Compositions and methods for modifying antibody effector functions

Abstract

Feline, canine, and equine antibody variants and uses thereof, as well as compositions and methods for improving therapeutic monoclonal antibodies by modulating immune effector functions and antibody variants using those methods and compositions.

Description

FIELD OF THE DISCLOSURE

[0001] The present application relates to feline, canine, and equine antibody variants and uses thereof, as well as compositions and methods for modifying therapeutic veterinary monoclonal antibodies by modulating immune effector functions and antibody variants using those methods and compositions. BACKGROUND OF THE INVENTION:

[0002] Feline, canine, and equine IgG monoclonal antibodies (mAbs) and modified antibodies can be effective therapeutics in veterinary medicine. However, there has been limited work done to improve the characteristics of feline, canine, and equine IgGs.

[0003] Feline IgG monoclonal antibodies (mAbs) are being developed as effective therapeutics in veterinary medicine. Several years ago, feline IgG subclasses were identified and characterized (Strietzel et al., 2014, Vet Immunol Immunopathol., vol. 158(3-4), pages 214-223). Canine IgG monoclonal antibodies (mAbs) are also being developed as effective therapeutics in veterinary medicine. Several years ago, four canine IgG subclasses were identified and characterized (Bergeron et al., 2014, Vet Immunol Immunopathol., vol. 157(1-2), pages 31-41). The seven equine IgG subclasses are well known in the art and are fully described in, for example, Wagner et al., 2004, J. Immunol., vol. 173, pages 3230-3242; Wagner, 2006, Dev. Comp. Immunol., vol. 30, pages 155-164; Sheoran et al., 2000, Am. J. Vet. Res., vol 61, pages 1099-1105; and Wagner et al., 1998, Immunobiology, vol. 199 (1), pages 105-118

[0004] Through a recycling mechanism, the neonatal Fc receptor (FcRn) prolongs the halflife of an IgG in a pH-dependent interaction with its fragment crystallizable (Fc) region. Specifically, the Fc region spanning the interface of CH2 and CH3 domains interacts with the FcRn on the surface of cells to regulate IgG homeostasis. This interaction is favoured by an acidic interaction after IgG pinocytosis and thus IgG is protected from degradation. The endocytosed IgG is then recycled back to the cell surface and released into the blood stream at a slightly alkaline pH thereby maintaining sufficient serum IgG for proper function. Accordingly, the pharmacokinetic profile of IgGs depend on the structural and functional properties of their Fc regions.

[0005] Fc regions are also responsible for antibody immune effector functions, such as complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). These effector functions rely on the interactions of antibody Fc regions with FcyRs. Therefore, engineering Fc regions to tune their interactions with FcyRs and Clq has emerged as a promising approach for enhancing the activity of therapeutic antibodies.

[0006] Accordingly, there exists a need for novel IgG Fc region mutations to improve various characteristics of feline, canine, and equine IgGs for use as safe therapeutic mAbs, specifically mutations that reduce, abolish or enhance immune effector function in cell-based CDC, ADCC, and ADCP assays. SUMMARY OF THE INVENTION:

[0007] The ability to mediate cytotoxic and phagocytic effector functions are potent mechanisms by which antibodies destroy targeted cells. The Fc region links the recognition domain of antibodies to these effector functions through an interaction with Fc receptors and ligands. Manipulation of these effector functions by alteration of the Fc region has important implications in the treatment of numerous medical conditions, for example cancer, autoimmune diseases and infectious diseases.

[0008] The Fc domain can be modified to obtain beneficial gain-of-function modifications, such as enhancing immune effector function on therapeutic mAbs targeting cancer cells, bacteria or viruses in order to augment killing / clearing of the cells / virus particles. However, in some cases, it can be beneficial to reduce or abolish antibody Fc function. These situations include antibodies that are used as receptor agonists to crosslink receptors and induce signaling, (receptor antagonists to block receptor:ligand interactions to prevent signaling, or drug delivery vehicles to deliver drugs or a drug to antigen-expressing target cells. In these instances, Fc engagement of receptors on effector cells or engagement of Cl q is not wanted, because it can lead to undesired killing of biologically-important cells expressing the receptor or recruitment of drug-conjugated antibodies to off-target cells. Effector function null IgG is thus important for a number of antibody mechanisms of action in a wide range of disease areas. In addition, this is also important in Fc-fusion proteins and alternative antibody formats such as bi- or multi-specific antibodies Several strategies to manipulate FcyRs binding of human antibodies and complement protein Clq binding, including changes to Fc sequence and glycosylation, are described in Saunders, Front Immunol., Article 1 296, Volume 10, 7 June 2019.

[0009] The invention provides modified canine, modified feline, and modified equine Fc regions (compared to wt) with advantageous properties. It is known that canine the IgGl and IgG4 subclasses are effector function deficient, while IgG2 and IgG3 are effector function proficient. However, IgG2 is the predominant IgG currently being used in therapeutic antibodies due to its other characteristics such as protein A binding capacity and relatively long half-life compared to other canine IgGs. Therefore, generating effector function null canine IgG2 is attractive for certain therapeutic areas. Similarly, feline IgGl has been the preferred IgG subclass for the development of therapeutic mAbs, such that generating effector function null feline IgGl is also attractive for certain therapeutic areas. There are seven reported equine IgG subclasses, IgGl - IgG7, each with different characterization Protein A binding, pharmacokinetics, and effector function profiles, although IgG4 and IgG7 are very similar. All seven equine IgG subclasses possess some capability of triggering effector function(s), with the possible exception of IgG2. Thus, the different subclasses may require different sets of mutations to reduce effector functions.

[0010] Accordingly, the invention relates to modified recombinant feline, canine, and equine IgGs that exhibit desired characteristics, relative to wild-type IgGs. Specifically, the inventors of the instant application have found that substituting an amino acid residue at positions described herein with another amino acid surprisingly and unexpectedly produced a desired effect. In an exemplary embodiment, the unexpected, desired effects include, but are not limited to, enhanced affinity to FcRn, reduced complement-dependent cytotoxicity (CDC); reduced antibodydependent cellular cytotoxicity (ADCC); reduced antibody-dependent cellular phagocytosis (ADCP); reduced or enhanced binding to Fc gamma receptor (bFcyR); or a combination thereof.

[0011] In one aspect, an Fc region of feline IgGla may comprise one or more substitutions at positions 234, 235, 235, 236, 237, 237, 239, 329, 330, 331, 345, 265, 267, 268, 270, 270, 297, 298, 322, 324, 329, 330, 331, 332, 333, and / or 334 using EU Index numbering as in Kabat.

[0012] In another aspect, an Fc region of feline IgGla may comprise one or more substitutions selected from among M234A, L235A, L235Y, G236A, G237A, G237W, S239D, P329G, P329S, S330A, P331A, P331S, E345R, D265A, G267E, P268F, D270G, D270S, N297G, S298A, K322A, N324T, P329G, S330A, S330L, P331A, P331S, I332E, E333A, and R334A using EU Index numbering.

[0013] In another aspect, various combinations of substitutions in a Fc region of feline IgGla can include one or more of the following, referred to herein by short designations: WIN (M234A, L235A, G237A), GSP (P329G), PSS (P331S), GAP (P329G, S33OA), DANG (D265A, N297G), KA (K322A), PA (P331A), KAPA (K322A, P331A), WIN PA (M234A, L235A, G237A, P331A), WIN_KA (M234A, L235A, G237A, K322A), WIN_KAPA (M234A, L235A, G237A, K322A, P331A), WIN GAP (M234A, L235A, G237A, P329G, S330A), DANG-GAP (D265A, N297G, P329G, S33OA), WIN DANG (M234A, L235A, G237A, D265A, N297G), WIN DANG GAP (M234A, L235A, G237A, D265A, N297G, P329G, S330A), DANG PSS (D265A, N297G, P331S), WIN_PSA (M234A, L235A, G237A, P331A), WINDANGPSS (M234A, L235A, G237A, D265A, N297G, P331S), WIN_PSS (M234A, L235A, G237A, P331S), WINGSA (M234A, L235A, G237A, P329G, P331A), WIN_GSP (M234A, L235A, G237A, P329G), WIN_GSS (M234A, L235A, G237A, P329G, P331S), WIN_SAS (M234A, L235A, G237A, P329S, S330A, P331S), AAA (S298A, E333A, R334A), DLE (S239D, S330L, I332E), DE (S239D, I332E), DAE (G236A, S239D, I332E), YWA (L235Y, G237W, S298A), EFT (G267E, P268F, N324T), D270G (D270G), D270S (D270S), and E345R (E345R).

[0014] In another aspect, an Fc region of feline IgGlb may also comprise one or more substitutions or combinations of substitutions in positions corresponding to the positions in IgGla described above. Of course, one will also recognize that canine and equine IgG of any subclass could contain one or more corresponding substitutions or combinations of substitutions in positions corresponding to the positions in feline IgGla described above and that the short designations used above may be used in identifying those corresponding substitutions.

[0015] In another aspect, an Fc region of canine IgG2 may comprise one or more mutations, such at locations including 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 294, 295, 296, 297, 298, 299, 300, 301, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 422 423, and / or 441, using EU Index numbering as in Kabat.

[0016] In another aspect, an Fc region of canine IgG2 may comprise one or more substitutions listed in Table 5, and / or substitutions selected from among M234A, L235A, L235Y, G236A, G237A, G237W, S239D, D265A, D270G, D270S, N297G, G298A, K322A, P329G, S330A, S330L, P331 A, I332E, E333A, R334A, using EU Index numbering as in Kabat.

[0017] In another aspect, an Fc region of canine IgG2 may comprise various combinations of substitutions in a Fc region of canine IgG2, which can include one or more of the following, referred to herein by short designations: GSP (P329G), GAP (P329G, S330A), DANG (D265A, N297G), DANG-GAP (D265A, N297G; P329G, S330A), WIN (M234A, L235A, G237A), KA (K322A), PA (P331A), KAPA (K322A, P331A), WIN_PA (M234A, L235A, G237A, P331A), WIN KA (M234A, L235A, G237A, K322A), WIN_KAPA (M234A, L235A, G237A, K322A, P331A), WINDANGGAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A), AAA (G298A, E333A, R334A), DE (S239D, I332E), DLE (S239D, S33OL, I332E), DAE (G236A, S239D, I332E), YWA (L235Y, G237W, G298A), EFT (D267E, P268F, N324T), Q345R (Q345R), D270G (D270G), and D270S (D270S).

