75–80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137, ILA, TNFRSF21 (DR6), TNFRSF22 (DCTRAIL R2 TNFRH2), TNFRST23 (DCTRAIL R1TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL).Apo-2 ligand (TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α Conectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfering receptor, TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-associated carbohydrate, TWEAK, TXB2, Ung, UPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VI M, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth factors.

[0102] In some embodiments, the binding domain specifically binds to one or more therapeutic targets or antigens in cats, for example, but not limited to ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, Angiotensin type 1 (AT1) receptor, Angiotensin type 2 (AT2) receptor, Atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, Follicle-stimulating hormone, GDF-8 (Myostatin), GLP1, GLP2, GnRH, Growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL -15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL -1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1Examples include bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta R1 (ALK-5), TGF-beta R11, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, TNF, TNF-alpha, TNF-alpha-beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR, and VEGFR-3 (flt-4).

[0103] In some embodiments, one or more polypeptides may include proteins, where the protein is a therapeutic protein, such as EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, or a thrombopoietin-binding peptide. In some embodiments, the therapeutic protein may be ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, or activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, Angiotensin type 1 (AT1) receptor, Angiotensin type 2 (AT2) receptor, Atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, Follicle-stimulating hormone, GDF-8 (Myostatin), GLP1, GLP2, GnRH, Growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL -15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL -1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1These include bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta R1 (ALK-5), TGF-beta R11, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, TNF, TNF-alpha, TNF-alpha-beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR, or VEGFR-3 (flt-4).

[0104] Pharmaceutical composition To prepare pharmaceutical or sterile compositions of one or more polypeptides described herein, one or more polypeptides may be mixed with pharmaceutically acceptable carriers or excipients. (See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984)).

[0105] Therapeutic and diagnostic formulations can be prepared, for example, in the form of lyophilized powders, slurries, aqueous solutions, or suspensions by mixing with acceptable carriers, excipients, or stabilizers (e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY (see reference). In one embodiment, one or more polypeptides of the present invention are diluted to an appropriate concentration with a sodium acetate solution at pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0106] The toxicity and therapeutic efficacy of polypeptide compositions administered alone or in combination with other drugs are determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (A lethal dose for 50% of the population) and ED 50This can be determined by a procedure for determining the therapeutically effective dose (LD50%) in the population. The dose-to-toxicity ratio is the therapeutic index (LD50). 50 / ED 50 In certain embodiments, one or more polypeptides exhibiting a high therapeutic index are desirable. Data obtained from these cell culture assays and animal studies can be used to derive a range of dosages for use in cats. Doses of such compounds are preferably ED with little or no toxicity. 50 This falls within the circulating concentration range. The dosage may vary within this range depending on the dosage form and route of administration used.

[0107] The mode of administration can vary. Preferred routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, inhalation, topical, cutaneous, transdermal, or intraarterial. In some embodiments, one or more polypeptides may be administered by an invasive route such as injection. In further embodiments, one or more polypeptides may be administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery.

[0108] The pharmaceutical compositions disclosed herein may be administered by infusion. Examples of well-known forms of implants and modules for administering pharmaceutical compositions include U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,447,233 disclosing a drug infusion pump for delivering a drug at a precise infusion rate; U.S. Patent No. 4,447,224 disclosing an implantable variable flow rate infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196 disclosing a permeable drug delivery system having a multi-chamber compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0109] Alternatively, one or more polypeptides may be administered topically rather than systemically, for example, by direct injection of antibodies, often in depot or sustained-release formulations, into joints or pathogen-induced lesions of arthritis characterized by immunopathology. Furthermore, one or more polypeptides may be administered via a targeted drug delivery system, for example, by liposomes coated with tissue-specific antibodies, targeting joints or pathogen-induced lesions of arthritis characterized by immunopathology. The liposomes are targeted to the affected tissue and selectively taken up by the affected tissue.

[0110] The administration regimen depends on several factors, but is not limited to, the age, weight, and physical condition of the cat being treated, the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of one or more therapeutic polypeptides, and the accessibility of target cells in the biological matrix. In some implementations, the administration regimen delivers one or more therapeutic polypeptides sufficient to bring about improvement in the targeted disease state while simultaneously minimizing undesirable side effects. Therefore, the amount of biological agent delivered depends in part on one or more specific therapeutic polypeptides and the severity of the condition being treated. Guidance on selecting the appropriate dose of therapeutic antibodies is available (see, for example, Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)).

[0111] Determining the appropriate dose of one or more polypeptides is done by those skilled in the art, for example, using parameters or factors known or estimated to influence the treatment in the art. Generally, administration is started at a dose somewhat lower than the optimal dose and then increased in small increments until the desired or optimal effect is achieved against any negative side effects. Important diagnostic indicators include, for example, symptoms of inflammation or the level of inflammatory cytokines produced.

[0112] Nucleic acids, vectors, host cells, and methods for producing them. This disclosure also includes one or more nucleic acids encoding one or more polypeptides described herein, one or more vectors comprising one or more nucleic acids, and host cells comprising one or more nucleic acids or one or more vectors.

[0113] One or more polypeptides described herein may be produced in bacterial or eukaryotic cells. Some polypeptides, e.g., Fab', can be produced in bacterial cells, e.g., E. coli cells. Polypeptides can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS, 293T, Hela). In addition, polypeptides (e.g., scFv) can be expressed in yeast cells such as Pichia (e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces. To produce the antibody of interest, one or more polynucleotides encoding one or more polypeptides are constructed, introduced into one or more expression vectors, and then expressed in suitable host cells. To improve expression, the nucleotide sequence of the gene can be recoded without altering (or making minimal changes to, e.g., removal of C-terminal residues of the heavy or light chain) the amino acid sequence. Regions that may be subject to recoding include those related to translation initiation, codon usage frequency, and unintended mRNA splicing candidates. The polynucleotides encoding the Fc region variants described herein will be readily apparent to those skilled in the art.

