Compositions and methods of use for extending half-life of therapeutic agents in felines

Abstract

The present invention relates to compositions and methods of use for extending the half-life of therapeutic agents in felines. Compositions for extending the half-life of one or more polypeptides in felines and methods of their use are provided. The composition relates to a variant feline IgG Fc region.

Description

[0001] This application is a divisional application of the patent application "Compositions and methods of use for extending the half-life of therapeutic agents in felines" with an international application date of June 23, 2021 and application number 202180056036.X (international application number PCT / US2021 / 038714).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 050,535 filed on July 10, 2020 and U.S. Provisional Application No. 63 / 143,720 filed on January 29, 2021, the contents of each of which are incorporated by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 23, 2021, named "47406-0016WO1_SL.txt" and is 25KB in size. Technical Field

[0006] The present disclosure generally relates to polypeptides (e.g., fusion polypeptides such as polypeptide-Fc region fusions; or binding molecules such as ligand binding portions of antibodies or receptor-Fc fusions) having extended half-lives in felines compared to their wild-type counterparts. Background Art

[0007] The Fc region of an antibody plays many functional roles, including but not limited to protecting the antibody from degradation by the lysosomal pathway and mediating antibody effector functions. With the increasing use of feline antibodies as therapeutic agents, attention has increased not only in selecting the best Fab, but also in combining it with the appropriate Fc to obtain the desired half-life and effector functions.

[0008] There is little guidance in the art regarding extending the half-life of polypeptide therapeutics (eg, antibodies) for cats. The present disclosure remedies this deficiency by providing Fc region variants that improve the serum persistence of polypeptides (eg, antibodies) in felines. Summary of the invention

[0009] Provided herein are feline Fc (e.g., feline IgG Fc region variants) or feline FcRn binding fragments thereof that can be used for therapeutic polypeptides. The disclosure is characterized in that the polypeptide increases the binding to feline FcRn compared to a control polypeptide (e.g., a wild-type counterpart IgG feline Fc region). In some cases, at pH 5.5, pH 6.0, and / or pH 6.5, these polypeptides increase the binding to feline FcRn compared to a control polypeptide. In some cases, compared to at neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5), these polypeptides can, for example, bind to feline FcRn at a higher level at acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5). In some cases, compared to at pH 7.4, these polypeptides bind to feline FcRn at a higher level at pH 5.5 and / or 6.0. The present disclosure relates in part to polypeptides with extended half-life in felines compared to their wild-type counterparts. For example, binding molecules (e.g., ligand binding portions of antibodies or receptors) are provided, which have extended half-life relative to versions of these binding molecules not connected to Fc regions or their feline FcRn binding regions disclosed herein. Enzyme-Fc region fusions, ligand-Fc region fusions, nanobody-Fc fusions, and peptide-Fc region fusions are also provided, wherein the fusions have extended half-life compared to their wild-type counterparts. In addition to one or more substitutions (relative to wild-type feline Fc regions) with extended half-life, the Fc region may also include other substitutions, such as increasing effector function, reducing effector function, increasing binding to protein A, and / or reducing the heterogeneity of polypeptides (e.g., by removing one or more post-translational modifications in the Fc region). The feline Fc region sequence may be from any feline antibody. In some cases, the feline Fc region sequence is from a feline IgG (eg, IgG1a, IgG1b, IgG2).

[0010] The present disclosure features a recombinant protein comprising (1) a binding domain or fragment thereof that specifically binds to a ligand or protein epitope, wherein the binding domain is linked to (2) a domain comprising an Fc region (CH2+CH3 region) or a feline FcRn binding region thereof as disclosed herein. In some cases, the binding domain comprises (i) six complementary determining regions (CDRs) of, for example, a feline or human / humanized antibody; (ii) a nanobody; (iii] a soluble receptor binding domain that binds to a ligand or a ligand binding fragment thereof, and (iv) an extracellular domain of a feline receptor protein.

[0011] The present disclosure also provides a composition comprising: (1) a first polypeptide comprising a first Fc region (e.g., a CH2 region, a CH3 region, a CH2+CH3 region), the first Fc region comprising a feline IgG Fc region variant described herein; and (2) a second polypeptide comprising a second Fc region, the second Fc region comprising a feline IgG Fc region variant described herein. The first polypeptide and the second polypeptide can be associated through the first Fc region and the second Fc region. In some cases, the amino acid sequences of the first Fc region and the second Fc region are the same. In other cases, the amino acid sequences of the first Fc region and the second Fc region are different (e.g., there are 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 amino acids that are different). In some cases, the Fc region variant is a variant of a feline IgG1a antibody Fc region. In some cases, the Fc region variant is a variant of a feline IgG1b antibody Fc region. In some cases, the Fc region variant is a variant of a feline IgG2 antibody Fc region.

[0012] Also disclosed are fusion molecules comprising feline IgG Fc region variants and polypeptides disclosed herein. In some cases, feline IgG Fc region variants are covalently linked to polypeptides (e.g., through hinge regions or joints). In some cases, the polypeptide is a ligand binding domain of a feline receptor protein, an extracellular domain of a feline receptor protein, or an antigen binding domain. In some cases, the polypeptide is selected from a ligand binding domain or an extracellular domain of a feline IL-13Rα1 or IL-13Rα2, a feline EPO, a feline CTLA4, a feline LFA3, a feline VEGFR1 / VEGFR3, a feline IL-1R, a feline GLP-1 receptor agonist, and a feline thrombopoietin binding peptide. In some cases, the polypeptide is a scFv, a nanobody, or a single domain antibody. In some cases, IgG Fc region variants are variants of feline IgG1a antibody Fc regions. In some cases, the IgG Fc region variant is a variant of a feline IgG1b antibody Fc region. In some cases, the IgG Fc region variant is a variant of a feline IgG2 antibody Fc region.

[0013] In some aspects, the disclosure provides a polypeptide comprising a feline IgG Fc region variant or a feline FcRn binding region thereof, wherein the polypeptide comprises an amino acid substitution at at least one position selected from the group consisting of:

[0014] (i) a position corresponding to amino acid position 252 of a wild-type feline IgG, wherein the amino acid substitution is S252W;

[0015] (ii) a position corresponding to amino acid position 254 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S254R and S254K;

[0016] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V or L309Y;

[0017] (iv) a position corresponding to amino acid position 311 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of Q311R, Q311V, Q311L, and Q311K;

[0018] (v) a position corresponding to amino acid position 428 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428M, S428Y, S428H, and S428R; and

[0019] (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;

[0020] wherein the amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity for feline FcRn when compared to the Fc domain of wild-type feline IgG.

[0021] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 252 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 252 of a wild-type feline IgG is S252W.

[0022] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 254 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 254 of a wild-type feline IgG is S254R. In some embodiments, the amino acid substitution at position 254 of a wild-type feline IgG is S254K.

[0023] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 309 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 309 of a wild-type feline IgG is L309V. In some embodiments, the amino acid substitution at position 309 of a wild-type feline IgG is L309Y.

[0024] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 311 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311R. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311V. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311K. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311L.

[0025] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 428 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428M.

[0026] In some embodiments, the polypeptide comprises at least the amino acid substitution S428Y. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428Y. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428R. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428H.

[0027] In another embodiment, the polypeptide comprises an amino acid substitution 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. 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, and a conservative amino acid substitution of any of the foregoing. 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.

[0028] In another aspect, the disclosure provides a polypeptide comprising a feline IgG Fc region variant or a feline FcRn binding region thereof, wherein the polypeptide comprises two or more amino acid substitutions, wherein the two or more amino acid substitutions are selected from the group consisting of:

[0029] (i) an amino acid substitution at a position corresponding to amino acid position 252 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S252W, S252Y, S252F, and S252R;

[0030] (ii) an amino acid substitution at 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;

[0031] (iii) an amino acid substitution at a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of L309V, L309Y, and L309E;

[0032] (iv) an amino acid substitution at 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;

[0033] (v) an amino acid substitution at a position corresponding to amino acid position 428 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428L, S428M, S428Y, S428H, and S428R;

[0034] (vi) an amino acid substitution at one or more positions corresponding to amino acid positions 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type feline IgG; and

[0035] (vii) an amino acid substitution at a position corresponding to amino acid position 434 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S434F, S434W, S434H, S434R, and S434Y;

[0036] wherein the amino acid positions are based on EU numbering, wherein the two or more amino acid substitutions are at different positions, and wherein the polypeptide has increased binding affinity for feline FcRn when compared to (a) an Fc domain of a wild-type feline IgG and (b) a polypeptide comprising only one of the two or more amino acid substitutions.

[0037] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 286 of a wild-type feline IgG. In some embodiments, the amino acid substitution is selected from the group consisting of T286E and T286D.

[0038] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 289 of a wild-type feline IgG. In some embodiments, the amino acid substitution is selected from the group consisting of T289K and T289H.

[0039] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 301 of a wild-type feline IgG. In some embodiments, the amino acid substitution is R301L.

[0040] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 334 of a wild-type feline IgG. In some embodiments, the amino acid substitution is R334D.

[0041] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 426 of a wild-type feline IgG. In some embodiments, the amino acid substitution is selected from the group consisting of S426L and S426H.

[0042] In some embodiments, 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. In some embodiments, the amino acid substitution is T437L.