[0018] In another aspect, an Fc region of equine IgGl may comprise substitutions at one or more positions, including 229, 234, 235, 237, 329, 330, and / or 331, using EU Index numbering as in Kabat.

[0019] In another aspect, an Fc region of equine IgGl may comprise one or more substitutions selected from among P229S, L234A, L235A, G237A, P329S, Q330A, Q330S, and P331S, using EU Index numbering as in Kabat.

[0020] In another aspect, an Fc region of equine IgGl may comprise, various combinations of substitutions in a Fc region, including one or more of the following, referred to herein by short designations: SAP (P329S, Q33OA), PAP (Q330A), SQP (P229S), WIN (L234A, L235A, G237A), KA (K322A), PA (P331A), KAPA (K322A, P331A), WIN PA (M234A, L235A, G237A, P331A), WIN_KA (M234A, L235A, G237A, K322A), WIN KAPA (M234A, L235A, G237A, K322A, P331A), PSS (Q330S, P331S), WIN_SAP (L234A, L235A, G237A; P329S, Q330A), and WIN_PSS (L234A, L235A, G237A; Q330S, P331S).

[0021] In another aspect, an Fc region of equine IgG4 may comprise one or more mutations and / or substitutions at positions selected from among 234, 235, 237, 239, 330, and / or 331.

[0022] In another aspect, an Fc region of equine IgG4 may comprise one or more substitutions selected from among L234A, Q235A, G237A, P239S, A330Q, A330S, P331S, using EU Index numbering as in Kabat.

[0023] In another aspect, an Fc region of equine IgG4 may comprise various combinations of substitutions in a Fc region of equine IgG4, which can include one or more of the following, referred to herein by short designations: PSS (A330S, P331S), SQP (P239S, A330Q), Win (L234A, Q235A, G237A), WinPSS (L234A, Q235A, G237A; A330S, P331S), SAP (P329S), and WinSAP (L234A, Q235A, G237A; P329S).

[0024] In another aspect, an Fc region of equine IgG7 may comprise one or more mutations and / or substitutions at positions selected from among 234, 235, 236, 237, 239, 330, and / or 331 , using EU Index numbering as in Kabat.

[0025] In another aspect, an Fc region of equine IgG7 may comprise one or more substitutions selected from among L234A, S235A, V236G, G237A, P239S, P329S, A33OS, A330Q, P331S, using EU Index numbering as in Kabat.

[0026] In another aspect, an Fc region of equine IgG7 may comprise combinations of substitutions in a Fc region of equine IgG7, which can include one or more of the following, referred to herein by short designations: PSS (A330S, P331S), SAP (P329S), SQP (P239S, A330Q), WIN (L234A, S235A, V236G, G237A), WIN_PSS (L234A, S235A, V236G, G237A; A330S, P331S), and WIN SAP (L234A, S235A, V236G, G237A; P329S).

[0027] In another aspect, the invention provides a recombinant polypeptide comprising a modified recombinant feline, canine, or equine IgGFc region as described herein comprising one or more amino acid substitutions described herein.

[0028] In another aspect, the invention provides a recombinant antibody or a molecule comprising a feline, canine, or equine IgG Fc region as described herein comprising one or more amino acid substitutions described herein.

[0029] In another aspect, the invention provides a method for producing or manufacturing an antibody or a molecule, the method comprising: providing a vector or a host cell having a nucleic acid sequence that encodes an antibody, wherein said antibody comprises a feline, canine, or equine IgG constant domain comprising one or more amino acid substitutions described herein.

[0030] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The patent or application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] FIG. 1 : Cell-based complement-dependent cytotoxicity (CDC) activity of feline IgGla, IgGlb and IgG2 Fc subclass CTLA4 fusion proteins.

[0033] FIGS. 2A & 2B - 4: Cell-based CDC activity of feline wild type Fc IgGla subclass CTLA4 fusion proteins and Fc mutants of that subclass

[0034] FIGS. 5-9: Cell-based antibody-dependent cellular phagocytosis (ADCP) activity of feline wild type IgGla Fc subclass CTLA4 fusion protein and Fc mutants of that subclass.

[0035] FIGS. 10-12: Cell-based antibody-dependent cellular cytotoxicity (ADCC) activity of feline wild type IgGla Fc subclass CTLA4 fusion protein and Fc mutants of that subclass.

[0036] FIG. 13: Feline IgGla Fc mutations investigated by the ADCC assay.

[0037] FIGS. 14A - 14C: Protein modeling of Feline IgGla and IgGlb Fc regions. Fig. 14A shows an overlay of protein models of variations with a zoomed-in sub-panel showing amino acid residue positions in ball-and-stick form. Fig 14B shows root mean square deviation (RMSD) comparisons of wild-type constructs of feline IgGla and IgGlb. Fig. 14C shows an overlay of protein models with G236, G267, P268, S298, N324, E333, R334 and E345 variations with a zoomed-in sub-panel showing the amino acid residue positions in ball-and-stick form.

[0038] FIG. 15: Cell-based CDC activity of canine IgGl and IgG2 Fc subclass CTLA4 fusion proteins.

[0039] FIG. 16: Cell-based CDC activity of canine wild type Fc IgG2 subclass CTLA4 fusion proteins and Fc mutants of that subclass.

[0040] FIGS. 17-21: Cell-based ADCP activity of canine wild type IgG2 Fc subclass CTLA4 fusion protein and Fc mutants of that subclass.

[0041] FIGS. 22-24: Cell-based antibody-dependent cellular cytotoxicity (ADCC) activity of canine wild type IgG2 Fc subclass CTLA4 fusion protein and Fc mutants of that subclass.

[0042] FIG. 25: Canine IgG2 Fc mutations investigated by the ADCC assay.

[0043] FIG. 26: Cell-based CDC activity of equine wild type Fc subclass CTLA4 fusion proteins.

[0044] FIG. 27: Cell-based CDC activity of equine wild type Fc IgGl subclass CTLA4 fusion proteins and Fc mutants of that subclass.

[0045] FIG. 28: Cell-based CDC activity of equine wild type Fc IgG4 subclass CTLA4 fusion proteins and Fc mutants of that subclass.

[0046] FIG. 29: Cell-based CDC activity of equine wild type Fc IgG7 subclass CTLA4 fusion proteins and Fc mutants of that subclass.

[0047] FIG. 30: Cell-based ADCP activity of equine wild type Fc IgGl subclass CTLA4 fusion proteins and Fc mutants of that subclass.

[0048] FIGS. 31-32: Cell-based ADCP activity of equine wild type Fc IgG4 subclass CTLA4 fusion proteins and Fc mutants of that subclass.

[0049] FIGS. 33-34: Cell-based ADCP activity of equine wild type Fc IgG7 subclass CTLA4 fusion proteins and Fc mutants of that subclass.

[0050] FIG 35: Cell-based antibody-dependent cell-mediated cytotoxicity activity of equine wild type Fc subclass CTLA4 fusion proteins.

[0051] FIGS. 36-37: Cell-based antibody-dependent cell-mediated cytotoxicity activity of equine wild type Fc IgGl subclass CTLA4 fusion protein and and Fc mutants of that subclass.

[0052] FIG. 38: Cell-based antibody-dependent cell-mediated cytotoxicity activity of equine wild type Fc IgG4 subclass CTLA4 fusion protein and mutations of that Fc subclass.

[0053] FIG. 39: Cell-based antibody-dependent cell-mediated cytotoxicity activity of equine wild type Fc IgG7 subclass CTLA4 fusion protein and mutations of that Fc subclass.

[0054] FIG. 40: Protein modeling of equine FcRn and IgGl Fc regions with a zoomed-in sub-panel showing amino acid residue positions are shown in ball-and-stick form.

[0055] FIG 41: Protein modeling of equine FcRn, IgG4a-Fc and IgG4b-Fc regions with a zoomed-in sub-panel showing amino acid residue positions are shown in ball-and-stick form.

[0056] FIG. 42: Protein modeling of Equine FcRn, IgG7a-Fc and IgG7b-Fc regions with a zoomed-in sub-panel showing amino acid residue positions are shown in ball-and-stick form.

[0057] FIG 43: Root Mean Square Deviation (RMSD) comparisons of wild-type constructs of equine IgGl, IgG4a, IgG4b, IgG7a and IgG7b.

[0058] FIG. 44: Cell-based CDC activity of feline wild type Fc IgGla subclass and Fc mutants of that subclass.

[0059] FIGS. 45 and 46: Cell-based CDC activity of feline wild type Fc IgGlb subclass and Fc mutants of that subclass.

[0060] FIG. 47: Cell-based CDC activity of feline wild type Fc IgG2 subclass and Fc mutants of that subclass.

[0061] FIGS. 48 and 49: Cell-based CDC activity of feline wild type Fc IgG3 subclass and Fc mutants of that subclass.

[0062] FIG. 50: Cell-based CDC activity of feline IgGla variants.

[0063] FIGS. 51-61: Cell-based CDC activity of canine wild type Fc IgG2 subclass and Fc mutants of that subclass.

[0064] FIGS. 62 and 63: Cell-based CDC activity of canine wild type Fc IgG3 subclass and Fc mutants of that subclass.

[0065] FIG. 64: Cell-based ADCP activity of canine wild type anti canine CD20 chimeric IgG mAb and Fc mutants of that subclass.

[0066] FIGS. 65-92: Cell-based ADCP activity of canine wild type IgG2 Fc subclass CTLA4 fusion protein and Fc mutants of that subclass.

[0067] FIG. 93: Cell-based ADCP activity of canine wild type anti canine CD20 chimeric IgG mAb and Fc mutants of that subclass.

[0068] FIG. 94: A schema for an ADCC assay is illustrated.

[0069] FIGS. 95-98: Cell-based canine FcRIII binding of canine wild type IgG2 Fc subclass antibody and Fc mutants of that subclass. BRIEF DESCRIPTION OF THE SEQUENCES

[0070] The contents of the electronic sequence listing (sequencelisting_ST26.xml; Size: 82,432 bytes; and Date of Creation: February 1, 2024) is herein incorporated by reference in its entirety.

[0071] SEQ ID NO: 1 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a GSP (P329G) mutation in the Fc region.

[0072] SEQ ID NO:2 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a GSP (P329G) mutation.

[0073] SEQ ID NO:3 is a sequence for feline IgGla heavy chain comprising a rituximab variable region and a GAP (P329G,S330A) mutation in the Fc region.

[0074] SEQ ID NO:4 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a DANG-GAP (D265A, N297G; P329G, S330A) mutation in the Fc region.

[0075] SEQ ID NO:5 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a WIN DANG GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) mutation in the Fc region.