[0114] Using standard molecular biology techniques, recombinant expression vectors (or multiple vectors) can be prepared, host cells can be transfected, transformants can be selected, host cells can be cultured, and polypeptides (e.g., antibodies) can be recovered.

[0115] When one or more polypeptides are expressed in bacterial cells (e.g., E. coli), the expression vector must have features that allow for the amplification of the vector in the bacterial cells. Furthermore, when E. coli such as JM109, DH5α, HB101, or XL1-Blue is used as the host, the vector must have a promoter capable of enabling sufficient expression in E. coli, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter. Examples of such vectors include, for example, the M13 vector, the pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. In the case of production into the periplasm of E. coli, the pelB signal sequence (Lei et al.) al., J. Bacteriol., 169:4379 (1987)) can be used as a signal sequence for antibody secretion. In the case of bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0116] When one or more polypeptides are expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector includes promoters necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)) (e.g., early Simian virus 40 promoter), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter (e.g., human cytomegalovirus pre-early promoter). In addition to the nucleic acid sequence encoding the Fc region variant, the recombinant expression vector may contain additional sequences such as sequences that regulate vector replication in the host cell (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced (e.g., U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, typically, selection marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Examples of vectors containing selection markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0117] In some embodiments, one or more polypeptides are produced in mammalian cells. Exemplary mammalian host cells for expressing one or more polypeptides include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, and used with a DHFR selection marker, for example, as described in Kaufman and Sharp (1982) Mol. Biol. 159:601621), human fetal kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte cell lines, for example, NS0 myeloma cells and SP2 cells, and cells derived from transgenic animals, for example, transgenic mammals. For example, cells are mammary epithelial cells.

[0118] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy and light chains is introduced into dhfr-CHO cells via calcium phosphate transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are operably ligated to enhancer / promoter regulatory elements (e.g., those derived from SV40, CMV, adenovirus, etc., e.g., a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of gene transcription. The recombinant expression vector also contains the DHFR gene, which allows for the selection of vector-transfected CHO cells using methotrexate selection / amplification. The selected transformed host cells are cultured to express the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0119] Treatment method One or more polypeptides disclosed herein can be used to treat or prevent any disease or disorder in cats in need. The present invention is particularly useful for treating chronic conditions requiring repeated administration. The increased half-life of the protein therapeutic agent may allow for a reduction in the frequency of administration and / or the dose level.

[0120] In some embodiments, the diseases, disorders, conditions, or symptoms treated or prevented are allergic diseases, chronic pain, acute pain, inflammatory diseases, autoimmune diseases, endocrine disorders, gastrointestinal disorders, skeletal / musculoskeletal disorders, cardiovascular diseases, neurological disorders, kidney diseases, metabolic disorders, immune disorders, genetic / hereditary disorders, reproductive function disorders, infections, or cancer. In certain embodiments, the diseases or disorders treated or prevented are atopic dermatitis, allergic dermatitis, food allergies, osteoarthritis pain, perioperative pain, toothache, cancer pain, arthritis, anemia, obesity, or diabetes.

[0121] Antibodies can be used not only to treat or prevent diseases, but also to regulate normal biological functions, for example, to manage reproductive capacity or behavior.

[0122] diagnosis One or more polypeptides disclosed herein can also be used for various diagnostic applications, for example, to determine whether a cat has any particular disease or disorder. In some embodiments, the one or more polypeptides can include a binding domain. The binding domain can specifically bind to a protein, subunit, domain, motif, and / or epitope described herein (e.g., a marker of cancer cells). In some embodiments, the one or more polypeptides further include a labeling group. Generally, labeling groups are classified into various types depending on the assay in which they are detected: a) isotope labels that can be radioactive isotopes or heavy element isotopes; b) magnetic labels (e.g., magnetic particles); c) oxygen reduction active moieties; d) optical dyes; enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is attached to the antibody via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used in practicing the present invention.

[0123] In some embodiments, the labeling group is a probe, a dye (e.g., a fluorescent dye), or a radioactive isotope (e.g., 3 H, 14 C, 22 Na, 36 Cl, 35 S, 33 P, or 125 I).

[0124] Certain labels also include optical dyes, including, but not limited to, chromophores, phosphors, and fluorophores, the latter of which are often specific. Fluorophores can be either "small molecule" phosphors or proteinaceous phosphors.

[0125] A fluorescent label can be any molecule that can be detected through its intrinsic fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, rhodamine, and Texas This includes, but is not limited to, Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, and Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable optical dyes, including fluorophores, are described in the Molecular Probes Handbook by Richard P. Haugland, which is incorporated herein by reference in its entirety.

[0126] Suitable protein-based fluorescent labels also include green fluorescent protein (including GFP from Renilla, Ptilosarcus, or Aequorea species) (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), improved yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), and luciferase (Ichiki et al. al., 1993, J.Immunol. 150:5408-5417), β-galactosidase (Nolan et al.) This includes, but is not limited to, U.S. Patent Nos. al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607, and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / 49019, U.S. Patent Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, and 5,925,558). All references cited above in this paragraph are expressly incorporated herein by reference in their entirety.

[0127] Assay Fc γ RI and FcγRIII binding: Binding to FcγRI and FcγRIII is an indicator of the ability of an antibody to mediate ADCC. To evaluate this property of an antibody, assays measuring the binding of the antibody to FcγRI and FcγRIII can be performed using methods known in the art.

[0128] C1q binding: Binding to C1q, the first component of complement, is an indicator of an antibody's ability to mediate complement-dependent cell-mediated cytotoxicity (CDC). To evaluate this property of an antibody, assays measuring the binding of an antibody to C1q can be performed using methods known in the art.