[0043] In some embodiments, the two or more amino acid substitutions are selected from the group consisting of:

[0044] (i) a combination of S252Y and Q311R and / or Q311L;

[0045] (ii) a combination of S434Y and one or more of S254R, S254K, L262E, T286D, T286E, T289K, E293D, E293K, L309V, L309E, K326D and Q347L;

[0046] (iii) combination of S434F and E380D;

[0047] (iv) a combination of S428L and one or more of S252R, T286E, Q311V, Q311K, D312T, I377V, I383L and N389cR;

[0048] (v) S428L, E380D and S434R;

[0049] (vi) S428L, E380T and S434R;

[0050] (vii) a combination of S252R and L262Q;

[0051] (viii) T260E, L309E and Q355L;

[0052] (ix) Combination of S290V and R344D;

[0053] (x) R301L, E380V and T437L;

[0054] (xi) a combination of T286E and S428H;

[0055] (xii) a combination of R334D and one or more of S428R, T437L and R301L;

[0056] (xiii) a combination of S426L and T289H and / or S428H;

[0057] (xiv) a combination of S428Y and one or more of Q311V, S254R, L309V, T286E and E380T; and

[0058] (xv) Combination of S428H and T289H.

[0059] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

[0060] In some embodiments, the wild-type feline IgG is a feline IgG1a comprising an Fc domain having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:1, a feline IgG1b comprising an Fc domain having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:2, or a feline IgG2 comprising an Fc domain having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:3.

[0061] In other embodiments, the wild-type feline IgG is a feline IgG1a comprising an Fc domain having the amino acid sequence of SEQ ID NO: 1. In other embodiments, the wild-type feline IgG is a feline IgG1b comprising an Fc domain having the amino acid sequence of SEQ ID NO: 2. In other embodiments, the wild-type feline IgG is a feline IgG2 comprising an Fc domain having the amino acid sequence of SEQ ID NO: 3.

[0062] In some embodiments, the polypeptide further comprises a binding domain. In some embodiments, the binding domain comprises (i) six complementary 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 a complement system complex.

[0063] In some embodiments, the polypeptide further comprises a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, and a thrombopoietin binding peptide.

[0064] In some embodiments, the polypeptide binds to feline FcRn at a higher level at acidic pH than at neutral pH. In some embodiments, the polypeptide binds to feline FcRn at a higher level at pH 5.5 than at pH 7.4. In some embodiments, the polypeptide binds to feline FcRn at a higher level at pH 6.0 than at pH 7.4.

[0065] In some embodiments, the polypeptide has: (1) an extended half-life in cats compared to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except that they have a corresponding wild-type feline IgG Fc region instead of an IgG Fc region variant; and / or (2) increased binding to feline FcRn compared to the control polypeptides; and wherein the amino acid positions are based on EU numbering.

[0066] In some aspects, the present disclosure provides pharmaceutical compositions comprising (i) a polypeptide described herein and (ii) a pharmaceutically acceptable excipient.

[0067] In some aspects, the disclosure provides one or more nucleic acids encoding the polypeptides described herein.

[0068] In some aspects, the disclosure provides one or more expression vectors comprising one or more nucleic acids described herein.

[0069] In some aspects, the disclosure provides a host cell comprising one or more nucleic acids described herein or one or more expression vectors described herein.

[0070] In some aspects, the present disclosure provides methods of preparing one or more polypeptides, the methods comprising:

[0071] (a) providing one or more nucleic acids described herein;

[0072] (b) expressing the one or more nucleic acids in host cell culture, thereby producing the polypeptide; and

[0073] (c) collecting the polypeptide produced in (b) from the host cell culture.

[0074] In some embodiments, the method further comprises formulating the polypeptide into a pharmaceutical preparation.

[0075] In some aspects, the present disclosure provides methods of treating a feline disease or condition in a cat in need thereof, the method comprising administering to the cat an effective amount of a composition comprising a pharmaceutical composition described herein.

[0076] In some aspects, the present disclosure provides a method of preventing a feline disease or condition in a cat in need thereof, the method comprising administering to the cat an effective amount of a composition comprising a pharmaceutical composition described herein.

[0077] In some embodiments, the disease or disorder is an allergic disease, chronic pain, acute pain, an inflammatory disease, an autoimmune disease, an endocrine disease, a gastrointestinal disease, a cardiovascular disease, a kidney disease, a fertility-related disorder, an infectious disease, or cancer.

[0078] In some embodiments, the disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present application (including definitions) shall prevail. The materials, methods and examples are illustrative only and are not intended to be limiting.

[0080] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1Depicted are the Biacore sensorgrams of S428L, S428M, S428Y, S434F, S434W and S434H feline IgG1a Fc variants from the NNK library. The brighter lines in each figure represent the measured data, and the darker lines represent the fitted curves using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0082] Figure 2 Depicted are the Biacore sensorgrams of S252W, S252Y and S252F feline IgG1a Fc variants from the NNK library. Also depicted are the Biacore sensorgrams of the wild type (WT). The brighter lines in each figure represent the measured data, and the darker lines represent the fitted curves using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0083] Figure 3 The amino acid sequences of the wild-type feline IgG1a Fc region (SEQ ID NO: 1) and the wild-type feline IgG1b Fc region (SEQ ID NO: 2) are shown in alignment with the putative wild-type feline IgG2 Fc region (SEQ ID NO: 3). The hinge region is located between the triangles. The arrows indicate that the cysteine ​​residues in the hinge region may be involved in the disulfide bond between the two heavy chains (from Strietzel et al., 2014, Vet. Immunol. Immunopathol., 158: 214-223).

[0084] Figure 4 An alignment of the amino acid sequences of wild-type feline IgG1a Fc (SEQ ID NO: 1) and human IgG1 Fc regions based on EU numbering is shown. The 55 amino acid positions used in the generation of the phage library described in Example 2 are highlighted and underlined.

[0085] Figure 5A and 5B Depicted are the Carterra LSA sensorgrams of the S252Y feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0086] Fig. 6A and 6BDepicted are the Carterra LSA sensorgrams of the S252Y+Q311R feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0087] Fig. 7A and 7B Depicted are Carterra LSA sensorgrams of the S252Y+Q311K feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0088] Fig. 8A and 8B Depicted are the Carterra LSA sensorgrams of the S252Y+Q311V feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0089] Fig.9A and 9B Depicted are the Carterra LSA sensorgrams of the S252Y+Q311L feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0090] Fig. 10A and 10B Depicted are the Carterra LSA sensorgrams of the S434Y feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0091] Fig.11A and 11BDepicted are Carterra LSA sensorgrams of the S434Y+S254R feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0092] Fig. 12A and 12B Depicted are Carterra LSA sensorgrams of the S434Y+S254K feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0093] Fig.13A and 13B Depicted are the Carterra LSA sensorgrams of the S434Y+L262E feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0094] Fig.14A and 14B Depicted are Carterra LSA sensorgrams of the S434Y+T286D feline IgG1a Fc variant from a phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. Irregular lines represent measured data and smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0095] Fig.15A and 15B Depicted are Carterra LSA sensorgrams of the S434Y+T286E feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0096] Fig.16A and 16BDepicted are Carterra LSA sensorgrams of the S434Y+T289K feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0097] Fig.17A and 17B Depicted are the Carterra LSA sensorgrams of the S434Y+E293D feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0098] Fig.18A and 18B Depicted are Carterra LSA sensorgrams of the S434Y+E293K feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0099] Fig.19A and 19B Depicted are Carterra LSA sensorgrams of the S434Y+L309V feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0100] Fig. 20A and 20B Depicted are Carterra LSA sensorgrams of the S434Y+L309E feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0101] Fig.21A and 21BDepicted are the Carterra LSA sensorgrams of the S434Y+K326D feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0102] Fig.22A and 22B Depicted are Carterra LSA sensorgrams of the S434Y+Q347L feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0103] Fig.23A and 23B Depicted are Carterra LSA sensorgrams of the S434Y+S426L feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0104] Fig.24A and 24B Depicted are the Carterra LSA sensorgrams of the S434F feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0105] Fig.25A and 25B Depicted are Carterra LSA sensorgrams of the S434F+E380D feline IgG1a Fc variant from a phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. Irregular lines represent measured data and smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0106] Fig.26A and 26BDepicted are Carterra LSA sensorgrams of the S428L+T286E feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0107] Fig.27A and 27B Depicted are the Carterra LSA sensorgrams of the S428L+Q311V feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0108] Fig.28A and 28B Depicted are Carterra LSA sensorgrams of the S428L+Q311K feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0109] Fig.29A and 29B Depicted are Carterra LSA sensorgrams of the S428L+D312T feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0110] Fig. 30A and 30B Depicted are Carterra LSA sensorgrams of the S428L+I377V feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0111] Fig.31A and 31BDepicted are Carterra LSA sensorgrams of the S428L+I383L feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0112] Fig.32A and 32B Depicted are Carterra LSA sensorgrams of the S428L+N389cR feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0113] Fig.33A and 33B Depicted are Carterra LSA sensorgrams of the S428L+E380D+S434R feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0114] Fig.34A and 34B Depicted are Carterra LSA sensorgrams of the S428L+E380T+S434R feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0115] Fig.35A and 35B Depicted are the Carterra LSA sensorgrams of the Q311R feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0116] Fig.36A and 36BDepicted are the Carterra LSA sensorgrams of the R392E feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0117] Fig.37A and 37B Depicted are Carterra LSA sensorgrams of the S252R+L262Q feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0118] Fig.38A and 38B Depicted are Carterra LSA sensorgrams of the S252R+A378E feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0119] Fig.39A and 39B Depicted are Carterra LSA sensorgrams of the T260E+L309E+Q355L feline IgG1a Fc variant from a phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. Irregular lines represent measured data and smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0120] Fig.40A and 40B Depicted are Carterra LSA sensorgrams of the T286E+S428R feline IgG1a Fc variant from a phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. Irregular lines represent measured data and smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0121] Fig.41A and 41BDepicted are Carterra LSA sensorgrams of the S290V+R334D feline IgG1a Fc variant from a phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. Irregular lines represent measured data and smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0122] Fig.42A and 42B Depicted are Carterra LSA sensorgrams of the R301L+E380V+T437L feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. Irregular lines represent measured data and smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0123] Fig.43A and 43B Depicted are Carterra LSA sensorgrams of the S428L feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0124] Fig.44A and 44B Depicted are Carterra LSA sensorgrams of the S428L+S252R feline IgG1a Fc variant from the phage display library and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0125] Fig.45A and 45B Depicted are the Carterra LSA sensorgrams of the wild-type feline IgG1a Fc variant (SEQ ID NO: 1) and its interaction with feline FcRN at pH 6.0 and pH 7.4. The irregular lines represent the measured data and the smooth lines are the curves fitted using a 1:1 interaction model. Y-axis: resonance units (RU); X-axis: time (seconds).