[0076] SEQ ID NO:6 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a DANG (D265A, N297G) mutation in the Fc region.

[0077] SEQ ID NO:7 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a AAA (S298A, E333A, R334A)mutation in the Fc region.

[0078] SEQ ID NO:8 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a DLE (S239D, S330L, I332E) mutation in the Fc region.

[0079] SEQ ID NO:9 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a DE (S239D, I332E) mutation in the Fc region.

[0080] SEQ ID NO: 10 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a DAE (G236A, S239D, I332E) mutation in the Fc region.

[0081] SEQ ID NO: 1 lisa sequence for a feline IgGla heavy chain comprising a rituximab variable region and a YWA (L235Y, G237W, S298A) mutation in the Fc region.

[0082] SEQ ID NO: 12 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a EFT (G267E, P268F, N324T) mutation in the Fc region.

[0083] SEQ ID NO: 13 is a sequence for a feline IgGla heavy chain comprising a rituximab variable region and a E345R (E345R) mutation in the Fc region.

[0084] SEQ ID NO: 14 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_DANG (M234A, L235A, G237A; D265A, N297G) mutation.

[0085] SEQ ID NO: 15 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_DANG_PSS (M234A, L235A, G237A; D265A, N297G; P331S) mutation.

[0086] SEQ ID NO: 16 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a DANG PSS (D265A, N297G; P33 IS) mutation.

[0087] SEQ ID NO: 17 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN (M234A, L235A, G237A) mutation.

[0088] SEQ ID NO: 18 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a D270G mutation.

[0089] SEQ ID NO: 19 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a D270S mutation.

[0090] SEQ ID NO:20 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_PSA (M234A, L235A, G237A; P331A) mutation.

[0091] SEQ ID NO:21 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_GSA (M234A, L235A, G237A; P329G, P331A) mutation.

[0092] SEQ ID NO:22 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_GSP (M234A, L235A, G237A; P329G) mutation.

[0093] SEQ ID NO:23 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_PSS (M234A, L235A, G237A; P331S) mutation.

[0094] SEQ ID NO:24 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a PSS (P331S) mutation.

[0095] SEQ ID NO:25 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_GSS (M234A, L235A, G237A; P329G, P331S) mutation.

[0096] SEQ ID NO:26 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_GAP (M234A,L235A,G237A; P329G, S330A) mutation.

[0097] SEQ ID NO:27 is a sequence for a feline IgGla heavy chain comprising a canine CTLA-4 peptide fused to a feline Fc region having a WIN_GAP (M234A,L235A,G237A; P329G, S330A) mutation.

[0098] SEQ ID NO:28 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a GSP (P329G) mutation in the Fc region.

[0099] SEQ ID NO:29 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a GAP (P329G, S330A) mutation in the Fc region.

[00100] SEQ ID NO:30 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a DANG-GAP (D265A, N297G; P329G, S330A) mutation in the Fc region.

[00101] SEQ ID NO:3 lisa sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a KAPA (K322A, P331 A) mutation in the Fc region.

[00102] SEQ ID NO:32 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a WIN DANG GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) mutation in the Fc region.

[00103] SEQ ID NO:33 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a DANG (D265A, N297G) mutation in the Fc region.

[00104] SEQ ID NO:34 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a AAA (G298A, E333A, R334A) mutation in the Fc region.

[00105] SEQ ID NO:35 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a DLE (S239D, S330L, I332E) mutation in the Fc region.

[00106] SEQ ID NO:36 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a DE (S239D, I332E) mutation in the Fc region.

[00107] SEQ ID NO:37 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a DAE (G236A, S239D, I332E) mutation in the Fc region.

[00108] SEQ ID NO:38 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a YWA (L235Y, G237W, G298A) mutation in the Fc region.

[00109] SEQ ID NO:39 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a EFT (D267E, P268F, N324T) mutation in the Fc region.

[00110] SEQ ID NQ:40 is a sequence for a canine IgG2 heavy chain comprising a rituximab variable region and a Q345R mutation in the Fc region.

[00111] SEQ ID NO:41 is a sequence for a canine IgG2 heavy chain comprising a canine CTLA-4 peptide fused to a canine Fc region having a D270G mutation.

[00112] SEQ ID NO:42 is a sequence for a canine IgG2 heavy chain comprising a canine CTLA-4 peptide fused to a canine Fc region having a D270S mutation.

[00113] SEQ ID NO:43 is a sequence for a canine IgG2 heavy chain comprising a canine CTLA-4 peptide fused to a canine Fc region having a WIN (M234A, L235A, G237A) mutation.

[00114] SEQ ID NO:44 is a sequence for a canine IgG2 heavy chain comprising a canine CTLA-4 peptide fused to a canine Fc region having a WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) mutation.

[00115] SEQ ID NO:45 is a sequence for a equine IgGl heavy chain comprising a rituximab variable region and a wild type Fc region.

[00116] SEQ ID NO:46 is a sequence for a equine IgGl heavy chain comprising a rituximab variable region and a SAP (P329S, Q330A) mutation in the Fc region.

[00117] SEQ ID NO:47 is a sequence for a equine IgGl heavy chain comprising a rituximab variable region and a WIN (L234A, L235A, G237A) mutation in the Fc region.

[00118] SEQ ID NO:48 is a sequence for a equine IgGl heavy chain comprising a rituximab variable region and a PSS (Q330S, P331S) mutation in the Fc region.

[00119] SEQ ID NO:49 is a sequence for a equine IgGl heavy chain comprising a rituximab variable region and a WIN_SAP (L234A, L235A, G237A; P329S, Q330A) mutation in the Fc region.

[00120] SEQ ID NO:50 is a sequence for a equine IgGl heavy chain comprising a rituximab variable region and a WIN_PSS (L234A, 235A, G237A; Q330S, P331S) mutation in the Fc region.

[00121] SEQ ID NO:51 is a sequence for an equine IgGl heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a PAP (Q330A) mutation.

[00122] SEQ ID NO:52 is a sequence for an equine IgGl heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a SQP (P229S) mutation.

[00123] SEQ ID NO:53 is a sequence for an equine IgG4 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a PSS (A330S, P331S) mutation.

[00124] SEQ ID NO:54 is a sequence for an equine IgG4 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a SQP (P239S, A330Q) mutation.

[00125] SEQ ID NO:55 is a sequence for an equine IgG4 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a WIN PSS (L234A, Q235A, G237A; A330S, P331S) mutation.

[00126] SEQ ID NO:56 is a sequence for an equine IgG4 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a WIN_SAP (L234A, Q235A, G237A; P329S) mutation.

[00127] SEQ ID NO:57 is a sequence for an equine IgG4 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a SAP (P329S) mutation.

[00128] SEQ ID NO:58 is a sequence for an equine IgG4 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a WIN (L234A, Q235A, G237A) mutation.

[00129] SEQ ID NO:59 is a sequence for an equine IgG7 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a PSS (A330S, P331S) mutation.

[00130] SEQ ID NO:60 is a sequence for an equine IgG7 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a SAP (P329S) mutation.

[00131] SEQ ID NO:61 is a sequence for an equine IgG7 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a SQP (P239S, A330Q) mutation.

[00132] SEQ ID NO:62 is a sequence for an equine IgG7 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a WIN (L234A, S235A, V236G, G237A) mutation.

[00133] SEQ ID NO:63 is a sequence for an equine IgG7 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a WINPSS (L234A, S235A, V236G, G237A; A330S, P331S) mutation.

[00134] SEQ ID NO:64 is a sequence for an equine IgG7 heavy chain comprising a canine CTLA-4 peptide fused to an equine Fc region having a WIN SAP (L234A, S235A, V236G, G237A; P329S) mutation. DETAILED DESCRIPTION:

[00135] The present subject matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions, or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

[00136] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00137] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a molecule” or “a compound” is a reference to one or more of such molecules or compounds and equivalents thereof known to those skilled in the art, and so forth. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[00138] In the specification and claims, the numbering of the amino acid residues in an immunoglobulin heavy chain is that of the Eu index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The “Eu index as in Kabat” refers to the residue numbering of the IgG antibody.

[00139] The term “isolated” when used in relation to a nucleic acid is a nucleic acid that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is in a form or setting different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide encoded therein where, for example, the nucleic acid molecule is in a plasmid or a chromosomal location different from that of natural cells. The isolated nucleic acid may be present in single-stranded or double-stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand, but may contain both the sense and anti-sense strands (i.e., may be double-stranded).

[00140] A nucleic acid molecule is “operably linked” or “operably attached” when it is placed into afunctional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to a coding sequence of nucleic acid if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence of nucleic acid if it is positioned so as to facilitate translation. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof which does not, as it exists in nature, comprise an Fc region) if it is positioned such that the expressed fusion protein comprises the heterologous protein or functional fragment thereof adjoined either upstream or downstream to the variant Fc region polypeptide; the heterologous protein may by immediately adjacent to the variant Fc region polypeptide or may be separated therefrom by a linker sequence of any length and composition. Likewise, a polypeptide (used synonymously herein with “protein”) molecule is “operably linked” or “operably attached” when it is placed into a functional relationship with another polypeptide.

[00141] As used herein the term “functional fragment” when in reference to a polypeptide or protein (e.g., a variant Fc region, or a monoclonal antibody) refers to fragments of that protein which retain at least one function of the full-length polypeptide. The fragments may range in size from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. A functional fragment of a variant Fc region polypeptide of the present invention retains at least one “amino acid substitution” as herein defined. A functional fragment of a variant Fc region polypeptide retains at least one function known in the art to be associated with the Fc region (e.g., ADCC, CDC, Fc receptor binding, Clq binding, down regulation of cell surface receptors or may, e.g., increase the in vivo or in vitro half-life of a polypeptide to which it is operably attached).

[00142] The term “purified” or “purify” refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody may be purified by complete or substantial removal (at least 90%, 91%, 92%, 93%, 94%, 95%, or more preferably at least 96%, 97%, 98% or 99%) of at least one contaminating nonimmunoglobulin protein; it may also be purified by the removal of immunoglobulin protein that does not bind to the same antigen. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulins that do not bind a particular antigen results in an increase in the percent of antigen-specific immunoglobulins in the sample. In another example, a polypeptide (e.g., an immunoglobulin) expressed in bacterial host cells is purified by the complete or substantial removal of host cell proteins; the percent of the polypeptide is thereby increased in the sample.