[0129] Half-life: Methods for measuring the half-life of antibodies are well known in the art. See, for example, Booth et al., MAbs, 10(7):1098-1110 (2018). As an example, the half-life of an antibody (e.g., a feline antibody) can be measured by injecting the antibody into an animal model (e.g., a feline model) and measuring the antibody level in the serum over a certain period of time. Exemplary animal models include non-human primate models and transgenic mouse models. Transgenic mouse models (e.g., Tg32 or Tg276 transgenic mice) lack the mouse FcRn alpha chain and can express the human FcRn alpha transgene (e.g., under the control of a constitutive promoter). The human FcRn alpha chain can pair with the mouse β2-microglobulin protein in vivo to form a functional chimeric FcRn heterodimer. [Examples]

[0130] Example 1: Generation of an NNK saturated mutation library at a selected location and analysis of individual variants. Wild-type (wt) sequences of the CH2 and CH3 domains of feline IgG1a (SEQ ID NO: 1) were synthesized and used as templates for NNK mutagenesis. NNK saturated mutagenesis is an effective strategy for generating all 20 possible amino acids at the desired position (Hogrefe et al., Biotechniques. 33:1158-1165

[2002] ). Individual NNK libraries were generated at positions 252, 428, and 434 (EU numbering). NNK (N=A / C / G / T, K=G / T) primers at specific positions were used with the QuikChange Site-Directed Mutagenesis Kit (Agilent). PCR products were subcloned into GenScript FASEBA plasmids, transformed into E. coli, and sequenced for the presence of variants. Downstream of the CH3 domain is a SASA (single-domain antibody against serum albumin) tag with pM affinity for albumin (Zhang, J.; Wu, S.; Liu, J. Methods and systems for increasing protein stability. Patent application number US2013 / 0129727A1). The SASA antibody enables the capture of Fc to the sensor chip surface as described below. The PelB (pectic acid lyase B) signal peptide is located at the N-terminus and promotes the secretion of Fc into the culture medium. CH2-CH3 protein expression was controlled by the Lac promoter. The supernatant from the conditioned medium was analyzed using surface plasmon resonance (SPR) for variant binding to feline FcRn (GenBank KF773786 [IgG receptor FcRn large subunit p51] and European Nucleotide Archive AY829266.1 [feline beta-2-microglobulin]) at pH 6.0.

[0131] The supernatants of 90 individual transformants obtained from each library were assayed for binding to feline FcRn at pH 6.0 using the Biacore method as described below.

[0132] In SPR analysis using Biacore 8K, bovine serum albumin (BSA) was immobilized on a CM5 sensor tip. The sensor tip surfaces of flow cells 1 and 2 were activated over 420 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Subsequently, BSA diluted with 10 mM sodium acetate (pH 4.5) was injected into flow cell 2 to achieve conjugation, and flow cell 1 was used as a blank. After the amine coupling reaction, the active coupling site remaining on the tip surface was blocked by injecting 1 mM ethanolamine hydrochloride for 420 seconds. The running buffer for the coupling experiment was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 5.5), and the experiment was performed at 25°C. The supernatant of the variant was injected onto the chip surface and captured by the immobilized BSA via a SASA tag over 60 seconds. 200 nM cat FcRn was injected for 120 seconds, and dissociation was performed with running buffer for 120 seconds. The flow rate during the BSA immobilization phase was 10 μl / min, and the flow rate during the association and dissociation phases was 30 μl / min. All data were processed using Biacore 8K evaluation software version 1.1. See Figures 1 and 2 for Biacore sensorgrams.

[0133] The tested variant showed increased binding affinity to feline FcRn at pH 6.0 compared to wild-type feline IgG1a Fc (SEQ ID NO: 1).

[0134] Introducing NNK mutations at amino acid positions 252, 428, and 434 resulted in mutants with increased FcRn binding at pH 6.0. Sequencing of all 90 clones generated at these three positions showed that the following six clones—S252H, S428E, S428C, S428F, S428W, and S434Y—were not generated. All other amino acid substitutions at these positions did not yield any experimental benefit. The results are summarized in Table 3 below. [Table 3]

[0135] Example 2: Scanning mutation introduction into feline IgG1a Fc Using a phage display library approach, we identified feline IgG1a Fc variants that increased affinity for feline FcRn at pH 6.0. Feline IgG1a Fc (Kanai et al., 2000. Vet. Immunol. Immunopathol. 73:53) was synthesized by Twist Bioscience to have variants at 55 different positions (Figure 4). Eight possible amino acid substitutions were made at each mutated position. These amino acids were arginine and lysine (positively charged side chains), aspartic acid and glutamic acid (negatively charged side chains), threonine and glutamine (polar uncharged side chains), and leucine and valine (hydrophobic side chains). An Fc DNA library was designed to have an average of two variants per Fc molecule. The complexity of this library is given by the formula: nCr = n! / r! * Using the formula (nr)! (where n represents the number of sites and r represents the number of variants per molecule), there were 95,040 possible combinations. Fc variants containing the desired site-specific mutations were printed as mutagenic oligonucleotides on Twist's silicon-based platform.

[0136] Next, oligonucleotides were ligated using assembly PCR to generate a full-length Fc gene fragment pool. The ligated Fc gene fragment pool was then cloned into the Sfi cleavage site of Antibody Design Labs' pADL-22c phagemide vector. The cloned DNA library was transformed into TG1 E. coli electrocompetent cells, resulting in 8 × 10⁶ cells. 10Experimental diversity of variants was created. Subsequently, phagemid-transformed E. coli cells were co-transfected with the M13K07 helper phage to create a phage pool for use in protein-based panning. The library was resuspended in 20 mM MES buffer (pH 6.0), 0.05% Tween 20, and 3% milk.