[0126] Fig.46Depicted is a flow chart of a two-compartment pharmacokinetic (PK) model with linear clearance using nonlinear mixed effects (NLME) modeling for describing serum concentrations of anti-NGF monoclonal antibody (mAb) variants.

[0127] Fig.47 Depicted are the observed individual serum concentrations of wild-type (WT) antibody and antibody variants S252W, S428Y, S428Y+L309V, S428Y+Q311V and S428Y+S254R (results from two animals per antibody / variant).

[0128] Fig.48 Depicted are predicted serum concentration curves for wild-type (WT) antibody and antibody variants S252W, S428Y, S428Y+L309V, S428Y+Q311V, and S428Y+S254R for a typical 2 kg cat receiving a single IV dose of 2 mg / kg antibody / variant.

[0129] Figures 49A-49L Biacore sensorgrams showing the binding of wild-type feline IgG1a and feline IgG1a variants are depicted. The lighter lines in each graph represent the measured data and the darker lines represent the fitted curves. Y-axis: resonance units (RU); X-axis: time (seconds).

[0130] Fig.50 Depicted are the observed individual serum concentrations of wild type and Fc variants, with two animals per variant.

[0131] Fig.51 Predicted serum concentration curves for monoclonal antibodies carrying wild-type (WT) IgG1a Fc or IgG1a Fc variants are depicted. DETAILED DESCRIPTION

[0132] With the increasing use of polypeptides (e.g., antibodies, ligand binding domains of receptors, enzymes, ligands, peptides) as therapeutic agents for the prevention and treatment of a variety of feline diseases, it is important to develop polypeptides with extended half-lives, particularly for the prevention or treatment of chronic diseases in which repeated administration of the polypeptide is necessary.

[0133] Thus, the disclosure features a feline immunoglobulin Fc region or a feline FcRn binding region thereof comprising mutations that enhance the half-life of one or more polypeptides containing these sequences. Also disclosed are polypeptides comprising these domains and methods of using them. These peptides can be used for a variety of therapeutic and diagnostic purposes.

[0134] Where values ​​are described in the form of ranges, it is to be understood that the description includes disclosure of all possible subranges within such ranges, as well as specific numerical values ​​falling within such ranges, regardless of whether specific numerical values ​​or specific subranges are explicitly stated. All numerical designations (e.g., pH, temperature, time, concentration, and molecular weight, including ranges) are approximate values, varying by (+) or (-) increments of 1.0 or 0.1, as the case may be, or alternatively by + / -15%, or alternatively by 10%, or alternatively by 5%, or alternatively by 2%. It is to be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about". It is also to be understood that, although not always explicitly stated, the agents described herein are exemplary only and equivalents of such agents are known in the art.

[0135] As used herein, the term "about" when referring to a measurable value such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0136] Feline Antibodies

[0137] Cats usually have three IgG heavy chains, called IgG1a, IgG1b and IgG2. These heavy chains represent three different subclasses of cat IgG. The amino acid and DNA sequences of these heavy chains can be obtained from Strietzel et al., 2014, Vet. Immunol. Immunopathol., 158: 214-223 and GENBANK database. For example, the amino acid sequence of the feline IgG1a heavy chain has GENBANK accession number BAA32229.1, the feline IgG1b heavy chain has GENBANK accession number BAA32230.1, and the feline IgG2 heavy chain has GENBANK accession number KF811175.1. Feline antibodies also include two types of light chains: κ and λ. The DNA and amino acid sequences of these light chains can also be obtained from the GENBANK database. For example, the feline κ light chain amino acid sequence has accession number AF198257.1, and the feline λ light chain has accession number E07339.1.

[0138] CH2 region of the feline Fc region:

[0139] The CH2 region of a feline antibody comprises or consists of amino acids 231 to 340 (according to EU numbering) of a feline IgG antibody. It should be understood that the CH2 region may include one to six (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N and / or C terminus.

[0140] The amino acid sequence of the CH2 region of feline IgG1a is provided as follows: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITW FVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAK (SEQ ID NO: 4)

[0141] The amino acid sequence of the CH2 domain of feline IgG1b is provided below:

[0142] PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQ ITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSPIERTISKDK(SEQ ID NO:5)

[0143] The amino acid sequence of the CH2 domain of feline IgG2 is provided below:

[0144] VPEIPGAPSVFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQI TWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEF KCKVNSKSLPSAMERTISKAK(SEQ ID NO:6)

[0145] CH3 region of the feline Fc region:

[0146] The CH3 region of a feline antibody comprises or consists of amino acids 341 to 447 (according to EU numbering) of a feline IgG antibody. It should be understood that the CH3 region may include one to six (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N and / or C terminus.

[0147] The amino acid sequence of the CH3 domain of feline IgG1a is provided below:

[0148] GQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEI TGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTC SVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:7)

[0149] The amino acid sequence of the CH3 domain of feline IgG1b is provided below:

[0150] GQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEI TGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCS VSHEALHSHHTQKSLTQSPGK(SEQ ID NO:8)

[0151] The amino acid sequence of the CH3 domain of feline IgG2 is provided below:

[0152] GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEI TGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCS VSHEALHSHHTQKSLTQSPGK(SEQ ID NO:9)

[0153] Fc region of feline Fc region:

[0154] The Fc region of a feline IgG antibody comprises or consists of amino acids 231 to 447 (according to EU numbering) of a feline IgG antibody.

[0155] The amino acid sequence of the Fc domain of feline IgG1a is provided below:

[0156] PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKA KGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK

[0157] (SEQ ID NO:1)

[0158] The amino acid sequence of the Fc domain of feline IgG1b is provided below:

[0159] PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKD KGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK

[0160] (SEQ ID NO:2)

[0161] The amino acid sequence of the Fc domain of feline IgG2 is provided below:

[0162] VPEIPGAPSVFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKG QPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK(SEQ ID NO:3)

[0163] 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:

[0164] Table 1:

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] Substitutions in Feline IgG Fc to Improve Half-Life

[0174] Increased serum persistence is a beneficial property of therapeutic polypeptides. The present disclosure is characterized by substitutions in wild-type feline IgG1a, IgG1b and IgG2 Fc regions that increase the half-life of one or more polypeptides comprising these Fc regions in cats relative to one or more control polypeptides, wherein the one or more control polypeptides are identical to the one or more polypeptides except for having a corresponding wild-type feline IgG Fc region instead of an IgG Fc region variant. Substitutions that extend the half-life can be carried out in one or more of the contexts of a feline CH2 region, a feline CH3 region or a feline Fc (e.g., CH2+CH3) region.

[0175] The present disclosure provides polypeptides comprising a feline IgG Fc region variant or a feline FcRn binding region thereof, wherein the polypeptide comprises an amino acid substitution at at least one position selected from the group consisting of:

[0176] (i) a position corresponding to amino acid position 252 of a wild-type feline IgG, wherein the amino acid substitution is S252W;

[0177] (ii) a position corresponding to amino acid position 254 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S254R and S254K;

[0178] (iii) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V or L309Y;

[0179] (iv) a position corresponding to amino acid position 311 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of Q311R, Q311V, Q311L, and Q311K;

[0180] (v) a position corresponding to amino acid position 428 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428M, S428Y, S428H, and S428R; and

[0181] (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;

[0182] wherein the amino acid positions are based on EU numbering, and wherein the polypeptide has increased binding affinity for feline FcRn when compared to the Fc domain of wild-type feline IgG.

[0183] In some embodiments, the polypeptide has increased binding affinity for feline FcRn at a pH of about 5.0 to about 6.5 (eg, about 5.5 or about 6.0) when compared to the Fc domain of wild-type feline IgG.

[0184] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 252 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 252 of a wild-type feline IgG is S252W.

[0185] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 254 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 254 of a wild-type feline IgG is S254R. In some embodiments, the amino acid substitution at position 254 of a wild-type feline IgG is S254K.

[0186] In some embodiments, the polypeptide comprises the amino acid substitution L309V or L309Y.

[0187] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 311 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311R. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311V. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311K. In some embodiments, the amino acid substitution at position 311 of a wild-type feline IgG is Q311L.

[0188] In some embodiments, the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 428 of a wild-type feline IgG. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428M.

[0189] In some embodiments, the polypeptide comprises at least the amino acid substitution S428Y. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428Y. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428R. In some embodiments, the amino acid substitution at position 428 of a wild-type feline IgG is S428H.

[0190] In another embodiment, the polypeptide comprises an amino acid substitution 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. 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, and a conservative amino acid substitution of any of the foregoing. 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.