[00143] The term “native” as it refers to a polypeptide (e.g., Fc region) is used herein to indicate that the polypeptide has an amino acid sequence consisting of the amino acid sequence of the polypeptide as it commonly occurs in nature or a naturally occurring polymorphism thereof. A native polypeptide (e.g, native Fc region) may be produced by recombinant means or may be isolated from a naturally occurring source.

[00144] The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.

[00145] As used herein, the term “host cell” refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E.coli, CHO cells, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in situ, or in vivo.

[00146] As used herein, the term “Fc region” refers to a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. Although the generally accepted boundaries of the Fc region of an immunoglobulin heavy chain might vary, the IgG heavy chain Fc region is usually defined to stretch, for example, from an amino acid residue at about position 231, to the carboxyl-terminus thereof.

[00147] In some embodiments, variants comprise only portions of the Fc region and can include or not include the carboxy-terminus. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In some embodiments, variants having one or more of the constant domains are contemplated. In other embodiments, variants without such constant domains (or with only portions of such constant domains) are contemplated.

[00148] The “CH2 domain” of an IgG Fc region usually extends, for example, from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule.

[00149] The “CH3 domain” of a feline IgG Fc region generally is the stretch of residues C- terminal to a CH2 domain in an Fc region extending, for example, from about amino acid residue 341 to about amino acid residue 447.

[00150] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. At least one effector function of a polypeptide comprising a variant Fc region of the present invention may be enhanced or diminished with respect to a polypeptide comprising a native Fc region or the parent Fc region of the variant. Examples of effector functions include, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-depended cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be operably linked to a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assay, ADCC assays, CDC assays, target cell depletion from whole or fractionated blood samples, etc.).

[00151] A “native sequence Fc region” or “wild type Fc region” refers to an amino acid sequence that is identical to the amino acid sequence of an Fc region commonly found in nature.

[00152] A “variant Fc region” comprises an amino acid sequence that differs from that of a native sequence Fc region (or fragment thereof) by virtue of at least one “amino acid substitution” as defined herein. In preferred embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or in the Fc region of a parent polypeptide, preferably 1, 2, 3, 4 or 5 amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. In an alternative embodiment, a variant Fc region may be generated according to the methods herein disclosed and this variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or a non- antibody polypeptide, e.g., binding domain of a receptor or ligand.

[00153] As used herein, the term “derivative” in the context of polypeptides refers to a polypeptide that comprises an amino acid sequence which has been altered by introduction of an amino acid residue substitution. The term “derivative” as used herein also refers to a polypeptide which has been modified by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative polypeptide may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative polypeptide possesses a similar or identical function as the polypeptide from which it was derived. It is understood that a polypeptide comprising a variant Fc region of the present invention may be a derivative as defined herein, preferably the derivatization occurs within the Fc region.

[00154] “Substantially of feline origin” as used herein in reference to a polypeptide (e.g., an Fc region or a monoclonal antibody), indicates the polypeptide has an amino acid sequence at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94% or even more preferably at least 95%, 95%, 97%, 98% or 99% homologous to that of a native feline amino polypeptide. “Substantially of canine origin” and “substantially of equine origin” are similarly defined with respect to a native canine or equine polypeptide, respectively.

[00155] The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to an Fc region (e.g., the Fc region of an antibody). The preferred FcR is a native sequence FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc gamma RI, Fc gamma RII, Fc gamma Rlii subclasses, including allelic variants and alternatively spliced forms of these receptors. Another preferred FcR includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

[00156] The phrases “complement-dependent cytotoxicity” and “CDC” refer to an effector function of IgG and IgM antibodies. When IgG are bound to surface antigen on target cell (e.g., bacterial or viral infected cell), the classical complement pathway is triggered by bonding protein Clq to these antibodies, resulting in formation of a membrane attack complex (MAC) and target cell lysis.

[00157] “Clq” is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. Clq together with two serine proteases, Clr and Cis, forms the complex Cl, the first component of the CDC pathway.

[00158] The phrase “antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., nonspecific) that express FcRs (e.g., Natural Killer (“NK”) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cells. The primary cells for mediating ADCC, NK cells, express Fc gamma RIII only, whereas monocytes express Fc gamma RI, Fc gamma RII and Fc gamma Rin.

[00159] As used herein, the phrase “effector cells” refers to leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc gamma Rlii and perform ADCC effector function. Examples of leukocytes which mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils. The effector cells may be isolated from a native source (e.g., from blood or PBMCs).

[00160] A variant polypeptide with “altered” FcRn binding affinity is one which has either enhanced (i.e., increased, greater or higher) or diminished (z.e., reduced, decreased or lesser) FcRn binding affinity compared to the variant’s parent polypeptide or to a polypeptide comprising a native Fc region when measured at pH 6.0. A variant polypeptide which displays increased binding or increased binding affinity to an FcRn binds FcRn with greater affinity than the parent polypeptide. A variant polypeptide which displays decreased binding or decreased binding affinity to an FcRn, binds FcRn with lower affinity than its parent polypeptide. Such variants which display decreased binding to an FcRn may possess little or no appreciable binding to an FcRn, e.g., 0-20% binding to the FcRn compared to a parent polypeptide. A variant polypeptide which binds an FcRn with "enhanced affinity" as compared to its parent polypeptide, is one which binds FcRn with higher binding affinity than the parent polypeptide, when the amounts of variant polypeptide and parent polypeptide in a binding assay are essentially the same, and all other conditions are identical. For example, a variant polypeptide with enhanced FcRn binding affinity may display from about 1.10 fold to about 100 fold (more typically from about 1.2 fold to about 50 fold) increase in FcRn binding affinity compared to the parent polypeptide, where FcRn binding affinity is determined, for example, in an ELISA assay or other method available to one of ordinary skill in the art.

[00161] As used herein, an “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a given amino acid sequence with another different “replacement” amino acid residue. The replacement residue or residues may be “naturally occurring amino acid residues” (i.e., encoded by the genetic code) and selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gin); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (He): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); praline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Vai). Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein. A “non-naturally occurring amino acid residue” refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues (s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202: 301-336 (1991).

[00162] The term “assay signal” refers to the output from any method of detecting protein-protein interactions, including but not limited to, SPR assays, absorbance measurements from colorimetric assays, fluorescent intensity, or disintegrations per minute. Assay formats could include ELISA, facs, SPR, or other methods. A change in the “assay signal” may reflect a change in cell viability and / or a change in the kinetic off-rate, the kinetic on-rate, or both. A “higher assay signal” refers to the measured output number being larger than another number (e.g, a variant may have a higher (larger) measured number in an ELISA assay as compared to the parent polypeptide). A “lower” assay signal refers to the measured output number being smaller than another number (e.g, a variant may have a lower (smaller) measured number in an ELISA assay as compared to the parent polypeptide).

[00163] The term “binding affinity” refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each Fc receptor-Fc binding interaction. The binding affinity is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, e.g, seconds-1) divided by the kinetic on-rate (generally reported in units of concentration per unit time, e.g, molar / second). In general it is not possible to unequivocally state whether changes in equilibrium dissociation constants are due to differences in on-rates, off-rates or both unless each of these parameters are experimentally determined (e.g., by BIACORE or SAPID YNE measurements).

[00164] As used herein, the term “hinge region” refers to the stretch of amino acids, for example, in feline IgGla (e.g., stretching from position 216 to position 230 of feline IgGla). Hinge regions of other IgG isotypes may be aligned with the IgG sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions.

[00165] As used herein, the term “antibody” is used interchangeably with “immunoglobulin” or “1g,” and is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or functional activity. Single chain antibodies, and chimeric, feline, or felinized antibodies, canine or caninized, or equine or equinized, as well as chimeric or CDR-grafted single chain antibodies, and the like, comprising portions derived from different species, are also encompassed by the present invention and the term “antibody”. The various portions of these antibodies can be joined together chemically by conventional techniques, synthetically, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or felinized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. No. 4,816,567; U.S. Pat. No. 4,816,397; WO 86 / 01533; U.S. Pat. No. 5,225,539; and U.S. Pat. Nos. 5,585,089 and 5,698,762. See also, Newman, R. et al. BioTechnology, 10: 1455-1460, 1993, regarding primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242:423-426, 1988, regarding single chain antibodies. It is understood that all forms of the antibodies comprising an Fc region (or portion thereof) are encompassed herein within the term “antibody.” Furthermore, the antibody may be labeled with a detectable label, immobilized on a solid phase and / or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

[00166] As used herein, the term “antibody fragments” refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc’ peptides, Fab and Fab fragments, and multispecific antibodies formed from antibody fragments. The antibody fragments preferably retain at least part of the hinge and optionally the CHI region of an IgG heavy chain. In other preferred embodiments, the antibody fragments comprise at least a portion of the CH2 region or the entire CH2 region.

[00167] As used herein, the term “functional fragment”, when used in reference to a monoclonal antibody, is intended to refer to a portion of the monoclonal antibody that still retains a functional activity. A functional activity can be, for example, antigen binding activity or specificity, receptor binding activity or specificity, effector function activity and the like. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab’; bivalent fragments such as F(ab’)2; single chain Fv (scFv); and Fc fragments. Such terms are described in, for example, Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed ), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The term functional fragment is intended to include, for example, fragments produced by protease digestion or reduction of a monoclonal antibody and by recombinant DNA methods known to those skilled in the art.

[00168] As used herein, the term “fragment” refers to a polypeptide comprising an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100 or more contiguous amino acid residues of the amino acid sequence of another polypeptide. In a preferred embodiment, a fragment of a polypeptide retains at least one function of the full-length polypeptide.

[00169] As used herein, the term “chimeric antibody” includes monovalent, divalent or polyvalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated through disulfide bridges with a chimeric light chain. A divalent chimeric antibody is a tetramer formed by two heavy chain-light chain dimers associated through at least one disulfide bridge. A chimeric feline heavy chain of an antibody comprises an antigen-binding region derived from the heavy chain of a nonfeline antibody, which is linked to at least a portion of a feline heavy chain constant region, such as CHI or CH2. A chimeric light chain of a feline antibody comprises an antigen binding region derived from the light chain of a non-feline antibody, linked to at least a portion of a feline light chain constant region (CL). Chimeric canine and equine antibodies are similarly defined.

[00170] Antibodies, fragments or derivatives having chimeric heavy chains and light chains of the same or different variable region binding specificity, can also be prepared by appropriate association of the individual polypeptide chains, according to known method steps. With this approach, hosts expressing chimeric heavy chains are separately cultured from hosts expressing chimeric light chains, and the immunoglobulin chains are separately recovered and then associated. Alternatively, the hosts can be cocultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin or fragment or both the heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, e.g., U.S. Pat. Nos. 6,284,471; 5,807,715; 4,816,567; and 4,816,397).