[0137] Library quality was determined by selecting 96 random phage clones, which were then sequenced using the Sanger method. The number of variants per phage is shown in Table 4 below. [Table 4]

[0138] In the initial phage selection, a protein A capture step was used to remove any Fc variants that had lost protein A binding. For this selection, the phage library was captured on protein A beads and washed with PBS (pH 7.4). The phages were eluted with 0.1 M glycine (pH 2.7) and immediately neutralized with 1 M Tris-HCl (pH 7.5). The neutralized phages were precipitated with polyethylene glycol / NaCl and centrifuged. The pelletized phages were resuspended in 20 mM MES (pH 6.0), 0.05% Tween 20, and 3% milk.

[0139] The following phage selection was based on the protocol described in Borrok et al., 2015, J Biol. Chem., 290:4282. Briefly, Nunc 96 multiwell plates were coated with Nutroavidin and then blocked with 5% bovine serum albumin PBS (pH 7.4). Biotinylated feline FcRn (FCN-F82W3, Acrobiosystems) was immobilized in wells in PBS (pH 6.0) at a concentration of 0.31 μg / ml. The phage library in PBS (pH 6.0) was incubated with the immobilized feline FcRn and then washed with PBS (pH 6.0), 0.05% Tween 20, and 0.3 M NaCl. The phages were eluted by incubation with PBS (pH 7.4) at 37°C for 30 minutes. The eluted phages were depleted with 0.31 μg / ml feline FcRn at pH 7.4. Unbound phages were grown in TG1 cells. The exact conditions and results for each round are shown in Table 5 below. [Table 5]

[0140] A total of 768 phage clones were isolated as a result of the third selection round, and another 768 phage clones were isolated in the fourth selection round. The clones were sequenced using next-generation sequencing with Illumina MiSeq.

[0141] Sequencing revealed numerous clones containing a substituted tyrosine at position 252 and a substituted tyrosine or phenylalanine at position 434 (see table below), and some substitutions were shown to contain residues other than the intended eight amino acid substitutions. Unique variants were reformatted to IgG using the variable domain previously described by Gearing et al. (2016, J Vet Intern Med, 30:1129). Miniprep plasmid DNA was transfected into Expi293 cells using ExpiFectamine293 transfection reagent. IgG variants were purified from conditioned medium by protein A chromatography and adjusted to 43 mM sodium citrate, 130 mM sodium bicarbonate, pH 6.0.

[0142] To determine the affinity of the IgG variant for feline FcRn, binding kinetics were determined using a Carterra instrument. The antibody (approximately 5 μg / ml) was amine-coupled to an HC30M sensor chip by EDC / NHS activation followed by quenching with ethanolamine HCl.

[0143] Feline FcRn (FCN-F82W3, Acrobiosystems) at various concentrations (333nM, 111nM, 37nM, 12.3nM, 4.1nM, 1.37nM, 0.45nM) were flowed onto a sensor tip in HBSTE (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween-20), 0.5% bovine serum albumin, pH 6.0, and their kinetics at pH 6.0 were determined. The same strategy was used to determine the binding kinetics to feline FcRn at pH 7.4, except that the pH of the HBSTE buffer was adjusted to 7.4.

[0144] The feline FcRn-binding kinetics of IgG variants are shown in Table 6 below. [Table 6]

[0145] Example 3: Scanning mutation introduction into feline IgG1a Fc Further sets of anti-nerve growth factor (NGF) feline IgG1a antibody variants were synthesized by Twist Bioscience using the variable domain previously described by Gearing et al. (2016, J Vet Intern Med, 30:1129). Modifications to the Fc region of these antibodies are described in Tables 8 and 9 below, compared to the reference sequence of the wild-type feline IgG1a Fc domain described by Kanai et al. (2000, Vet. Immunol. Immunopathol. 73:53). The feline IgG1a construct was subcloned into a mammalian expression vector and transfected into Expi293 cells using the ExpiFectamine293 transfection reagent. IgG1a Fc variants were purified from the conditioned medium by protein A chromatography and prepared in 43 mM sodium citrate, 130 mM sodium bicarbonate, pH 6.0. Next, the binding affinity of IgG Fc variants to feline FcRn was determined using a Carterra instrument. The antibody variant (approximately 5 μg / ml) was amine-coupled to an HC30M sensor chip by EDC / NHS activation followed by quenching with ethanolamine HCl. Feline FcRn (FCN-F82W3, Acrobiosystems) at concentrations of 333 nM, 111 nM, 37 nM, 12.3 nM, 4.1 nM, 1.37 nM, or 0.45 nM was flowed onto the sensor chip in HBSTE (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20), 0.5% bovine serum albumin, at pH 6.0 to determine the kinetics at pH 6.0. The same strategy was used to determine the binding affinity of Fc variants to feline FcRn at pH 7.4. However, the pH of the HBSTE buffer was adjusted to 7.4.

[0146] The feline FcRn-binding kinetics of IgG variants are shown in Table 7 below.

[0147] [Table 7]

[0148] Table 8 below provides a list of amino acid substitutions identified to increase the binding of feline IgG1a Fc variants to feline FcRN: [Table 8]

[0149] Example 4: Pharmacokinetic studies of feline IgG1a Fc variant with increased FcRn binding and wild-type feline IgG1a. Pharmacokinetic (PK) studies were conducted using 12 male and female cats. Feline IgG1a Fc variants were prepared, including antibodies that retain the Fc domain of wild-type feline IgG1a, using the anti-NGF variable domain previously described by Gearing et al. (2016, J Vet Intern Med, 30:1129).