[0191] In another aspect, the disclosure provides a polypeptide comprising a feline IgG Fc region variant or a feline FcRn binding region thereof, wherein the polypeptide comprises two or more amino acid substitutions, wherein the two or more amino acid substitutions are selected from the group consisting of:

[0192] (i) an amino acid substitution at a position corresponding to amino acid position 252 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S252W, S252Y, S252F, and S252R;

[0193] (ii) an amino acid substitution at 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;

[0194] (iii) an amino acid substitution at a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of L309V, L309Y, and L309E;

[0195] (iv) an amino acid substitution at 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;

[0196] (v) an amino acid substitution at a position corresponding to amino acid position 428 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S428L, S428M, S428Y, S428H, and S428R;

[0197] (vi) an amino acid substitution at one or more positions corresponding to amino acid positions 262, 286, 289, 290, 293, 301, 312, 326, 334, 347, 355, 377, 380, 383, 389c, 392, 426, and 437 of wild-type feline IgG; and

[0198] (vii) an amino acid substitution at a position corresponding to amino acid position 434 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S434F, S434W, S434H, S434R, and S434Y;

[0199] wherein the amino acid positions are based on EU numbering, wherein the two or more amino acid substitutions are at different positions, and wherein the polypeptide has increased binding affinity for feline FcRn when compared to (a) an Fc domain of a wild-type feline IgG and (b) a polypeptide comprising only one of the two or more amino acid substitutions.

[0200] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 286 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of T286E and T286D.

[0201] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 289 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of T289K and T289H.

[0202] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 301 of a wild-type feline IgG, wherein the amino acid substitution is R301L.

[0203] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 334 of a wild-type feline IgG, wherein the amino acid substitution is R334D.

[0204] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 426 of a wild-type feline IgG, wherein the amino acid substitution is selected from the group consisting of S426L and S426H.

[0205] In some embodiments, the two or more amino acid substitutions include an amino acid substitution at a position corresponding to amino acid position 437 of a wild-type feline IgG, wherein the amino acid substitution is T437L.

[0206] In some embodiments, the two or more amino acid substitutions are selected from the group consisting of:

[0207] (i) a combination of S252Y and Q311R and / or Q311L;

[0208] (ii) a combination of S434Y and one or more of S254R, S254K, L262E, T286D, T286E, T289K, E293D, E293K, L309V, L309E, K326D and Q347L;

[0209] (iii) S434F and E380D;

[0210] (iv) a combination of S428L and one or more of S252R, T286E, Q311V, Q311K, D312T, I377V, I383L, and N389cR;

[0211] (v) S428L, E380D and S434R;

[0212] (vi) S428L, E380T and S434R;

[0213] (vii) a combination of S252R and L262Q;

[0214] (viii) T260E, L309E and Q355L;

[0215] (ix) S290V and R344D; and

[0216] (x)R301L, E380V and T437L.

[0217] 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.

[0218] In some cases, the disclosure provides a feline IgG CH2 region variant comprising 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 set forth in any one of SEQ ID NOs: 4 to 6. Also provided are feline IgG CH2 region variants comprising an amino acid sequence that differs from SEQ ID NOs: 4 to 6 by 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids.

[0219] In other cases, the disclosure features a feline IgG CH3 region variant comprising 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 set forth in any one of SEQ ID NOs: 7 to 9. It also features a feline IgG CH3 region variant comprising an amino acid sequence that differs from SEQ ID NOs: 7 to 9 by 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acids.

[0220] In some cases, the disclosure features a feline IgG Fc region variant comprising 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 set forth in any one of SEQ ID NOs: 1 to 3. Also disclosed are feline IgG Fc region variants comprising an amino acid sequence that differs from SEQ ID NOs: 1 to 3 by 1 to 20 (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.

[0221] In some embodiments, one or more polypeptides are provided comprising a feline IgG Fc CH2 region variant comprising 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 shown in any one of SEQ ID NOs: 4 to 6.

[0222] In some embodiments, it is characterized by comprising one or more polypeptides of 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 shown in any one of SEQ ID NOs: 7 to 9.

[0223] In some embodiments, it is characterized by comprising one or more polypeptides of a feline IgG Fc region variant, 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 shown in any one of SEQ ID NOs: 1 to 3.

[0224] As noted elsewhere, in some embodiments, the polypeptide further comprises at least one additional amino acid substitution in a region corresponding to amino acid positions 250-256, amino acid positions 285-288, amino acid positions 307-315; amino acid positions 376-380, amino acid positions 383 to 392; or amino acid positions 428-437 of a wild-type feline IgG, wherein the amino acid positions are numbered based on EU, and wherein the polypeptide has increased binding to feline FcRn compared to the Fc domain of a wild-type feline IgG.

[0225] In some embodiments, the polypeptide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) additional amino acid substitutions selected from those disclosed in Table 2 below.

[0226] Table 2: List of amino acid substitutions (Group 1 and Group 2) that increase binding of feline IgG1a Fc variants to feline FcRN.

[0227]

[0228]

[0229] Amino acid substitutions may be made on one or both chains of a CH2 domain, a CH3 domain, or an Fc domain. In some cases, the substitutions on both chains of a CH2 domain, a CH3 domain, or an Fc domain are identical. In some cases, the substitutions on both chains of a CH2 domain, a CH3 domain, or an Fc domain are not identical. In some cases, the Fc region comprises one or more additional substitutions that increase or decrease effector function and / or improve product heterogeneity.

[0230] Other Substitutions That Can Be Combined with Half-Life Enhancing Substitutions

[0231] The development of therapeutic polypeptides / proteins (e.g., monoclonal antibodies) is a complex process that requires coordination of a complex set of activities to produce the desired polypeptide / protein. These include optimization of specificity, affinity, functional activity, expression levels in engineered cell lines, long-term stability, elimination or enhancement of effector functions, and development of commercially viable manufacturing and purification methods. The present disclosure encompasses any additional substitutions that promote any one or more of the above goals.

[0232] In some embodiments, substitutions are introduced to reduce the effector function of the feline Fc region. Such substitutions will be familiar to those skilled in the art and may be located at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) positions of feline IgG. Illustrative examples include WO 2019 / 035010 A1.

[0233] In some embodiments, substitutions are introduced into the wild-type feline IgG Fc region to enhance binding to protein A, thereby facilitating purification by protein A chromatography. Such substitutions will be familiar to those skilled in the art and may be located at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) positions of feline IgG. Illustrative examples include WO 2019 / 035010 A1.

[0234] In some embodiments, additional amino acid substitutions may be made to alter the binding affinity to FcRn compared to a parent polypeptide or wild-type polypeptide (eg, to increase or decrease the binding affinity to FcRn).

[0235] In some embodiments, the polypeptide comprises a hinge region of a feline antibody. In some embodiments, the hinge region of a feline antibody can be modified to extend half-life.

[0236] Polypeptides comprising feline IgG Fc variants

[0237] The present disclosure encompasses any polypeptide that may benefit from half-life extension in cats. To extend half-life, these polypeptides are designed to include the Fc region variants disclosed above (eg, CH2 region, CH3 region, CH2+CH3 region).

[0238] Exemplary polypeptides include, but are not limited to, whole antibodies, scFv, nanobodies, ligand binding portions of receptors, cytokines, growth factors, enzymes, and peptides. For example, the CH3 domain variants disclosed above can be linked to scFv nanobodies, ligand binding portions of receptors (e.g., ligand binding portions of feline IL-13Rα1 or IL-13Rα2), cytokines, growth factors, enzymes, or peptides. As used herein, the terms "nanoantibody," "VHH," "VHH antibody fragment," and "single domain antibody" are used interchangeably herein to represent the variable domains of single heavy chains of those types of antibodies found in Camelidae (which are usually naturally found to lack light chains). Those skilled in the art will be familiar with suitable nanobodies, illustrative examples of which include nanobodies of camels, dromedaries, llamas, and alpacas. Alternatively, the Fc region variants disclosed above can be linked to these polypeptides. In another embodiment, feline or feline-derived antibodies are modified to include Fc region variants disclosed herein.

[0239] In some embodiments, the polypeptides of the present disclosure include an antibody hinge region. The hinge region can be located between the antigen or ligand binding domain of the polypeptide and the Fc region variant. In some cases, the hinge region is connected to the C-terminus of a cytokine, a growth factor, an enzyme or a peptide, and the hinge region is connected to the N-terminus of the Fc region variant. Exemplary hinge region sequences are provided below.

[0240] IgG1a: KTDHPPGPKPCDCPKCP (SEQ ID NO: 10);

[0241] IgG1b: KTDHPPGPKPCDCPKCP (SEQ ID NO: 11);

[0242] IgG2: KTASTIESKTGEGPKCP (SEQ ID NO: 12);

[0243] The hinge region in the recombinant protein of the present disclosure, if used, includes zero to six (i.e., 0, 1, 2, 3, 4, 5, or 6) amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NOs: 10-12. In some cases, the hinge region used in the recombinant protein of the present 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 set forth in any one of SEQ ID NOs: 10-12.