[00171] As used herein, “felinized” forms of non-feline (e.g., murine) antibodies (i.e., felinized antibodies) are antibodies that contain minimal sequence, or no sequence, derived from non-feline immunoglobulin. For the most part, felinized antibodies are feline immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a nonfeline species (donor antibody) such as mouse, rat, rabbit, human or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the feline immunoglobulin are replaced by corresponding non-feline residues. Furthermore, felinized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are generally made to further refine antibody performance. In general, the felinized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-feline immunoglobulin and all or substantially all of the FR residues are those of a feline immunoglobulin sequence. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a feline immunoglobulin.

[00172] As used herein, “cananized” forms of non-canine (e.g., murine) antibodies (i.e., caninized antibodies) are antibodies that contain minimal sequence, or no sequence, derived from non-canine immunoglobulin. For the most part, caninized antibodies are canine immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-canine species (donor antibody) such as mouse, rat, rabbit, human or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. Furthermore, caninized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are generally made to further refine antibody performance. In general, the caninized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-canine immunoglobulin and all or substantially all of the FR residues are those of a canine immunoglobulin sequence. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin. [00173J As used herein, “equinized” forms of non-equine (e.g., murine) antibodies (z.e., equinized antibodies) are antibodies that contain minimal sequence, or no sequence, derived from non-equine immunoglobulin. For the most part, equinized antibodies are equine immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-equine species (donor antibody) such as mouse, rat, rabbit, human or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the equine immunoglobulin are replaced by corresponding non-equine residues. Furthermore, equinized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are generally made to further refine antibody performance. In general, the equinized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-equine immunoglobulin and all or substantially all of the FR residues are those of a equine immunoglobulin sequence. The equinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a equine immunoglobulin.

[00174] As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding domain of a heterologous “adhesin” protein (e.g., a receptor, ligand or enzyme) with an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e., is “heterologous”) with an immunoglobulin constant domain sequence.

[00175] As used herein, the term “ligand binding domain” refers to any native receptor or any region or derivative thereof retaining at least a qualitative ligand binding ability of a corresponding native receptor. In certain embodiments, the receptor is from a cell-surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergenefamily. Other receptors, which are not members of the immunoglobulin supergenefamily but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L-, and P-selectins).

[00176] As used herein, the term “receptor binding domain” refers to any native ligand for a receptor, including, e.g., cell adhesion molecules, or any region or derivative of such native ligand retaining at least a qualitative receptor binding ability of a corresponding native ligand.

[00177] As used herein, an “isolated” polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the isolated polypeptide is purified (1) to greater than 95% by weight of polypeptides as determined by the Lowry method, and preferably, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-page under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide’s natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[00178] As used herein, the term “disorder” and “disease” are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the invention), including chronic and acute disorders or diseases (e.g., pathological conditions that predispose a patient to a particular disorder).

[00179] As used herein, the term “receptor” refers to a polypeptide capable of binding at least one ligand. The preferred receptor is a cell-surface or soluble receptor having an extracellular ligand-binding domain and, optionally, other domains (e.g, transmembrane domain, intracellular domain and / or membrane anchor). A receptor to be evaluated in an assay described herein may be an intact receptor or a fragment or derivative thereof (e.g., a fusion protein comprising the binding domain of the receptor fused to one or more heterologous polypeptides). Moreover, the receptor to be evaluated for its binding properties may be present in a cell or isolated and optionally coated on an assay plate or some other solid phase or labeled directly and used as a probe. Feline IgGs

[00180] The inventors of the instant application have found that by substituting one or more amino acid residues in the Fc region of feline IgGl, advantageous characteristics may be introduced into feline IgGs, for example reducing or completely knocking out CDC, ADCC and ADCP activities.

[00181] Accordingly, an Fc region of feline IgGl a may comprise one or more mutations or substitutions at positions including amino acid 234, 235, 236, 237, 239, 329, 265, 267, 268, 270, 297, 298, 330, 331, 332, 333, 334, and / or 345, referring to positions numbered according to the EU index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[00182] In various embodiments, an Fc region of feline IgGla may comprise one or more substitutions selected from among M234A, L235A, L235Y, G236A, G237A, G237W, S239D, D265A, G267E, P268F, D270G, D270S, N297G, S298A, K322A, N324T, P329G, P329S, S330A, S330L, P331A, P331S, P331A, I332E, E333A, R334A, and E345R using EU Index numbering. For example, various combinations of substitutions in aFc region of feline IgGla can include one or more of the following, which are referred to herein by short designations: WIN (M234A, L235A, G237A), GSP (P329G), PSS (P331S), GAP (P329G, S330A), DANG (D265A, N297G), KA (K322A), PA (P331A), KAPA (K322A, P331A), WIN KA (M234A, L235A, G237A, K322A), WIN_PA (M234A, L235A, G237A, P331A), WIN_KAPA (M234A, L235A, G237A, K322A, P331A), WIN_GAP (M234A, L235A, G237A, P329G, S330A), DANG-GAP (D265A, N297G, P329G, S330A), WINDANG (M234A, L235A, G237A, D265A, N297G), WINDANGGAP (M234A, L235A, G237A, D265A, N297G, P329G, S330A), DANGPSS (D265A, N297G, P331S), WIN_PSA (M234A, L235A, G237A, P331A), WIN_DANG_PSS (M234A, L235A, G237A, D265A, N297G, P331S), WIN_PSS (M234A, L235A, G237A, P331S), WIN_GSA (M234A, L235A, G237A, P329G, P331A), WIN_GSP (M234A, L235A, G237A, P329G), WIN GSS (M234A, L235A, G237A, P329G, P331S), WINSAS (M234A, E235A, G237A, P329S, S330A, P331S), AAA (S298A, E333A, R334A), DLE (S239D, S330L, I332E), DE (S239D, I332E), DAE (G236A, S239D, I332E), YWA (L235Y, G237W, S298A), EFT (G267E, P268F, N324T), D270G (D270G), D270S (D270S), and E345R (E345R). Due to apparent sequence and structural similarities, an Fc region of feline IgGlb may also comprise one or more corresponding substitutions or combinations of substitutions in positions corresponding to the positions in IgGla described above.