[0150] Animals were randomly divided into six groups, with one male and one female in each group. Each animal received a single intravenous dose of 2 mg / kg of antibody. Approximately 0.5 ml of whole blood was collected at the following time points: 0 (before administration), 4 hours after injection, and on days 1, 2, 4, 6, 10, 14, 18, 22, 30, 34, 38, and 42. Serum was separated from the whole blood and assayed for the presence of antibodies by ELISA specific to anti-NGF antibodies. Serum concentrations of the six anti-NGF monoclonal antibody (mAb) variants were described using a two-compartment pharmacokinetic (PK) model with linear clearance, employing a nonlinear mixed-effects (NLME) model (Figure 46). Population parameters were estimated using the stochastic expectation maximization (SAEM) algorithm implemented in Monolix Suite 2019R1 (Monolix version 2019R1. Antony, France: Lixoft SAS, 2019). Each parameter was modeled as a random variable following a log-normal distribution. The PK parameter depends on body weight (BW), and the coefficient (β) is specific to mAb. BW,Cl =0.75, β BW,V1 =βBW,V2 =1, β BW,Q The individual parameters φ were used (=2 / 3). i The formula for this is as follows (Dong et al. 2011. Clin Pharmacokinet, 50:131).

number

[0151] Antibody variants were identified using categorical covariates related to clearance, inter-compartmental transport coefficient, and peripheral volume, according to the following formula.

number

[0152] Figure 47 shows the serum concentrations of variants WT, S252W, S428Y, S428Y+L309V, S428Y+Q311V, and S428Y+S254R, observed individually in two animals per variant.

[0153] The estimated PK parameters are shown in Table 9 below. Figure 48 shows the predicted serum concentration profiles of monoclonal antibodies holding wild-type (WT) IgG1a Fc or IgG1a Fc variants S252W, S428Y, S428Y+L309V, S428Y+Q311V, and S428Y+S254R in a typical 2kg cat receiving a single IV dose of 2 mg / kg. These pharmacokinetic studies not only confirm the benefits of substitutions at two or more amino acid positions, but more importantly, show that amino acid modifications to the feline IgG Fc domain, which confer enhanced FcRn binding in vitro, were also sufficient to extend the in vivo half-lives of these IgG Fc variants. [Table 9]

[0154] Example 5: Binding kinetics of feline IgG1a variant to feline FcRn using a C1 biosensor The binding kinetics of several feline IgG1a variants (S252W, L309V, Q311V, S428Y, S428Y+Q311V, S428Y+254R, S428Y+L309V, S428Y+Q311V+T286E, S428Y+Q311V+E380T, S428Y+L309V+T286E, and S428Y+L309V+E380T) to feline FcRn (GenBank KF773786 [feline FcRn large subunit p51] and European Nucleotide Archive AY829266.1 [feline beta-2-microglucurin]) were evaluated at pH 5.9. EU numbering was used for location identification (Figure 4). In this study, feline Fc variants containing a single amino acid substitution or a combination of amino acid substitutions were synthesized into feline IgG1a (Kanai et al., 2000, Vet.Immunol.Immunopathol.73:53) using a variable domain described by Gearing DP et al. (2016, J Vet Intern Med, 30:1129). The synthesized feline IgGa variant DNA was subcloned into a mammalian expression vector and transiently transfected into CHO cells. The conditioned medium was purified using protein A chromatography.

[0155] For feline FcRn binding experiments, all assays were performed at 25°C using a Biacore 8K+ system. In this experimental setup, antibodies were immobilized on Series S C1 sensor tips using standard amine coupling reagents. The surface was activated by injecting a mixture of 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mmol / L N-hydroxysuccinimide (NHS) for 420 seconds. Then, antibodies were injected at a concentration of 0.5–2 μg / ml in 10 mM sodium acetate (pH 5.0) for 120 seconds. Finally, 1 M ethanolamine was injected for 420 seconds. The running buffer was 1X PBS-P+ (Cytiva, Cat#28995084) adjusted to pH 5.9.

[0156] To evaluate the binding affinity of feline IgG1a variants to feline FcRn at pH 5.9, feline FcRn in the concentration range of 1.56 - 2000 nM was selected and injected in single cycle mode. The concentrations of feline FcRn tested for each variant are shown in Table 10 below. [Table 10]

[0157] Four concentrations of the antibody were injected at 5 μl / min for 90 seconds, followed by dissociation for 180 seconds. Each concentration series was injected three times in this format, and at least three buffer-only cycles were performed for subtraction of appropriate references. 1X PBS-P+(pH 7.4) was injected twice for 30 seconds each, followed by a 60-second wait command to regenerate the surface. Three startup cycles were included to stabilize the surface before analysis.

[0158] Data was evaluated using Insight Evaluation Software by fitting to a 1:1 kinetic interaction model or to steady-state affinity. Quality metrics including U values and T values were used to select acceptable parameters. U values less than 15 were considered acceptable as reaction rate constants, and T values greater than 100 were considered acceptable as reaction rate constants. If these values were outside that range, steady-state affinity parameters were considered acceptable.

[0159] The kinetic data of feline IgG1a variants are shown in Table 11 below, and sensorgrams are shown in Figures 49A - 49L. [Table 11]

[0160] Example 6: Pharmacokinetic studies of feline IgG1a Fc variants containing two or three Fc substitutions and wild-type feline IgG1a. Pharmacokinetic (PK) studies were conducted using 14 male and female cats. Feline IgG1a Fc variants were prepared, including antibodies retaining the Fc domain of wild-type feline IgG1a, using the anti-NGF variable domain previously described by Gearing et al. (2016, J Vet Intern Med, 30:1129). The feline IgG1a variants tested in this study included S428Y+L309V, S428Y+Q311V, S428Y+Q311V+T286E, S428Y+L309V+E380T, S428Y+Q311V+E380T, S428Y+L309V+T286E, and wild-type.