[0244] One or more polypeptides disclosed herein may comprise a binding domain. The binding domain may specifically bind to a protein, subunit, domain, motif and / or epitope of a selected target as described herein. In some embodiments, one or more polypeptides (e.g., a fusion polypeptide) may comprise a protein, wherein the protein is a therapeutic protein as described herein. In some embodiments, the target (e.g., for a target of a binding domain) or a therapeutic protein (e.g., for a 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, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APR IL, AR, IgE, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, ARC, ART, Artemin, anti-Id, aspartate (ASPARTIC), atrial natriuretic peptide, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, B CA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP-2a, BMP-3 osteogenin, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB(A LK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, 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), cancer-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, CC L25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CC R2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11 a. CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD2 9. CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD49a, CD52, CD 54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, 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-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNA enzyme, Dpp, DPPIV / CD26, Dtk, ECAD, EDA,EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMPRIN, ENA, endothelin receptor, neprilysin, eNOS, Eot, eotaxin 1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, Fibroblast activation 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, Gas 6. GCP-2, GCSF, 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-α1, GFR-α2, GFR-α3, GITR, GLP1, GLP2, glucagon, Glut 4, 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, HCMV gB 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, HIVIIIB 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, I L-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptor (e.g., IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15 R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), interferon (INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 1 2. 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), Potential TGF-1, Potential 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, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, 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,Mullerian inhibitory substance, Mug, MuSK, NAIP, NAP, NAV 1.7, NCAD, N-cadherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4 or -6, Neurturin, Neuron growth factor (NGF), NGFR, NGF-β, 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 , PDGF, 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, RSV Fgp, 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis P LAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βR1(ALK-5), TGF-βR11, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, 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(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY CROWN), TNFRSF19L(RELT), TNFRSF1A(TNF R1CD120a, p55-60), TNFRSF1B(TNF RIICD120b, 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). R2TNFRH2、TNFRST23(DCTRAIL R1TNFRH1)、TNFRSF25(DR3Apo-3、LARD、TR-3、TRAMP、WSL-1)、TNFSF10(TRAIL Apo-2 division, TL2, TNFSF11 (TRANCE / RANK division ODF, OPG division), TNFSF12 (TWEAK Apo-3 division, DR3 division), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF). LIGHT、TALL1、THANK、TNFSF20)、TNFSF14(LIGHT Whether LTg, TNFSF15 (TL1A / VEGI), TNFSF18 (GITR, AITR, TL6), TNFSF1A (TNF-binding factor (Conectin), DIF, TNFSF2), TNFSF1B (TNF-b). 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, transfer receptor, TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expression Lewis Y-related 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, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, vascular vascular disease 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. 、

[0245] In some embodiments, the binding domain specifically binds one or more therapeutic targets or antigens in felines, such as, 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 peptide, 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-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-2R, 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), LAP (TGF-1), potential TGF-1, potential TGF-1bp1, LFA-1, neuronal growth factor (NGF) F), NGFR, NGF-β, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, TG F-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9(4-1BB CD137, ILA), VEFGR-1(fit-1), VEGF, VEGFR or VEGFR-3(flt-4).

[0246] In some embodiments, the one or more polypeptides may comprise a protein, wherein the protein is a therapeutic protein, for example, EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonist, and thrombopoietin binding peptide.In some embodiments, the therapeutic protein is 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 peptide, 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-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-2R, 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), LAP (TGF-1), potential TGF-1, potential TGF-1bp1, LFA-1, neuronal growth factor (NGF) F), NGFR, NGF-β, OX40L, OX40R, PD1, PDL1, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βR1 (ALK-5), TGF-βR11, TGF-βRIIb, TG F-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9(4-1BB CD137, ILA), VEFGR-1(fit-1), VEGF, VEGFR or VEGFR-3(flt-4).

[0247] Pharmaceutical composition

[0248] To prepare a pharmaceutical or sterile composition of one or more polypeptides described herein, the one or more polypeptides may be mixed with a pharmaceutically acceptable carrier or excipient (see, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984)).

[0249] Formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions (see, 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: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, one or more polypeptides of the invention are diluted to an appropriate concentration in a sodium acetate solution at pH 5-6, and NaCl or sucrose is added to adjust tonicity. Additional agents (such as polysorbate 20 or polysorbate 80) may be added to enhance stability.

[0250] Toxicity and therapeutic efficacy of a polypeptide composition administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50(the dose that is lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index (LD 50 / ED 50 ). In certain aspects, it is desirable for one or more polypeptides to exhibit a high therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range for felines. The dosage of such compounds is preferably within the range that includes the ED 50 The dosage may vary within this range depending on the dosage form employed and the route of administration employed.

[0251] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, directly intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial. In some embodiments, one or more polypeptides can be administered by an invasive route such as by injection. In other embodiments, administration is by intravenous, subcutaneous, intramuscular, intraarterial, intratumoral or by inhalation, aerosol delivery.

[0252] The pharmaceutical composition disclosed herein can also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0253] Alternatively, one can administer one or more polypeptides in a local rather than systemic manner, for example by injecting the antibody directly into an arthritic joint or a pathogen-induced lesion characterized by immunopathology, typically in a depot or sustained release formulation. In addition, one can administer one or more polypeptides in a targeted drug delivery system (e.g., in liposomes coated with tissue-specific antibodies, targeted to, for example, an arthritic joint or a pathogen-induced lesion characterized by immunopathology). The liposomes will target the diseased tissue and be selectively taken up by the diseased tissue.

[0254] The administration regimen depends on several factors, including but not limited to the age, weight and physical condition of the feline being treated, serum or tissue turnover of the therapeutic antibody, symptom level, immunogenicity of one or more therapeutic polypeptides and accessibility of target cells in the biomatrix. In some embodiments, the administration regimen delivers enough one or more therapeutic polypeptides to achieve improvement in the target disease state while minimizing undesirable side effects. Therefore, the amount of biological delivery depends in part on the severity of the specific therapeutic polypeptide and the condition being treated. Guidance for selecting appropriate doses of therapeutic antibodies is available (see, e.g., 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)).

[0255] One skilled in the art, for example, uses parameters or factors known in the art or suspected of affecting treatment to determine the appropriate dosage of one or more polypeptides. Typically, the dosage is started with an amount slightly less than the optimal dosage and thereafter increased in small increments until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include those measures such as inflammatory symptoms or levels of inflammatory cytokines produced.

[0256] Nucleic acid, vector, host cell and preparation method

[0257] The present disclosure also encompasses one or more nucleic acids encoding one or more polypeptides described herein, one or more vectors comprising the one or more nucleic acids, and host cells comprising the one or more nucleic acids or the one or more vectors.

[0258] One or more polypeptides described herein can be produced in bacteria 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 (Pichia) (see, e.g., Powers et al., J Immunol Methods. 251: 123-35 (2001)); Hanseula or Saccharomyces. In order to produce antibodies of interest, one or more polynucleotides encoding one or more polypeptides are constructed, introduced into one or more expression vectors, and then expressed in a suitable host cell. In order to improve expression, the nucleotide sequence of the gene can be recoded without changing (or minimally changing - for example, removing the C-terminal residue of the heavy chain or light chain) the amino acid sequence. Potentially recoded regions include regions associated with translation initiation, codon usage, and possible unintended mRNA splicing. Polynucleotides encoding the Fc region variants described herein are readily apparent to the skilled artisan.

[0259] Standard molecular biology techniques can be used to prepare one or more recombinant expression vectors, transfect the host cells, select for transformants, culture the host cells and recover the polypeptide (eg, antibody).

[0260] If the one or more polypeptides are to be expressed in bacterial cells (e.g., E. coli), the expression vector should have features that allow the vector to be amplified in the bacterial cells. In addition, when E. coli (such as JM109, DH5α, HB101 or XL1-Blue) is used as a host, the vector must have a promoter, such as the lacZ promoter (Ward et al., 341:544-546 (1989), the araB promoter (Better et al., Science, 240:1041-1043 (1988)) or a T7 promoter that can allow efficient expression in E. coli. Examples of such vectors include, for example, the M13 series vectors, the pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP and pET (when using such expression vectors, the host is preferably a T7 promoter that expresses the T7 promoter). The expression vector may contain a signal sequence for antibody secretion. For production in the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) may be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector may be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0261] If the one or more polypeptides are expressed in animal cells such as CHO, COS and NIH3T3 cells, the expression vector includes a promoter 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), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)) or CMV promoter (e.g., human cytomegalovirus immediate early promoter). In addition to the nucleic acid sequence encoding the Fc region variant, the recombinant expression vector may carry additional sequences such as sequences that regulate replication of the vector in host cells (e.g., replication origin) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically, the selectable marker gene confers resistance to drugs such as G418, hygromycin or methotrexate to a host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

[0262] In some embodiments, the 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), which are used together with the DHFR selection marker described in the following documents: Kaufman and Sharp (1982) Mol. Biol. 159: 601621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte lines (e.g., NS0 myeloma cells and SP2 cells) and cells from transgenic animals (e.g., transgenic mammals). For example, the cell is a mammary epithelial cell.

[0263] In the exemplary system for antibody expression, the recombinant expression vector of the antibody heavy chain and antibody light chain of encoding antibody is introduced into dhfr-CHO cell by calcium phosphate-mediated transfection.In the recombinant expression vector, antibody heavy chain and light chain gene are each operably connected to enhancer / promoter regulatory element (for example, be derived from SV40, CMV, adenovirus etc., such as CMV enhancer / AdMLP promoter regulatory element or SV40 enhancer / AdMLP promoter regulatory element) to drive the high level transcription of gene.The recombinant expression vector also carries DHFR gene, and it allows to select the CHO cell of carrier transfected using methotrexate selection / amplification.Cultivate selected transformant host cell to allow the expression of antibody heavy chain and light chain, and reclaim the antibody from culture medium.

[0264] Treatment

[0265] One or more polypeptides disclosed herein can be used to treat or prevent any disease or condition in a cat in need thereof. The present invention is particularly useful for treating chronic conditions that require repeated dosing. Due to the extended half-life of protein therapeutics, less frequent dosing and / or reduced dosage levels may be possible.

[0266] In some embodiments, the disease, disorder, condition or symptom treated or prevented is allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, skeletal / musculoskeletal disease, cardiovascular disease, neurological disease, renal disease, metabolic disease, immune disease, gene / genetic disease, fertility-related disorder, infectious disease or cancer. In certain embodiments, the disease or condition treated or prevented is atopic dermatitis, allergic dermatitis, food allergy, osteoarthritis pain, perioperative pain, dental pain, cancer pain, arthritis, anemia, obesity or diabetes.

[0267] Antibodies can be used not only to treat or prevent disease, but also to regulate normal biological functions, such as managing fertility or behavior.