[00183] As further examples, feline IgGla Fc can be comprised within a recombinantly modified protein, e.g., a feline IgG heavy chain comprising a rituximab variable region and a mutated constant region, or a feline IgGla Fc fused to another functional peptide such as a CTLA-4 polypeptide fragment. Examples of such proteins include the following sequences: Rituximab Feline IgGla GSP (P329G) SEQ ID NO: 1 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPSI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNSTY RWSVLPILH QDWLKGKEFK CKVNSKSLGS PIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla GAP (P329G,S330A) SEQ ID NO: 2 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPSI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNSTY RWSVLPILH QDWLKGKEFK CKVNSKSLGA PIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla DANG-GAP (D265A, N297G; P329G, S330A) SEQ ID NO: 3 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPSI FIFPPKPKDT LSISRTPEVT CLWALGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFGSTY RWSVLPILH QDWLKGKEFK CKVNSKSLGA PIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla KAPA (K322A, P331A) SEQ ID NO: 4 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY  60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPSI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNSTY RWSVLPILH QDWLKGKEFK CAVNSKSLPS AIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) SEQ ID NO: 5 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEAAGAPSI FIFPPKPKDT LSISRTPEVT CLWALGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFGSTY RWSVLPILH QDWLKGKEFK CKVNSKSLGA PIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla DANG (D265A, N297G) SEQ ID NO: 6 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPSI FIFPPKPKDT LSISRTPEVT CLWALGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFGSTY RWSVLPILH QDWLKGKEFK CKVNSKSLPS PIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla AAA (S298A, E333A, R334A) SEQ ID NO: 7 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPSI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNATY RWSVLPILH QDWLKGKEFK CKVNSKSLPS PIAATISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla DLE (S239D, S330L, I332E) SEQ ID NO: 8 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPDI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNSTY RWSVLPILH QDWLKGKEFK CKVNSKSLPL PEERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla DE (S239D, I332E) SEQ ID NO: 9 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPDI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNSTY RWSVLPILH QDWLKGKEFK CKVNSKSLPS PEERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla DAE (G236A, S239D, I332E) SEQ ID NO: 10 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLAGPDI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNSTY RWSVLPILH QDWLKGKEFK CKVNSKSLPS PEERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla YWA (L235Y, G237W, S298A) SEQ ID NO: 11 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMYGWPSI FIFPPKPKDT LSISRTPEVT CLWDLGPDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNATY RWSVLPILH QDWLKGKEFK CKVNSKSLPS PIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla EFT (G267E, P268F, N324T) SEQ ID NO: 12 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SVSSASTTAP SVFPLAPSCG TTSGATVALA CLVLGYFPEP VTVSWNSGAL TSGVHTFPAV 180 LQASGLYSLS SMVTVPSSRW LSDTFTCNVA HPPSNTKVDK TVRKTDHPPG PKPCDCPKCP 240 PPEMLGGPSI FIFPPKPKDT LSISRTPEVT CLWDLEFDD SDVQITWFVD NTQVYTAKTS 300 PREEQFNSTY RWSVLPILH QDWLKGKEFK CKVTSKSLPS PIERTISKAK GQPHEPQVYV 360 LPPAQEELSR NKVSVTCLIK SFHPPDIAVE WEITGQPEPE NNYRTTPPQL DSDGTYFVYS 420 KLSVDRSHWQ RGNTYTCSVS HEALHSHHTQ KSLTQSPGK                        459 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Rituximab Feline IgGla E345R (E345R) SEQ ID NO: 13 MEWSWVFLFF LSVTTGVHSQ VQLQQPGAEL VKPGASVKMS CKASGYTFTS YNMHWVKQTP  60 GRGLEWIGAI YPGNGDTSYN QKFKGKATLT ADKSSSTAYM QLSSLTSEDS AVYYCARSTY 120 YGGDWYFNVW GAGTTVTVSS VSSASTTAPS VFPLAPSCGT TSGATVALAC LVLGYFPEPV 180 TVSWNSGALT SGVHTFPAVL QASGLYSLSS MVTVPSSRWL SDTFTCNVAH PPSNTKVDKT 240 VRKTDHPPGP KPCDCPKCPP PEMLGGPSIF IFPPKPKDTL SISRTPEVTC LWDLGPDDS 300 DVQITWFVDN TQVYTAKTSP REEQFNSTYR WSVLPILHQ DWLKGKEFKC KVNSKSLPSP 360 IERTISKAKG QPHRPQVYVL PPAQEELSRN KVSVTCLIKS FHPPDIAVEW EITGQPEPEN 420 NYRTTPPQLD SDGTYFVYSK LSVDRSHWQR GNTYTCSVSH EALHSHHTQK SLTQSPGK   478 where amino acids 1-116 of this sequence comprise a rituximab heavy chain fragment and amino acids 117-459 comprise a heavy chain fragment containing the constant domain of feline IgGla. Canine CTLA-4 Feline IgGla fusion WIN_DANG (M234A, L235A, G237A; D265A, N297G) SEQ ID NO: 14 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDRKTDH PPGPKPCDCP KCPPPEAAGA PSIFIFPPKP 180 KDTLSISRTP EVTCLWALG PDDSDVQITW FVDNTQVYTA KTSPREEQFG STYRWSVLP 240 ILHQDWLKGK EFKCKVNSKS LPSPIERTIS KAKGQPHEPQ VYVLPPAQEE LSRNKVSVTC 300 LIKSFHPPDI AVEWEITGQP EPENNYRTTP PQLDSDGTYF VYSKLSVDRS HWQRGNTYTC 360 SVSHEALHSH HTQKSLTQSP GK                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion WIN_DANG_PSS (M234A, L235A, G237A; D265A, N297G; P331S) SEQ ID NO: 15 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDRKTDH PPGPKPCDCP KCPPPEAAGA PSIFIFPPKP 180 KDTLSISRTP EVTCLWALG PDDSDVQITW FVDNTQVYTA KTSPREEQFG STYRWSVLP 240 ILHQDWLKGK EFKCKVNSKS LPSSIERTIS KAKGQPHEPQ VYVLPPAQEE LSRNKVSVTC 300 LIKSFHPPDI AVEWEITGQP EPENNYRTTP PQLDSDGTYF VYSKLSVDRS HWQRGNTYTC 360 SVSHEALHSH HTQKSLTQSP GK                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion DANG_PSS (D265A, N297G; P331S) SEQ ID NO: 16 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDRKTDH PPGPKPCDCP KCPPPEMLGG PSIFIFPPKP 180 KDTLSISRTP EVTCLWALG PDDSDVQITW FVDNTQVYTA KTSPREEQFG STYRWSVLP 240 ILHQDWLKGK EFKCKVNSKS LPSSIERTIS KAKGQPHEPQ VYVLPPAQEE LSRNKVSVTC 300 LIKSFHPPDI AVEWEITGQP EPENNYRTTP PQLDSDGTYF VYSKLSVDRS HWQRGNTYTC 360 SVSHEALHSH HTQKSLTQSP GK                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion WIN (M234A, L235A, G237A) SEQ ID NO: 17 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclvvdlg pddsdvqitw fvdntqvyta ktspreeqfn styrwsvlp 240 ilhqdwlkgk efkckvnSks Ipspiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion D270G  (D270G) SEQ ID NO: 18 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppemlgg psififppkp 180 kDtlsisrtp evtclvvdlg pdGsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks Ipspiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqLdsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion D270S  (D270S) SEQ ID NO: 19 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppemlgg psififppkp 180 kDtlsisrtp evtclvvdlg pdSsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks Ipspiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqLdsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion WIN_PSA (M234A, L235A, G237A; P331A) SEQ ID NO: 20 METDTLLLVJV LLLWPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclvvdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks IpsAiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion Win_GSA(M234A, L235A, G237A; P329G, P331A) SEQ ID NO: 21 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclvvdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks IGsAiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion WIN_GSP (M234A, L235A, G237A; P329G) SEQ ID NO: 22 METDTLLLVJV LLLWPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclvvdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks IGspiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion Win_PSS (M234A, L235A, G237A; P331S) SEQ ID NO: 23 METDTLLLWV LLLTaP / PGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclvvdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks IpsSiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqLdsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion PSS (P331S) SEQ ID NO: 24 METDTLLLWV LLLWPGSTG DMHVAQPAW' LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKWLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppemlgg psififppkp 180 kDtlsisrtp evtclwdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks IpsSiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion Win_GSS (M234A, L235A, G237A; P329G, P331S) SEQ ID NO: 25 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclwdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks IGsSiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion WIN_GAP (M234A, L235A, G237A; P329G, S330A) SEQ ID NO: 26 METDTLLLVJV LLLWPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclvvdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks IGApiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhshh tqksltqsp gk 382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine CTLA-4 Feline IgGla fusion WIN_SAS (M234A, L235A, G237A;P329S , S330A, P331S) SEQ ID NO: 27 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDrktdh ppgpkpcdcp kcpppeAAgA psififppkp 180 kDtlsisrtp evtclvvdlg pddsdvqitw fvdntqvyta ktspreeqfn styrvvsvlp 240 ilhqdwlkgk efkckvnSks ISASiertis kakgqphepq vyvlppaqee Isrnkvsvtc 300 liksfhppdi aveweitgqp epennyrttp pqldsdgtyf vysklsvdrs hwqrgntytc 360 svshealhsh htqksltqsp gk                                            382 where amino acids 21-145 comprise a canine CTLA-4 sequence and amino acids 146-382 comprise a fragment of feline IgGla heavy chain containing the Fc region. Canine IgGs

[00184] The inventors of the instant application have surprisingly found that by substituting one or more amino acid residues in the Fc region of canine IgG2, advantageous characteristics may be introduced into canine IgGs, for example reducing or completely knocking out CDC, ADCC and ADCP activities or increasing or decreasing binding affinity to canine Fey receptor and Clq.

[00185] Accordingly, in various embodiments, an Fc region of canine IgG2 may comprise one or more mutations or substitutions, such as at locations listed in Table 4, for example the substitutions listed in Table 5, and / or substitutions selected from among M234A, L235A, L235Y, G236A, G237A, G237W, S239D, D265A, D270G, D270S, N297G, G298A, K322A, P329G, S330A, S330L, P331 A, I332E, E333A, R334A, using EU Index numbering as in Kabat. For example, various combinations of substitutions in a Fc region of canine IgG2 can include one or more of the following, referred to herein by short designations: GSP (P329G), GAP (P329G, S330A), DANG (D265A, N297G), DANG-GAP (D265A, N297G; P329G, S330A), WIN (M234A, L235A, G237A), WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A), AAA (G298A, E333A, R334A), DE (S239D, I332E), DLE (S239D, S330L, I332E), DAE (G236A, S239D, I332E), YWA (L235Y, G237W, G298A), EFT (D267E, P268F, N324T), Q345R (Q345R), D270G (D270G), and D270S (D270S).

[00186] As further examples, mutated canine IgG2 Fc can be comprised within a recombinantly modified protein, e.g., a canine IgG heavy chain comprising a rituximab variable region and a mutated constant region, or a fusion protein comprising another effector region (such as a CTLA-4 sequence) fused to a canine IgG heavy chain comprising a mutated constant region. Examples of such proteins include the following sequences: Rituximab Canine IgG2 GSP (P329G) SEQ ID NO: 28 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCKV NNKALGSPIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 GAP (P329G, S330A) SEQ ID NO: 29 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCKV NNKALGAPIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 DANG-GAP (D265A, N297G; P329G, S330A) SEQ ID NO: 30 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VALDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFGGTYRW SVLPIGHQDW LKGKQFTCKV NNKALGAPIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 KAPA (K322A, P331A) SEQ ID NO: 31 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCAV NNKALPSAIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) SEQ ID NO: 32 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 AAGAPSVFIF PPKPKDTLLI ARTPEVTCW VALDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFGGTYRW SVLPIGHQDW LKGKQFTCKV NNKALGAPIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 DANG D265A, N297G) SEQ ID NO: 33 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VALDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFGGTYRW SVLPIGHQDW LKGKQFTCKV NNKALPSPIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 AAA(G2 98A, E333A, R334A) SEQ ID NO: 34 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNATYRW SVLPIGHQDW LKGKQFTCKV NNKALPSPIA ATISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 DLE (S239D, S330L, I332E) SEQ ID NO: 35 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPDVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCKV NNKALPLPEE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 DE (S239D, I332E) SEQ ID NO: 36 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPDVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCKV NNKALPSPEE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 DAE (G236A, S239D, I332E) SEQ ID NO: 37 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLAGPDVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCKV NNKALPSPEE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 YWA (L235Y, G237W, G298A) SEQ ID NO: 38 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MYGWPSVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNATYRW SVLPIGHQDW LKGKQFTCKV NNKALPSPIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 EFT (D267E, P268F, N324T) SEQ ID NO: 39 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VDLEFEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCKV TNKALPSPIE RTISKARGQA HQPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Rituximab Canine IgG2 Q345R  (Q345R) SEQ ID NO: 40 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 AASTTAPSVF PLAPSCGSTS GSTVALACLV SGYFPEPVTV SWNSGSLTSG VHTFPSVLQS 180 SGLYSLSSMV TVPSSRWPSE TFTCNVAHPA SKTKVDKPVP KRENGRVPRP PDCPKCPAPE 240 MLGGPSVFIF PPKPKDTLLI ARTPEVTCW VDLDPEDPEV QISWFVDGKQ MQTAKTQPRE 300 EQFNGTYRW SVLPIGHQDW LKGKQFTCKV NNKALPSPIE RTISKARGQA HRPSVYVLPP 360 SREELSKNTV SLTCLIKDFF PPDIDVEWQS NGQQEPESKY RTTPPQLDED GSYFLYSKLS 420 VDKSRWQRGD TFICAVMHEA LHNHYTQESL SHSPGK                           456 where amino acids 1-116 comprise a fragment of heavy chain variable region of rituximab and amino acids 117-456 comprise a mutated canine IgG2 heavy chain containing the constant region. Canine CTL4 - Canine IgG2 Fusion D270G (D270G) SEQ ID NO: 41 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDPKREN GRVPRPPDCP KCPAPEMLGG PSVFIFPPKP 180 KDTLLIARTP EVTCVWDLD PEGPEVQISW FVDGKQMQTA KTQPREEQFN GTYRWSVLP 24 0 IGHQDWLKGK QFTCKVNNKA LPSPIERTIS KARGQAHQPS VYVLPPSREE LSKNTVSLTC 300 LIKDFFPPDI DVEWQSNGQQ EPESKYRTTP PQLDEDGSYF LYSKLSVDKS RWQRGDTFIC 360 AVMHEALHNH YTQESLSHSP GK                                          382 where amino acids 1-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218) and amino acids 146-382 comprise a fragment of canine IgG2 heavy chain containing the constant region. Canine CTL4 - Canine IgG2 Fusion D270S (D270S) SEQ ID NO: 42 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDPKREN GRVPRPPDCP KCPAPEMLGG PSVFIFPPKP 180 KDTLLIARTP EVTCVWDLD PESPEVQISW FVDGKQMQTA KTQPREEQFN GTYRWSVLP 24 0 IGHQDWLKGK QFTCKVNNKA LPSPIERTIS KARGQAHQPS VYVLPPSREE LSKNTVSLTC 300 LIKDFFPPDI DVEWQSNGQQ EPESKYRTTP PQLDEDGSYF LYSKLSVDKS RWQRGDTFIC 360 AVMHEALHNH YTQESLSHSP GK                                          382 where amino acids 1-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218) and amino acids 146-382 comprise a fragment of canine IgG2 heavy chain containing the constant region. Canine CTL4 - Canine IgG2 Fusion WIN (M234A, L235A, G237A) SEQ ID NO: 43 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDPKREN GRVPRPPDCP KCPAPEAAGA PSVFIFPPKP 180 KDTLLIARTP EVTCVWDLD PESPEVQISW FVDGKQMQTA KTQPREEQFN GTYRWSVLP 24 0 IGHQDWLKGK QFTCKVNNKA LPSPIERTIS KARGQAHQPS VYVLPPSREE LSKNTVSLTC 300 LIKDFFPPDI DVEWQSNGQQ EPESKYRTTP PQLDEDGSYF LYSKLSVDKS RWQRGDTFIC 360 AVMHEALHNH YTQESLSHSP GK                                          382 where amino acids 1-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218) and amino acids 146-382 comprise a fragment of canine IgG2 heavy chain containing the constant region. Canine CTL4 - Canine IgG2 Fusion WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) SEQ ID NO: 44 METDTLLLWV LLLTaWPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA ELDDSTCTGT SSGNKWLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDPKREN GRVPRPPDCP KCPAPEAAGA PSVFIFPPKP 180 KDTLLIARTP EVTCVWALD PEDPEVQISW FVDGKQMQTA KTQPREEQFG GTYRWSVLP 24 0 IGHQDWLKGK QFTCKVNNKA LGAPZERTIS KARGQAHQPS VYVLPPSREE LSKNTVSLTC 300 LIKDFFPPDI DVEWQSNGQQ EPESKYRTTP PQLDEDGSYF LYSKLSVDKS RWQRGDTFIC 360 AVMHEALHNH YTQESLSHSP GK where amino acids 1-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218) and amino acids 146-382 comprise a fragment of canine IgG2 heavy chain containing the constant region. Equine IgG