[0161] Animals were randomly divided into seven groups, with males and females assigned to each group. Each animal received a single intravenous dose of 2 mg / kg of antibody. Approximately 0.5 ml of whole blood was collected at the following time points: 0 (before administration), 4 hours after injection, and on days 1, 2, 4, 6, 10, 14, 18, 22, 30, 34, 38, and 42. Serum was separated from the whole blood and assayed for the presence of the antibody by ELISA specific to feline anti-NGF antibodies. Serum concentrations of the seven anti-NGF monoclonal antibody (mAb) variants were described using a two-compartment pharmacokinetic (PK) model with linear clearance, employing a nonlinear mixed-effects (NLME) model (population parameters were estimated using the stochastic expectation maximization (SAEM) algorithm implemented in Monolix Suite 2019R1 (Monolix version 2019R1. Antony, France: Lixoft SAS, 2019)). The serum concentrations of S428Y+Q311V, S428Y+L309V, and wild-type PK in the PK test described in Example 4 were modeled as described above and included in their calculations. Each parameter was modeled as a random variable following a log-normal distribution. The PK parameters depend on body weight (BW), and the mAb-specific coefficient (β) was used. BW,Cl =0.75, β BW,V1 =β BW,V2 =1, β BW,Q The individual parameters φ were used (=2 / 3). iThe formula for this is as follows (Dong et al. 2011. Clin Pharmacokinet, 50:131).

number

[0162] φ pop η is a typical parameter of the population, and η is a random variable containing mean 0 and standard deviation ω, BW i This is the weight of animal i, BW ref The reference weight was 2 kg.

[0163] Antibody variants were identified using categorical covariates related to clearance, inter-compartmental transport coefficient, and peripheral volume, according to the following formula.

number

[0164] In the formula, if the covariates of each variant belong to that category, then Ω i = 1, otherwise Ω i = 0. A wild-type (WT) mAb was used as a reference.

[0165] The estimated PK parameters are shown in Table 12 below. [Table 12]

[0166] Figure 50 shows the serum concentrations of the wild-type and Fc variants S428Y+Q311V, S428Y+Q311V+T286E, S428Y+Q311V+E380T, S428Y+L309V, S428Y+L309V+T286E, and S428Y+L309V+E380T, observed individually in two animals per variant.