[0268] diagnosis

[0269] One or more polypeptides disclosed herein can also be used for various diagnostic purposes, such as determining whether a cat suffers from any particular disease or condition. In some embodiments, the one or more polypeptides may include a binding domain. The binding domain can specifically bind to a protein, subunit, domain, motif and / or epitope (e.g., a marker of a cancer cell) as described herein. In some embodiments, the one or more polypeptides also include a labeling group. Typically, labeling groups are divided into multiple categories according to the assays they will be detected in: a) isotopic labels, which can be radioisotopes or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by a second reporter molecule (e.g., leucine zipper pair sequences, binding sites of second antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is coupled to the antibody by spacer arms of various lengths to reduce potential steric hindrance. Various methods of labeling proteins are known in the art and can be used in the practice of the present invention.

[0270] In some embodiments, the labeling group is a probe, a dye (e.g., a fluorescent dye), or a radioisotope (e.g., 3 H. 14 C. 22 Na, 36 Cl, 35 S. 33 P or 125 I).

[0271] Specific labels may include optical dyes, including but not limited to chromophores, phosphors and fluorophores, the latter being specific in many cases. Fluorophores may be "small molecule" fluorophores or proteinaceous fluorophores.

[0272] Fluorescent label can be any molecule that can be detected by its intrinsic fluorescence property. Suitable fluorescent label includes but is not limited to fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescent yellow, cascade blue (Cascade BlueJ), Texas red (Texas Red), IAEDANS, EDANS, BODIPYFL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dye (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 (CascadeBlue), Cascade Yellow and R-phycoerythrin (PE) (Molecular Biotechnology, Inc., New York, NY). Probes, Eugene, Oreg.), FITC, rhodamine and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable optical dyes, including fluorophores, are described in Richard P. Haugland's Molecular Probes Handbook, which is incorporated herein by reference in its entirety.

[0273] Suitable proteinaceous fluorescent markers also include, but are not limited to, 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 No. 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), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607), and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / 49019; U.S. Pat. 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, 5,925,558). All of the above-cited references in this paragraph are expressly incorporated herein by reference in their entirety.

[0274] Determination

[0275] Fc γ RI and FcγRIII binding:

[0276] Binding to FcγRI and FcγRIII is a measure of the ability of an antibody to mediate ADCC. To assess this property of an antibody, assays measuring binding of the antibody to FcγRI and FcγRIII can be performed using methods known in the art.

[0277] C1q binding:

[0278] Binding to the first component of complement, CIq, is a measure of the ability of an antibody to mediate complement dependent cytotoxicity (CDC).To assess this property of an antibody, assays measuring antibody binding to CIq can be performed using methods known in the art.

[0279] half life:

[0280] Methods for measuring antibody half-life 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 cat model) and measuring the level of the antibody 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) may be ineffective for mouse FcRnα chains and express human FcRnα transgenes (e.g., under the control of a constitutive promoter). Human FcRnα chains can be paired with mouse β2 microglobulin in vivo to form functional chimeric FcRn heterodimers.

[0281] Example

[0282] Example 1: Generation of NNK saturation mutagenesis libraries at selected positions and analysis of individual variants

[0283] The wild-type (wt) sequence of the CH2 and CH3 domains of feline IgG1a was synthesized (SEQ ID NO: 1) and used as a template for NNK mutagenesis. The NNK saturation mutagenesis method is an effective strategy for producing all 20 possible amino acids at the desired position (Hogrefe et al., Biotechniques. 33: 1158-1165

[2002] ). A single NNK library at positions 252, 428 and 434 (EU numbering) was generated. The NNK (N = A / C / G / T, K = G / T) primers at the specified position were used with the QuikChange site-directed mutagenesis kit (Agilent). The PCR product was subcloned into the GenScript FASEBA plasmid, transformed into Escherichia coli, and the presence of the variant was verified by sequencing. Downstream of the CH3 domain is a SASA (single domain antibody against serum albumin) tag (Zhang, J.; Wu, S.; Liu, J. Methods and systems for increasing proteinstability. Patent application number: US2013 / 0129727 A1), which has a pM affinity for albumin. The SASA antibody is able to capture Fc onto the surface of the sensor chip described below. The PelB (pectate lyase B) signal peptide is located at the N-terminus to promote Fc secretion into the culture medium. The expression of CH2-CH3 proteins is regulated by the Lac promoter. For the variants, the supernatant from the conditioned medium was analyzed for binding to feline FcRn (GenBank KF773786 [IgG receptor FcRn large subunit p51] and European nucleotide archive AY829266.1 [feline β2 microglobulin]) at pH 6.0 using surface plasmon resonance (SPR).

[0284] Supernatants from 90 individual transformants from each library were assayed for binding to feline FcRn using the Biacore method at pH 6.0 as described below.

[0285] For SPR analysis using Biacore 8K, bovine serum albumin (BSA) was fixed to a CM5 sensor chip. The sensor chip surfaces of flow cells 1 and 2 were activated by freshly mixed 50mmol / L N-hydroxysuccinimide and 200mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride for 420s (10 μL / min). Then, BSA diluted in 10mM sodium acetate (pH 4.5) was injected into flow cell 2 to achieve conjugation, while flow cell 1 was set to blank. After the amine coupling reaction, 1mM ethanolamine hydrochloride was injected for 420s to block the remaining active coupling sites on the chip surface. The running buffer used for the binding experiment was HBS-EP (10mM HEPES, 500mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 5.5) and it was run at 25°C. Supernatants from the variants were injected onto the chip surface and captured via the SASA tag onto immobilized BSA for 60 seconds. 200 nM feline FcRn was injected for 120 seconds and dissociation was completed with running buffer for 120 seconds. The flow rate for the stationary phase of BSA was 10 μl / min, and the flow rate for the binding and dissociation phases was 30 μl / min. All data were processed using Biacore 8K Evaluation Software version 1.1. For Biacore sensorgrams see Figure 1 and 2 .

[0286] The tested variants showed increased binding affinity to feline FcRn at pH 6.0 when compared to wild-type feline IgG1a Fc (SEQ ID NO: 1).

[0287] It is found that NNK mutagenesis at amino acid positions 252, 428 and 434 produces a mutant that increases the combination with FcRn at pH 6.0. The sequencing of all 90 clones produced at these 3 positions indicates that the following 6 clones, i.e. S252H, S428E, S428C, S428F, S428W and S434Y, are not produced. All other amino acid substitutions at these positions do not produce benefits to the test. The results are summarized in Table 3 below.

[0288] Table 3: Binding kinetics of IgG variants to FcRn.

[0289]

[0290] Example 2: Scanning mutagenesis of feline IgG1a Fc

[0291] A phage display library approach was used to identify feline IgG1a Fc variants with increased affinity for FcRn at pH 6.0. Feline IgG1a Fc (Kanai et al., 2000. Vet. Immunol. Immunopathol. 73:53) was synthesized by Twist Bioscience with variants at 55 different positions ( Figure 4 ). At each mutation position, 8 possible amino acids are substituted. These amino acids are 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). The Fc DNA library is designed to have an average of two variants per Fc molecule. The complexity of the library is 95,040 combinations using the following formula: nCr=n! / r! *(nr)!, where n represents the number of sites and r represents the number of variants per molecule. Fc variants with desired site-specific mutations are printed as mutagenic oligonucleotides on the Twist silicon-based platform.

[0292] Assembly PCR was then used to assemble the oligonucleotides to generate a full-length Fc gene fragment library. The assembled Fc gene fragment library was then cloned into the Sfi cleavage site in the pADL-22c phagemid vector from Antibody Design Labs. The cloned DNA library was transformed into electrocompetent TG1 E. coli cells to generate 8 x 10 10 The experimental diversity of variants was obtained. Phagemid-transformed E. coli cells were then co-transfected with M13K07 helper phage to generate a phage library, which was used for protein-based panning. The library was resuspended in 20 mM MES buffer (pH 6.0), 0.05% tween 20 and 3% milk.

[0293] The quality of the library was determined by picking 96 random phage clones and sequencing them by the Sanger method. The number of mutants for each phage is shown in Table 4 below.

[0294] Table 4: Number of mutants per phage.

[0295] Number of mutations Number of clones 0 5 1 19 2 44 3 19 4 5 5 1 6 2

[0296] For the first phage selection, protein A capture step was used to remove any Fc variants that lost protein A binding. For this selection, the phage library was captured on protein A beads and washed with PBS (pH 7.4). Phage was eluted with 0.1M glycine at pH 2.7, and the pH was immediately neutralized with 1M Tris-HCl (pH 7.5). Neutralized phage was precipitated with polyethylene glycol / NaCl and centrifuged. The precipitated phage was resuspended in 20mM MES (pH 6.0), 0.05% tween 20, 3% milk.

[0297] The following phage selection was based on the protocol described by Borrok et al., 2015, J Biol. Chem., 290: 4282. Briefly, Nunc96 multiwell plates were coated with Neutravidin and then blocked with 5% bovine serum albumin, PBS (pH 7.4). Biotinylated feline FcRn (FCN-F82W3, Acrobiosystems) was fixed in the wells at a concentration of 0.31 μg / ml in PBS at pH 6.0. The phage library in PBS at pH 6.0 was incubated with fixed feline FcRn and then washed with PBS (pH 6.0), 0.05% tween 20, 0.3M NaCl. Phages were eluted with PBS at pH 7.4 by incubation at 37°C for 30 minutes. Eluted phage were depleted with 0.31 μg / ml feline FcRn at pH 7.4. Unbound phage were amplified in TG1 cells. The exact conditions and results for each round are shown in Table 5 below.

[0298] Table 5: Washing conditions and depletion times used for phage selection.

[0299]

[0300] A total of 768 phage clones were isolated from the output of the third round of selection, and 768 phage clones were isolated from the fourth round of selection. The clones were sequenced by next generation sequencing using Illumina MiSeq.