[00187] The inventors of the instant application have surprisingly found that by substituting one or more amino acid residues in the Fc region of equine IgGl, advantageous characteristics may be introduced into equine IgGs, for example reducing or completely knocking out CDC, ADCC and ADCP activities or increasing or decreasing binding affinity to canine Fey receptor and Clq.

[00188] Accordingly, in various embodiments, an Fc region of equine IgGl may comprise one or more mutations or substitutions, including at locations 229, 234, 235, 237, 329, 330, and / or 331. In various embodiments, an Fc region of equine IgGl may comprise one or more mutations and / or substitutions selected from among P229S, L234A, L235A, G237A, P329S, Q330A, Q330S, and P331S, using EU Index numbering as in Kabat. For example, various combinations of substitutions in a Fc region of equine IgGl can include one or more of the following, referred to herein by short designations: SAP (P329S, Q33OA), PAP (Q330A), SQP (P229S), WIN (L234A, L235A, G237A), PSS (Q33OS, P331S), WIN_SAP (L234A, L235A, G237A; P329S, Q330A), and WIN_PSS (L234A, L235A, G237A; Q330S, P331S).

[00189] As further examples, mutated equine IgGl Fc can be comprised within a recombinantly modified protein, e.g., an equine IgG heavy chain comprising a rituximab variable region and a mutated constant region, or a fusion protein comprising an effector region (such as a CTLA-4 sequence) fused to an equine IgG heavy chain comprising a mutated constant region. Examples of such proteins include the following sequences: Rituximab Equine IgGl wt SEQ ID NO: 45 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SASTTAPKVF ALAPGCGTTS DSTVALGCLV SGYFPEPVKV SWNSGSLTSG VHTFPSVLQS 180 SGFYSLSSMV TVPASTWTSE TYICNWHAA SNFKVDKRIE PIPDNHQKVC DMSKCPKCPA 240 PELLGGPSVF IFPPNPKDTL MITRTPEVTC VWDVSQENP DVKFNWYMDG VEVRTATTRP 300 KEEQFNSTYR WSVLRIQHQ DWLSGKEFKC KVNNQALPQP IERTITKTKG RSQEPQVYVL 360 APHPDELSKS KVSVTCLVKD FYPPEINIEW QSNGQPELET KYSTTQAQQD SDGSYFLYSK 420 LSVDRNRWQQ GTTFTCGVMH EALHNHYTQK NVSKNPGK                         458 where amino acids 1-121 comprise a rituximab variable region and amino acids 122-458 comprise a wild type sequence of the equine IgGl heavy chain constant region (e.g., accession CAC44624). Rituximab Equine IgGl SAP (P32 9S, Q330A) SEQ ID NO: 46 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SASTTAPKVF ALAPGCGTTS DSTVALGCLV SGYFPEPVKV SWNSGSLTSG VHTFPSVLQS 180 SGFYSLSSMV TVPASTWTSE TYICNWHAA SNFKVDKRIE PIPDNHQKVC DMSKCPKCPA 240 PELLGGPSVF IFPPNPKDTL MITRTPEVTC VWDVSQENP DVKFNWYMDG VEVRTATTRP 300 KEEQFNSTYR WSVLRIQHQ DWLSGKEFKC KVNNQALSAP IERTITKTKG RSQEPQVYVL 360 APHPDELSKS KVSVTCLVKD FYPPEINIEW QSNGQPELET KYSTTQAQQD SDGSYFLYSK 420 LSVDRNRWQQ GTTFTCGVMH EALHNHYTQK NVSKNPGK                         458 where amino acids 1-121 comprise a rituximab variable region and amino acids 122-458 comprise a mutated sequence of the equine IgGl heavy chain constant region. Rituximab Equine IgGl WIN (L234A, L235A, G237A) SEQ ID NO:47 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SASTTAPKVF ALAPGCGTTS DSTVALGCLV SGYFPEPVKV SWNSGSLTSG VHTFPSVLQS 180 SGFYSLSSMV TVPASTWTSE TYICNWHAA SNFKVDKRIE PIPDNHQKVC DMSKCPKCPA 240 PEAAGAPSVF IFPPNPKDTL MITRTPEVTC VWDVSQENP DVKFNWYMDG VEVRTATTRP 300 KEEQFNSTYR WSVLRIQHQ DWLSGKEFKC KVNNQALPQP IERTITKTKG RSQEPQVYVL 360 APHPDELSKS KVSVTCLVKD FYPPEINIEW QSNGQPELET KYSTTQAQQD SDGSYFLYSK 420 LSVDRNRWQQ GTTFTCGVMH EALHNHYTQK NVSKNPGK                         458 where amino acids 1-121 comprise a rituximab variable region and amino acids 122-458 comprise a mutated sequence of the equine IgGl heavy chain constant region. Rituximab Equine IgGl PSS (Q330S, P331S) SEQ ID NO:48 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SASTTAPKVF ALAPGCGTTS DSTVALGCLV SGYFPEPVKV SWNSGSLTSG VHTFPSVLQS 180 SGFYSLSSMV TVPASTWTSE TYICNWHAA SNFKVDKRIE PIPDNHQKVC DMSKCPKCPA 240 PELLGGPSVF IFPPNPKDTL MITRTPEVTC VWDVSQENP DVKFNWYMDG VEVRTATTRP 300 KEEQFNSTYR WSVLRIQHQ DWLSGKEFKC KVNNQALPSS IERTITKTKG RSQEPQVYVL 360 APHPDELSKS KVSVTCLVKD FYPPEINIEW QSNGQPELET KYSTTQAQQD SDGSYFLYSK 420 LSVDRNRWQQ GTTFTCGVMH EALHNHYTQK NVSKNPGK                         458 where amino acids 1-121 comprise a rituximab variable region and amino acids 122-458 comprise a mutated sequence of the equine IgGl heavy chain constant region. Rituximab Equine IgGl WIN_SAP (L234A, L235A, G237A; P329S, Q330A) SEQ ID NO:49 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SASTTAPKVF ALAPGCGTTS DSTVALGCLV SGYFPEPVKV SWNSGSLTSG VHTFPSVLQS 180 SGFYSLSSMV TVPASTWTSE TYICNWHAA SNFKVDKRIE PIPDNHQKVC DMSKCPKCPA 240 PEAAGAPSVF IFPPNPKDTL MITRTPEVTC VWDVSQENP DVKFNWYMDG VEVRTATTRP 300 KEEQFNSTYR WSVLRIQHQ DWLSGKEFKC KVNNQALSAP IERTITKTKG RSQEPQVYVL 360 APHPDELSKS KVSVTCLVKD FYPPEINIEW QSNGQPELET KYSTTQAQQD SDGSYFLYSK 420 LSVDRNRWQQ GTTFTCGVMH EALHNHYTQK NVSKNPGK                         458 where amino acids 1-121 comprise a rituximab variable region and amino acids 122-458 comprise a mutated sequence of the equine IgGl heavy chain constant region. Rituximab Equine IgGl WIN_PSS (L234A, L235A, G237A; Q330S, P331S) SEQ ID NO:50 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 SASTTAPKVF ALAPGCGTTS DSTVALGCLV SGYFPEPVKV SWNSGSLTSG VHTFPSVLQS 180 SGFYSLSSMV TVPASTWTSE TYICNWHAA SNFKVDKRIE PIPDNHQKVC DMSKCPKCPA 240 PEAAGAPSVF IFPPNPKDTL MITRTPEVTC VWDVSQENP DVKFNWYMDG VEVRTATTRP 300 KEEQFNSTYR WSVLRIQHQ DWLSGKEFKC KVNNQALPSS IERTITKTKG RSQEPQVYVL 360 APHPDELSKS KVSVTCLVKD FYPPEINIEW QSNGQPELET KYSTTQAQQD SDGSYFLYSK 420 LSVDRNRWQQ GTTFTCGVMH EALHNHYTQK NVSKNPGK                         458 where amino acids 1-121 comprise a rituximab variable region and amino acids 122-458 comprise a mutated sequence of the equine IgGl heavy chain constant region. Canine CTLA-4 Equine IgGl fusion PAP (Q330A) SEQ ID NO: 51 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDEPIPD NHQKVCDMSK CPKCPAPELL GGPSVFIFPP 180 NPKDTLMITR TPEVTCVWD VSQENPDVKF NWYMDGVEVR TATTRPKEEQ FNSTYRWSV 24 0 LRIQHQDWLS GKEFKCKVNN QALPAPIERT ITKTKGRSQE PQVYVLAPHP DELSKSKVSV 300 TCLVKDFYPP EINIEWQSNG QPELETKYST TQAQQDSDGS YFLYSKLSVD RNRWQQGTTF 360 TCGVMHEALH NHYTQKNVSK NPGK                                        384 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218) and amino acids 146-384 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgGl fusion SQP (P229S) SEQ ID NO: 52 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDEPIPD NHQKVCDMSK CPKCPAPELL GGPSVFIFPP 180 NPKDTLMITR TPEVTCVWD VSQENPDVKF NWYMDGVEVR TATTRPKEEQ FNSTYRWSV 24 0 LRIQHQDWLS GKEFKCKVNN QALSQPIERT ITKTKGRSQE PQVYVLAPHP DELSKSKVSV 300 TCLVKDFYPP EINIEWQSNG QPELETKYST TQAQQDSDGS YFLYSKLSVD RNRWQQGTTF 360 TCGVMHEALH NHYTQKNVSK NPGK                                        384 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218) and amino acids 146-384 comprise a fragment of equine IgGl heavy chain containing mutated the constant region.