[0167] The predicted serum concentration profiles of monoclonal antibodies bearing wild-type (WT) IgG1a Fc or the IgG1a Fc variants S428Y+Q311V, S428Y+Q311V+T286E, S428Y+Q311V+E380T, S428Y+L309V, S428Y+L309V+T286E, and S428Y+L309V+E380T in a typical 2 kg cat receiving a single IV dose of 2 mg / kg are shown in Figure 51. These pharmacokinetic studies not only confirmed the benefits of substitutions at two or more amino acid positions, but more importantly, they showed that amino acid modifications to the feline IgG Fc domain that confer improved FcRn binding in vitro were also sufficient to extend the in vivo half-life of these IgG Fc variants. (Note) (Note 1) A polypeptide comprising a feline IgG Fc region variant or its feline FcRn binding region, wherein the polypeptide has an amino acid substitution at at least one position selected from the group consisting of: (i) The position corresponding to amino acid position 252 of wild-type cat IgG, wherein the amino acid substitution is S252W; (ii) The position corresponding to amino acid position 254 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S254R and S254K; (iii) The position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V or L309Y; (iv) The position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of Q311R, Q311V, Q311L, and Q311K; (v) The position corresponding to amino acid position 428 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428M, S428Y, S428H, and S428R; and (vi) One or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type cat IgG. Included in, Here, the amino acid positions are based on EU numbering, and the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG. (Note 2) The polypeptide according to Appendix 1, wherein the polypeptide contains the amino acid substitution at a position corresponding to amino acid position 252 of wild-type feline IgG. (Note 3) The polypeptide as described in Appendix 1, comprising the amino acid substitution S252W. (Note 4) The polypeptide according to any one of the appendices 1 to 3, wherein the polypeptide contains the amino acid substitution at a position corresponding to amino acid position 254 of wild-type feline IgG. (Note 5) The polypeptide according to Appendix 4, wherein the amino acid substitution at position 254 of the wild-type feline IgG is S254R. (Note 6) The polypeptide according to Appendix 4, wherein the amino acid substitution at position 254 of the wild-type feline IgG is S254K. (Note 7) The polypeptide according to any one of the appendices 1 to 6, wherein the polypeptide contains the amino acid substitution at a position corresponding to amino acid position 309 of wild-type feline IgG. (Note 8) The polypeptide according to Appendix 7, wherein the amino acid substitution at position 309 of the wild-type feline IgG is L309V. (Note 9) The polypeptide according to Appendix 7, wherein the amino acid substitution at position 309 of the wild-type cat IgG is L309Y. (Note 10) The polypeptide according to any one of the appendices 1 to 9, wherein the polypeptide contains the amino acid substitution at a position corresponding to amino acid position 311 of wild-type feline IgG. (Note 11) The polypeptide according to Appendix 10, wherein the amino acid substitution at position 311 of the wild-type feline IgG is Q311R. (Note 12) The polypeptide according to Appendix 10, wherein the amino acid substitution at position 311 of the wild-type feline IgG is Q311V. (Note 13) The polypeptide according to Appendix 10, wherein the amino acid substitution at position 311 of the wild-type feline IgG is Q311K. (Note 14) The polypeptide according to Appendix 10, wherein the amino acid substitution at position 311 of the wild-type feline IgG is Q311L. (Note 15) The polypeptide according to any one of the appendices 1 to 14, wherein the polypeptide contains the amino acid substitution at a position corresponding to amino acid position 428 of wild-type feline IgG. (Note 16) The polypeptide according to Appendix 15, wherein the amino acid substitution at position 428 of the wild-type feline IgG is S428M. (Note 17) The polypeptide according to Appendix 15, wherein the amino acid substitution at position 428 of the wild-type cat IgG is S428Y. (Note 18) The polypeptide according to Appendix 15, wherein the amino acid substitution at position 428 of the wild-type feline IgG is S428R. (Note 19) The polypeptide according to Appendix 15, wherein the amino acid substitution at position 428 of the wild-type feline IgG is S428H. (Note 20) The polypeptide according to any one of the appendices 1 to 19, wherein the polypeptide contains amino acid substitutions at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type feline IgG. (Note 21) The polypeptide described in Appendix 20, wherein the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293K, E293H, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L, as well as any of the aforementioned conservative amino acid substitutions. (Note 22) The polypeptide described in Appendix 21, wherein the amino acid substitution is selected from the group consisting of L262Q, L262E, T286E, T286D, T289K, S290V, S290Y, E293D, E293K, E293H, R301L, D312T, K326D, R334D, Q347L, Q355L, I377V, I377Y, E380D, E380V, E380T, I383L, N389c-R, R392E, S426L, S426H, and T437L. (Note 23) A polypeptide comprising a feline IgG Fc region variant or its feline FcRn binding region, wherein the polypeptide comprises two or more amino acid substitutions, and the two or more amino acid substitutions comprise the following group: (i) An amino acid substitution at the position corresponding to amino acid position 252 of wild-type cat IgG, selected from the group consisting of S252W, S252Y, S252F, and S252R; (ii) An amino acid substitution at the position corresponding to amino acid position 254 of wild-type feline IgG, selected from the group consisting of S254R and S254K; (iii) an amino acid substitution at the position corresponding to amino acid position 309 of wild-type feline IgG, selected from the group consisting of L309V, L309Y, and L309E; (iv) An amino acid substitution at the position corresponding to amino acid position 311 of wild-type feline IgG, selected from the group consisting of Q311R, Q311V, Q311L, and Q311K; (v) an amino acid substitution at the position corresponding to amino acid position 428 of wild-type feline IgG, selected from the group consisting of S428L, S428M, S428Y, S428H, and S428R; (vi) amino acid substitutions at one or more positions corresponding to amino acid positions selected from the group consisting of 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426 and 437 of wild-type cat IgG; and (vii) An amino acid substitution at the position corresponding to amino acid position 434 of wild-type feline IgG, selected from the group consisting of S434F, S434W, S434H, S434R, and S434Y. Selected from, Herein, the amino acid positions are based on EU numbering, the two or more amino acid substitutions are in different positions, and the polypeptide has increased binding affinity to feline FcRn compared to (a) the Fc domain of wild-type feline IgG and (b) a polypeptide containing only one of the two or more amino acid substitutions. (Note 24) The polypeptide according to Appendix 23, wherein the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 286 of wild-type feline IgG, and the amino acid substitutions are selected from the group consisting of T286E and T286D. (Note 25) The polypeptide according to Appendix 23 or Appendix 24, wherein the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 289 of wild-type feline IgG, and the amino acid substitutions are selected from the group consisting of T289K and T289H. (Note 26) The polypeptide according to any one of the appendices 23 to 25, wherein the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 301 of wild-type feline IgG, and the amino acid substitution is R301L. (Note 27) The polypeptide according to any one of the appendices 23 to 26, wherein the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 334 of wild-type feline IgG, and the amino acid substitution is R334D. (Note 28) The polypeptide according to any one of the appendices 23 to 27, wherein the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 426 of wild-type feline IgG, and the amino acid substitutions are selected from the group consisting of S426L and S426H. (Note 29) The polypeptide according to any one of the appendices 23 to 28, wherein the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 437 of wild-type feline IgG, and the amino acid substitution is T437L. (Note 30) The group consisting of the following two or more amino acid substitutions: (i) A combination of S252Y and Q311R and / or Q311L; (ii) A combination of S434Y and one or more of S254R, S254K, L262E, T286D, T286E, T289K, E293D, E293K, L309V, L309E, K326D, and Q347L; (iii) The combination of S434F and E380D; (iv) A combination of S428L and one or more of S252R, T286E, Q311V, Q311K, D312T, I377V, I383L, and N389cR; (v) S428L, E380D and S434R; (vi) S428L, E380T and S434R; (vii) The combination of S252R and L262Q; (viii) T260E, L309E and Q355L; (ix) S290V and R344D combination; (x) R301L, E380V and T437L; (xi) T286E and S428H combination; (xii) A combination of R334D and one or more of S428R, T437L, and R301L; (xiii) A combination of S426L and T289H and / or S428H; (xiv) A combination of S428Y and one or more of Q311V, S254R, L309V, T286E, and E380T; and A polypeptide as described in Appendix 23, selected from a combination of (xv)S428H and T289H. (Note 31) The polypeptide according to any one of the appendices 1 to 30, wherein the polypeptide contains an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3. (Note 32) The polypeptide according to any one of the appendices 1 to 30, wherein the wild-type feline IgG is feline IgG1a containing an Fc domain having the amino acid sequence of SEQ ID NO: 1. (Note 33) The polypeptide according to any one of the appendices 1 to 30, wherein the wild-type feline IgG is feline IgG1b containing an Fc domain having the amino acid sequence of Sequence ID No. 2. (Note 34) The polypeptide according to any one of the appendices 1 to 30, wherein the wild-type feline IgG is feline IgG2 containing an Fc domain having the amino acid sequence of Sequence ID No. 3. (Note 35) A polypeptide as described in any one of the appendices 1 to 34, further comprising a binding domain. (Note 36) The polypeptide as described in Appendix 35, wherein the binding domain comprises (i) six complementarity-determining regions (CDRs) of an immunoglobulin molecule; (ii) a ligand-binding domain of a feline receptor protein; (iii) a nanobody; or (iv) an extracellular domain of a feline receptor protein. (Note 37) The polypeptide according to Appendix 35 or Appendix 36, wherein the binding domain specifically binds to an antigen selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and the complement system complex. (Note 38) A polypeptide as described in any one of the appendices 1 to 37, further comprising a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonists, and thrombopoietin-binding peptides. (Note 39) The polypeptide according to any one of the appendices 1 to 38, wherein the polypeptide binds to feline FcRn at a higher level at an acidic pH than at a neutral pH. (Note 40) The polypeptide described in Appendix 39, wherein the polypeptide binds to feline FcRn at a higher level at pH 5.5 than at pH 7.4. (Note 41) The polypeptide described in Appendix 39, wherein the polypeptide binds to feline FcRn at a higher level at pH 6.0 than at pH 7.4. (Note 42) A pharmaceutical composition comprising (i) a polypeptide as described in any one of Appendix 1 to 41, and (ii) a pharmaceutically acceptable excipient. (Note 43) One or more nucleic acids encoding a polypeptide as described in any one of the appendices 1 to 41. (Note 44) One or more expression vectors comprising one or more nucleic acids as described in Appendix 43. (Note 45) A host cell containing one or more nucleic acids as described in Appendix 43 or one or more expression vectors as described in Appendix 44. (Note 46) A method for producing polypeptides, (a) To provide one or more nucleic acids as described in Appendix 43; (b) Expressing one or more nucleic acids in a host cell culture to produce the polypeptide; and (c)(b) The polypeptide produced in the above-mentioned method is recovered from the host cell culture. The method, including the method described above. (Note 47) The method according to Appendix 46, further comprising formulating the polypeptide as a pharmaceutical preparation. (Note 48) A method for treating a feline disease or disorder in a cat that is in need thereof, comprising administering to the cat an effective amount of a composition comprising the pharmaceutical composition described in Appendix 42. (Note 49) A method for preventing a feline disease or disorder in a cat that is in need thereof, comprising administering to the cat an effective amount of a composition containing the pharmaceutical composition described in Appendix 42. (Note 50) The method according to Appendix 48 or Appendix 49, wherein the disease or disorder of the cat is allergy, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disorder, gastrointestinal disorder, cardiovascular disease, kidney disease, reproductive dysfunction, infection, or cancer. (Note 51) The method according to Appendix 48 or Appendix 49, wherein the disease or disorder of the cat is atopic dermatitis, allergic dermatitis, osteoarthritis, arthritis, anemia, or obesity.