[0301] The sequencing results revealed that a large number of clones (see table below) contained substituted tyrosine at position 252 and substituted tyrosine or phenylalanine at position 434, indicating that some substitutions contained residues other than the expected eight amino acid substitutions. The unique variants were reformatted into IgG using the variable domains previously described by Gearing et al. (2016, J Vet Intern Med, 30: 1129). Small amounts of prepared plasmid DNA were transfected into Expi293 cells with ExpiFectamine 293 transfection reagent. IgG variants were purified from conditioned medium with protein A chromatography and formulated in 43 mM sodium citrate, 130 mM sodium bicarbonate (pH 6.0).

[0302] To determine the affinity of the IgG variants for feline FcRn, binding kinetics were determined using a Carterra instrument. Antibodies (approximately 5 μg / ml) were amine coupled to a HC30M sensor chip by EDC / NHS activation and then quenched with ethanolamine HCl.

[0303] Several concentrations (333nM, 111nM, 37nM, 12.3nM, 4.1nM, 1.37nM, 0.45nM) of feline FcRn (FCN-F82W3, Acrobiosystems) were flowed over a sensor chip in HBSTE (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween-20), 0.5% bovine serum albumin, pH 6.0 to determine the kinetics at pH 6.0. This 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.

[0304] The feline FcRn binding kinetics of the IgG variants are shown in Table 6 below.

[0305] Table 6: Binding kinetics of IgG Fc variants to FcRn.

[0306]

[0307] Example 3: Scanning mutagenesis of feline IgG1a Fc

[0308] A set of additional anti-nerve growth factor (NGF) feline IgG1a antibody variants were synthesized by Twist Bioscience using variable domains previously described by Gearing et al. (2016, J Vet Intern Med, 30: 1129). Tables 8 and 9 below list modifications to the Fc regions of these antibodies compared to the reference sequences of the wild-type feline IgG1a Fc domains described by Kanai et al. (2000, Vet. Immunol. Immunopathol. 73: 53). Feline IgG1a constructs were subcloned into mammalian expression vectors and transfected into Expi293 cells with ExpiFectamine 293 transfection reagent. IgG1a Fc variants were purified from conditioned medium using protein A chromatography and formulated in 43mM sodium citrate, 130mM sodium bicarbonate (pH 6.0). The binding affinity of IgG Fc variants to feline FcRn was then determined using a Carterra instrument. Antibody variants (about 5 μg / ml) were amine coupled to HC30M sensor chips by EDC / NHS activation and then quenched with ethanolamine HCl. 333nM, 111nM, 37nM, 12.3nM, 4.1nM, 1.37nM or 0.45nM of feline FcRn (FCN-F82W3, Acrobiosystems) were passed through the sensor chip in HBSTE (10mM HEPES, 150mM NaCl, 3mM 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, wherein the pH of the HBSTE buffer was adjusted to 7.4.

[0309] The feline FcRn binding kinetics of the IgG variants are shown in Table 7 below.

[0310] Table 7: Binding kinetics of IgG Fc variants to FcRn – II

[0311]

[0312] A list of amino acid substitutions identified as increasing binding of feline IgG1a Fc variants to feline FcRN is provided below in Table 8:

[0313] Table 8: Summary of amino acid substitutions of feline IgG1a Fc variants showing increased binding affinity to feline FcRn

[0314]

[0315]

[0316] Example 4: Pharmacokinetic studies of feline IgG1a Fc variants with increased FcRn binding and wild-type feline IgG1a.

[0317] Pharmacokinetic (PK) studies were performed with 12 male and female cats.Feline IgG1aFc variants, including antibodies carrying a wild-type feline IgG1a Fc domain, were prepared using anti-NGF variable domains as previously described by Gearing et al. (2016, J Vet Intern Med, 30: 1129).

[0318] Animals were randomly divided into six groups, each with one male and one female. A single intravenous dose of 2 mg / kg of the antibody was administered to each animal. Approximately 0.5 ml of whole blood was collected at the following time points: 0 hours (pre-dose), 4 hours after injection and 1 day, 2 days, 4 days, 6 days, 10 days, 14 days, 18 days, 22 days, 30 days, 34 days, 38 days, 42 days. Serum was separated from the whole blood, and the presence of antibodies was determined by ELISA specific for anti-NGF antibodies. The serum concentrations of six anti-NGF monoclonal antibody (mAb) variants were described using a two-compartment pharmacokinetic (PK) model with linear clearance using nonlinear mixed effects (NLME) modeling ( Fig.46 ). Population parameters were estimated using the stochastic approximation to maximum expectation (SAEM) algorithm implemented in Monolix Suite 2019R1 (Monolix version 2019R1. Antony, France: Lixoft SAS, 2019). Each parameter was modeled as a random variable with a log-normal distribution. mAb-typical coefficients (β BW,Cl =0.75, β BW,V1 =β BW,V2 =1,β BW,Q =2 / 3), PK parameters depend on body weight (BW). Single parameters The equation (Dong et al. 2011. Clin Pharmacokinet, 50:131) is:

[0319]

[0320] in is the population typical parameter, η is a random variable with mean 0 and standard deviation ω, BW i is the weight of animal i, and BW ref The reference weight is 2kg.

[0321] Antibody variants were differentiated according to the following equation by using categorical covariates for clearance, intercompartmental exchange coefficient, and peripheral volume:

[0322]

[0323] where if the individual variant covariates are in the categories, then Ω i =1; otherwise Ω i = 0. Wild-type (WT) mAb variants were used as reference.

[0324] exist Fig.47 The observed individual serum concentrations of variants WT, S252W, S428Y, S428Y+L309V, S428Y+Q311V, and S428Y+S254R are shown in Figure 5, with two animals for each variant.

[0325] The estimated PK parameters are given below in Table 9. The predicted serum concentration curves for monoclonal antibodies carrying wild-type (WT) IgG1a Fc or the IgG1a Fc variants S252W, S428Y, S428Y+L309V, S428Y+Q311V, and S428Y+S254R for a typical 2 kg cat receiving a single IV dose of 2 mg / kg are shown in Table 9. Fig.48 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 feline IgG Fc domains that confer enhanced in vitro FcRn binding are also sufficient to extend the in vivo half-life of these IgG Fc variants.

[0326] Table 9: PK parameter estimates for cats weighing 2 kg.

[0327]

[0328] Example 5: Binding kinetics of feline IgG1a variants to feline FcRn using the C1 biosensor

[0329] 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 β2 microglobulin]) were evaluated at pH 5.9. EU numbering was used to identify positions ( Figure 4 In this study, feline Fc variants carrying single or combined amino acid substitutions were synthesized as feline IgG1a (Kanai et al., 2000, Vet. Immunol. Immunopathol. 73:53) using variable domains 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. Conditioned medium was purified using protein A chromatography.

[0330] For feline FcRn binding experiments, all assays were completed on a Biacore 8K+ system at 25°C. In this set of experiments, antibodies were immobilized onto S-series C1 sensor chips using standard amine coupling reagents. A mixture of 200mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50mmol / L N-hydroxysuccinimide (NHS) was injected for 420 seconds to activate the surface. Then, the antibody was injected for 120 seconds at a concentration of 0.5 to 2μg / ml in 10mM sodium acetate pH 5.0. Finally, 1M ethanolamine was injected for 420 seconds. The running buffer was 1X PBS-P+ (Cytiva, catalog number 28995084) adjusted to pH 5.9.

[0331] To evaluate the binding affinity of feline IgG1a variants to feline FcRn at pH 5.9, a concentration range of 1.56-2000 nM of feline FcRn was selected and injected in single cycle mode. The concentrations of feline FcRn tested for each variant are shown in Table 10 below.

[0332] Table 10: Concentration of feline FcRn used for each feline IgG1a variant

[0333] Variants FcRn concentration [nM] wild type 31.25,125，500，2000 Feline IgG1a-S252W 1.56，6.25,25，100 Feline IgG1a-L309V 31.25,125，500，2000 Feline IgG1a-Q311V 31.25,125，500，2000 Feline IgG1a-S428Y 7.81，31.25，125，500 Feline IgG1a-S428Y-Q311V 7.81，31.25，125，500 Feline IgG1a-S428Y-S254R 7.81，31.25，125，500 Feline IgG1a-S428Y-L309V 7.81，31.25，125，500 Feline IgG1a-S428Y-Q311V-Y286E 1.56，6.25。25,100 Feline IgG1a-S428Y-Q311V-E380T 1.56，6.25，25,100 Feline IgG1a-S428Y-1309V-T286E 1.56，6.25，25,100 Feline IgG1a-S428Y-1309V-E380T 1.56，6.25,25，100

[0334] Four concentrations of each antibody were injected at 5 μl / min for 90 seconds, followed by 180 seconds of dissociation. Each concentration series was injected three times in this format, with at least three buffer-only cycles for appropriate reference subtraction. The surface was regenerated by two injections of 1X PBS-P+ (pH 7.4) for 30 seconds, followed by a 60 second wait command. Three priming cycles were included to stabilize the surface prior to analysis.

[0335] Data were evaluated using Insight Evaluation software by fitting to a 1:1 kinetic interaction model or by fitting to steady-state affinity. Acceptable parameters were selected using quality metrics including U and T values. For kinetic rate constants, U values ​​less than 15 were considered acceptable, while for kinetic rate constants, T values ​​greater than 100 were considered acceptable. In the case where these values ​​were outside of the ranges, steady-state affinity parameters were considered acceptable.

[0336] The kinetic data for the feline IgG1a variants are shown in Table 11 below and the sensorgrams are shown in Figures 49A-49L middle.

[0337] Table 11: Binding kinetics of feline IgG1a variants to feline FcRn

[0338] Variants ka kd Ko Methods for fitting data wild type 1.06E-06 Steady-state affinity Feline IgG1a-S252W 1.06E+06 4.24E-03 4.01E-09 1∶1 kinetic interaction model Feline IgG1a-L309V 4.27E-07 Steady-state affinity Feline IgG1a-Q311V 3.34E-07 Steady-state affinity Feline IgG1a-S428Y 9.02E+05 6.42E-02 7.18E-08 1∶1 kinetic interaction model Feline IgG1a-S428Y-Q311V 8.27E+05 2.66E-02 3.22E-08 1∶1 kinetic interaction model Feline IgG1a-S428Y-S254R 1.15E+06 9.31E-02 8.12E-08 1∶1 kinetic interaction model Feline IgG1a-S428Y-L309V 8.80E+05 3.10E-02 3.53E-08 1∶1 kinetic interaction model Feline IgG1a-S428Y-Q311V-T286E 1.27E+06 7.78E-03 5.11E-09 1∶1 kinetic interaction model Feline IgG1a-S42BY-Q311V-E380T 1.60E+06 3.22E-02 2.01E-08 1∶1 kinetic interaction model Feline IgG1a-S428Y-L309V-T286E 1.34E+06 7.02E-03 5.26E-09 1∶1 kinetic interaction model Feline IgG1a-S428Y-L309V-E380T 1.63E+06 3.34E-02 2.06E-08 1∶1 kinetic interaction model

[0339] Example 6: Pharmacokinetic study of feline IgG1a Fc variants with two or three Fc substitutions and wild-type feline IgG1a

[0340] Pharmacokinetic (PK) studies were performed with 14 male and female cats. Feline IgG1aFc variants (including antibodies carrying wild-type feline IgG1a Fc domains) were prepared using anti-NGF variable domains as previously described by Gearing et al. (2016, J Vet Intern Med, 30: 1129). Feline IgG1a variants tested in this study included: S428Y+L309V, S428Y+Q311V, S428Y+Q311V+T286E, S428Y+L309V+E380T, S428Y+Q311V+E380T, S428Y+L309V+T286E, wild type.

[0341] Animals were randomly divided into seven groups, with one male and one female in each group. A single intravenous dose of 2 mg / kg of antibody was administered to each animal. Approximately 0.5 ml of whole blood was collected at the following time points: 0 hours (before administration), 4 hours after injection and 1 day, 2 days, 4 days, 6 days, 10 days, 14 days, 18 days, 22 days, 30 days, 34 days, 38 days, 42 days. Serum was separated from whole blood, and the presence of antibodies was determined by ELISA specific for feline anti-NGF antibodies. The serum concentrations of seven anti-NGF monoclonal antibody (mAb) variants were described using a two-compartment pharmacokinetic (PK) model with linear clearance using nonlinear mixed effects (NLME) modeling (population parameters were estimated using the maximum expected value stochastic approximation (SAEM) algorithm implemented in Monolix Suite 2019R1 (Monolix version 2019R1. Antony, France: Lixoft SAS, 2019)). Serum concentrations of S428Y+Q311V, S428Y+L309V, and wild-type from the PK study described in Example 4 were modeled as described above and included in these calculations. Each parameter was modeled as a random variable with a log-normal distribution. The mAb-typical coefficients (β BW,Cl =0.75, β BW,V1 =β BW,V2 =1,β BW,Q =2 / 3), PK parameters depend on body weight (BW). Single parameters The equation (Dong et al. 2011. Clin Pharmacokinet, 50:131) is:

[0342]

[0343] in is the population typical parameter, η is a random variable with mean 0 and standard deviation ω, BW i is the weight of animal i, and BW ref The reference weight is 2kg.

[0344] Antibody variants were differentiated according to the following equation by using categorical covariates for clearance, intercompartmental exchange coefficient, and peripheral volume:

[0345]

[0346] where if the individual variant covariates are in the categories, then Ω i =1; otherwise Ω i = 0. Wild-type (WT) mAb was used as reference.

[0347] The estimated PK parameters are given in Table 12 below.

[0348] Table 12: PK parameter estimates for cats weighing 2 kg.

[0349]

[0350] Fig.50 The observed individual serum concentrations for wild type and Fc variants S428Y+Q311V, S428Y+Q311V+T286E, S428Y+Q311V+E380T, S428Y+L309V, S428Y+L309V+T286E, and S428Y+L309V+E380T are shown in Figure 5, with two animals for each variant.

[0351] The predicted serum concentration curves of monoclonal antibodies carrying 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 for a typical 2 kg cat receiving a single IV dose of 2 mg / kg are shown in Fig.51 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 feline IgG Fc domains that confer enhanced FcRn binding in vitro are also sufficient to extend the in vivo half-life of these IgG Fc variants.

[0352] Other Implementations

[0353] While the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of 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 comprises (i) an amino acid substitution at a position corresponding to amino acid position 428 of a wild-type feline IgG, wherein the amino acid substitution is S428Y, and (ii) an amino acid substitution at at least one position selected from the group consisting of: (a) The position corresponding to amino acid position 286 of wild-type feline IgG; (b) the position corresponding to amino acid position 301 of wild-type feline IgG; (c) the position corresponding to amino acid position 309 of wild-type feline IgG; (d) a position corresponding to amino acid position 311 of wild-type feline IgG; and (e) the position corresponding to amino acid position 377 of wild-type feline IgG; wherein the amino acid positions are based on EU numbering, wherein the polypeptide comprises 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, wherein the polypeptide has increased binding affinity for feline FcRn when compared to the Fc domain of said wild-type feline IgG, and wherein the polypeptide binds to feline FcRn at a higher level at pH 6.0 than at pH 7.

4.

2. The polypeptide of claim 1, wherein the polypeptide comprises one or more amino acid substitutions selected from the group consisting of: (a) a position corresponding to amino acid position 286 of wild-type feline IgG, wherein the amino acid substitution is T286D; (b) a position corresponding to amino acid position 301 of wild-type feline IgG, wherein the amino acid substitution is R301L; (c) a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V or L309E; (d) a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Q311L; and (e) a position corresponding to amino acid position 377 of wild-type feline IgG, wherein the amino acid substitution is I377Y.

3. The polypeptide of claim 2, wherein the polypeptide comprises an amino acid substitution at a position corresponding to amino acid position 309 of wild-type feline IgG, wherein the amino acid substitution is L309V. 4 . The polypeptide of claim 1 , wherein the wild-type feline IgG is a feline IgG1a comprising an Fc domain having the amino acid sequence of SEQ ID NO:

1. 5 . The polypeptide of claim 1 , wherein the wild-type feline IgG is a feline IgG1b comprising an Fc domain having the amino acid sequence of SEQ ID NO:

2.

6. The polypeptide of claim 1, wherein the wild-type feline IgG is a feline IgG2 comprising an Fc domain having the amino acid sequence of SEQ ID NO:

3.

7. The polypeptide of claim 1, further comprising a polypeptide selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-IR, IL-4R, GLP-1 receptor agonist, and thrombopoietin binding peptide.

8. A pharmaceutical composition comprising (i) the polypeptide of claim 1, and (ii) a pharmaceutically acceptable excipient.

9. One or more nucleic acids encoding the polypeptide of claim 1.

10. One or more expression vectors comprising one or more nucleic acids according to claim 9.

11. A host cell comprising one or more nucleic acids according to claim 9 or comprising one or more expression vectors containing the one or more nucleic acids.

12. A method for preparing a polypeptide, the method comprising: (a) providing one or more nucleic acids as described in claim 9; (b) expressing the one or more nucleic acids in host cell culture, thereby producing the polypeptide; as well as (c) collecting the polypeptide produced in (b) from the host cell culture.

13. The polypeptide of claim 1, further comprising a binding domain, wherein the binding domain comprises a ligand binding domain of a feline receptor protein.

14. The polypeptide of claim 1, further comprising a binding domain, wherein the binding domain comprises a nanobody.

15. The polypeptide of claim 1, further comprising a binding domain, wherein the binding domain comprises an extracellular domain of a feline receptor protein.

16. A polypeptide comprising a feline IgG Fc region variant, wherein the polypeptide comprises: (a) an amino acid substitution at a position corresponding to amino acid position 252 of wild-type feline IgG, wherein the amino acid substitution is S252Y; and (b) an amino acid substitution at a position corresponding to amino acid position 311 of a wild-type feline IgG, wherein the amino acid substitution is Q311V, Q311L, Q311R or Q311K; wherein the amino acid positions are based on EU numbering, wherein the polypeptide comprises 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, wherein the polypeptide has increased binding affinity for feline FcRn when compared to the Fc domain of said wild-type feline IgG, and wherein the polypeptide binds to feline FcRn at a higher level at pH 6.0 than at pH 7.

4.

17. A polypeptide comprising a feline IgG Fc region variant, wherein the polypeptide comprises: (i) an amino acid substitution at a position corresponding to amino acid position 428 of a wild-type feline IgG, wherein the amino acid substitution is S428L; and (ii) an amino acid substitution at at least one position selected from the group consisting of: (a) an amino acid substitution at a position corresponding to amino acid position 286 of wild-type feline IgG, wherein the amino acid substitution is T286E; (b) an amino acid substitution at a position corresponding to amino acid position 311 of wild-type feline IgG, wherein the amino acid substitution is Q311V or Q311K; (c) an amino acid substitution at a position corresponding to amino acid position 312 of wild-type feline IgG, wherein the amino acid substitution is D312T; (d) an amino acid substitution at a position corresponding to amino acid position 377 of wild-type feline IgG, wherein the amino acid substitution is I377Y; and (e) an amino acid substitution at a position corresponding to amino acid position 383 of wild-type feline IgG, wherein the amino acid substitution is I383L; wherein the amino acid positions are based on EU numbering, wherein the polypeptide comprises 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, wherein the polypeptide has increased binding affinity for feline FcRn when compared to the Fc domain of said wild-type feline IgG, and wherein the polypeptide binds to feline FcRn at a higher level at pH 6.0 than at pH 7.4.

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