[0168] Other Embodiments The present invention has been described in detail, but the above description is illustrative of the scope of the invention and is not intended to limit it. The present invention is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A polypeptide comprising a feline IgG Fc region variant, wherein the polypeptide has an amino acid substitution at at least one position selected from the group consisting of: (i) The position corresponding to amino acid position 286 of wild-type cat IgG, wherein the amino acid substitution is T286E; (ii) The position corresponding to amino acid position 311 of wild-type cat IgG, wherein the amino acid substitution is Q311V; (iii) The position corresponding to amino acid position 428 of wild-type cat IgG, wherein the amino acid substitution is S428Y; the position includes, The amino acid positions are based on EU numbering, the polypeptide contains an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3, the polypeptide has increased binding affinity to feline FcRn compared to the Fc domain of wild-type feline IgG, and the polypeptide binds to feline FcRn at a higher level at pH 6.0 than at pH 7.

4. The aforementioned wild-type cat IgG is (a) Feline IgG1a containing an Fc domain having the amino acid sequence of SEQ ID NO: 1, (b) Feline IgG1b containing an Fc domain having the amino acid sequence of Sequence ID No. 2, or (c) Feline IgG2 containing an Fc domain having the amino acid sequence of Sequence ID No. 3, Polypeptide.

2. The polypeptide according to claim 1, wherein the polypeptide comprises T286E, Q311V, and S428Y.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence that is at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

4. The polypeptide according to any one of claims 1 to 3, further comprising a binding domain, wherein the binding domain comprises (i) six complementarity-determining regions (CDRs) of an immunoglobulin molecule, (ii) a ligand-binding domain of a feline receptor protein, (iii) a nanobody, or (iv) an extracellular domain of a feline receptor protein.

5. The polypeptide according to claim 4, wherein the binding domain specifically binds to an antigen selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and the complement system complex.

6. The polypeptide according to any one of claims 1 to 5, further comprising a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1, VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonists, and thrombopoietin-binding peptides.

7. (i) a polypeptide according to any one of claims 1 to 6, and (ii) a pharmaceutically acceptable excipient.

8. One or more nucleic acids encoding a polypeptide according to any one of claims 1 to 6.

9. One or more expression vectors comprising one or more nucleic acids as described in claim 8.

10. A host cell comprising one or more nucleic acids according to claim 8 or one or more expression vectors according to claim 9.

11. A method for producing polypeptides, (a) To provide one or more nucleic acids as described in claim 8; (b) expressing one or more nucleic acids in a host cell culture to produce the polypeptide; and (c) recovering the polypeptide produced in (b) from the host cell culture.

12. The pharmaceutical composition according to claim 11, used for the treatment or prevention of a feline disease or disorder requiring treatment or prevention.

13. The aforementioned cat's disease or disorder The pharmaceutical composition according to claim 12, wherein (a) allergies, chronic pain, acute pain, inflammatory diseases, autoimmune diseases, endocrine diseases, gastrointestinal diseases, cardiovascular diseases, kidney diseases, reproductive function disorders, infections, cancer, or (b) atopic dermatitis, allergic dermatitis, osteoarthritis pain, arthritis, anemia, or obesity.

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