[00190] The inventors of the instant application have surprisingly found that by substituting one or more amino acid residues in the Fc region of equine IgG4, advantageous characteristics may be introduced into equine IgGs, for example reducing or completely knocking out CDC, ADCC and ADCP activities or increasing or decreasing binding affinity to canine Fey receptor and Clq.

[00191] Accordingly, in various embodiments, an Fc region of equine IgG4 may comprise one or more mutations or substitutions, including at locations 229, 234, 235, 237, 329, 330, and / or 331. In various embodiments, an Fc region of equine IgG4 may comprise one or more mutations and / or substitutions selected from among L234A, Q235A, G237A, P239S, A33OQ, A330S, P331S, using EU Index numbering as in Kabat. For example, various combinations of substitutions in a Fc region of equine IgG4 can include one or more of the following, referred to herein by short designations: PSS (A330S, P331S), SQP (P239S, A33OQ), Win (L234A, Q235A, G237A), WinPSS (L234A, Q235A, G237A; A330S, P331S), SAP (P329S), and WinSAP (L234A, Q235A, G237A; P329S).

[00192] As further examples, mutated equine IgG4 Fc can be comprised within a recombinantly modified protein, e.g., an equine IgG heavy chain comprising a rituximab variable region and a mutated constant region, or a fusion protein comprising an effector region (such as a CTLA-4 sequence) fused to an equine IgG heavy chain comprising a mutated constant region. Examples of such proteins include the following sequences: Canine CTLA-4 Equine IgG4 fusion PSS (A330S, P331S) SEQ ID NO: 53 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC NGGCPAECLQ VGPSVFIFPP KPKDVLMISR 180 TPTVTCVWD VGHDFPDVQF NWYVDGVETH TATTEPKQEQ FNSTYRWSV LPIQHKDWLS 24 0 GKEFKCKVNN KALPSSVERT ISKPTGQPRE PQVYVLAPHR DELSKNKVSV TCLVKDFYPT 300 DIDIEWKSNG QPEPETKYST TPAQLDSDGS YFLYSKLTVE TNRWQQGTTF TCAVMHEALH 360 NHYTEKSVSK SPGK                                                   374 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-374 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG4 fusion SQP (P239S, A330Q) SEQ ID NO:54 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC NGGCPAECLQ VGPSVFIFPP KPKDVLMISR 180 TPTVTCVWD VGHDFPDVQF NWYVDGVETH TATTEPKQEQ FNSTYRWSV LPIQHKDWLS 24 0 GKEFKCKVNN KALSQPVERT ISKPTGQPRE PQVYVLAPHR DELSKNKVSV TCLVKDFYPT 300 DIDIEWKSNG QPEPETKYST TPAQLDSDGS YFLYSKLTVE TNRWQQGTTF TCAVMHEALH 360 NHYTEKSVSK SPGK                                                   374 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-374 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG4 fusion WIN_PSS (L234A, Q235A, G237A; A330S, P331S) SEQ ID NO: 55 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC NGGCPAECAA GAPSVFIFPP KPKDVLMISR 180 TPTVTCVWD VGHDFPDVQF NWYVDGVETH TATTEPKQEQ FNSTYRWSV LPIQHKDWLS 24 0 GKEFKCKVNN KALPSSVERT ISKPTGQPRE PQVYVLAPHR DELSKNKVSV TCLVKDFYPT 300 DIDIEWKSNG QPEPETKYST TPAQLDSDGS YFLYSKLTVE TNRWQQGTTF TCAVMHEALH 360 NHYTEKSVSK SPGK                                                   374 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-374 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG4 fusion WIN_SAP (L234A, Q235A, G237A; P329S) SEQ ID NO: 56 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC NGGCPAECAA GAPSVFIFPP FIFPP KPKDVLMISR 180 TPTVTCVWD VGHDFPDVQF NWYVDGVETH TATTEPKQEQ FNSTYRWSV LPIQHKDWLS 24 0 GKEFKCKVNN KALSAPVERT ISKPTGQPRE PQVYVLAPHR DELSKNKVSV TCLVKDFYPT 300 DIDIEWKSNG QPEPETKYST TPAQLDSDGS YFLYSKLTVE TNRWQQGTTF TCAVMHEALH 360 NHYTEKSVSK SPGK                                                   374 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-374 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG4 fusion SAP (P329S) SEQ ID NO: 57 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC NGGCPAECLQ VGPSVFIFPP KPKDVLMISR 180 TPTVTCVWD VGHDFPDVQF NWYVDGVETH TATTEPKQEQ FNSTYRWSV LPIQHKDWLS 24 0 GKEFKCKVNN KALSAPVERT ISKPTGQPRE PQVYVLAPHR DELSKNKVSV TCLVKDFYPT 300 DIDIEWKSNG QPEPETKYST TPAQLDSDGS YFLYSKLTVE TNRWQQGTTF TCAVMHEALH 360 NHYTEKSVSK SPGK                                                   374 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-374 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG4 fusion WIN (L234A, Q235A, G237A) SEQ ID NO: 58 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC NGGCPAECAA GAPSVFIFPP KPKDVLMISR 180 TPTVTCVWD VGHDFPDVQF NWYVDGVETH TATTEPKQEQ FNSTYRWSV LPIQHKDWLS 24 0 GKEFKCKVNN KALPAPVERT ISKPTGQPRE PQVYVLAPHR DELSKNKVSV TCLVKDFYPT 300 DIDIEWKSNG QPEPETKYST TPAQLDSDGS YFLYSKLTVE TNRWQQGTTF TCAVMHEALH 360 NHYTEKSVSK SPGK                                                   374 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-374 comprise a fragment of equine IgGl heavy chain containing mutated the constant region.

[00193] The inventors of the instant application have surprisingly found that by substituting one or more amino acid residues in the Fc region of equine IgG7, advantageous characteristics may be introduced into equine IgGs, for example reducing or completely knocking out CDC, ADCC and ADCP activities or increasing or decreasing binding affinity to canine Fey receptor and Clq.

[00194] Accordingly, in various embodiments, an Fc region of equine IgG7 may comprise one or more mutations or substitutions, including at locations 229, 234, 235, 237, 329, 330, and / or 331. In various embodiments, an Fc region of equine IgG7 may comprise one or more mutations and / or substitutions selected from among L234A, S235A, V236G, G237A, P239S, P329S, A330S, A330Q, P331S, using EU Index numbering as in Kabat. For example, various combinations of substitutions in a Fc region of equine IgG7 can include one or more of the following, referred to herein by short designations: PSS (A330S, P331S), SAP (P329S), SQP (P239S, A330Q), WIN (L234A, S235A, V236G, G237A), WIN_PSS (L234A, S235A, V236G, G237A; A330S, P331S), and WIN_SAP (L234A, S235A, V236G, G237A; P329S).

[00195] As further examples, mutated equine IgG7 Fc can be comprised within a recombinantly modified protein, e.g., an equine IgG heavy chain comprising a rituximab variable region and a mutated constant region, or a fusion protein comprising an effector region (such as a CTLA-4 sequence) fused to an equine IgG heavy chain comprising a mutated constant region. Examples of such proteins include the following sequences: Canine CTLA-4 Equine IgG7 fusion PSS (A330S, P331S) SEQ ID NO: 59 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC GGCPTCPECL SVGPSVFIFP PKPKDVLMIS 180 RTPTVTCVW DVGHDFPDVQ FNWYVDGVET HTATTEPKQE QNNSTYRWS ILAIQHKDWL 240 SGKEFKCKVN NQALPSSVQK TISKPTGQPR EPQVYVLAPH RDELSKNKVS VTCLVKDFYP 300 TDIDIEWKSN GQPEPETKYS TTPAQLDSDG SYFLYSKLTV ETNRWQQGTT FTCAVMHEAL 360 HNHYTEKSVS KSPGK                                                    375 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-375 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG7 fusion SAP (P329S) SEQ ID NO: 60 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC GGCPTCPECL SVGPSVFIFP PKPKDVLMIS 180 RTPTVTCVW DVGHDFPDVQ FNWYVDGVET HTATTEPKQE QNNSTYRWS ILAIQHKDWL 240 SGKEFKCKVN NQALSAPVQK TISKPTGQPR EPQVYVLAPH RDELSKNKVS VTCLVKDFYP 300 TDIDIEWKSN GQPEPETKYS TTPAQLDSDG SYFLYSKLTV ETNRWQQGTT FTCAVMHEAL 360 HNHYTEKSVS KSPGK                                                    375 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-375 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG7 fusion SQP (P239S, A330Q) SEQ ID NO: 61 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC GGCPTCPECL SVGPSVFIFP PKPKDVLMIS 180 RTPTVTCVW DVGHDFPDVQ FNWYVDGVET HTATTEPKQE QNNSTYRWS ILAIQHKDWL 240 SGKEFKCKVN NQALSQPVQK TISKPTGQPR EPQVYVLAPH RDELSKNKVS VTCLVKDFYP 300 TDIDIEWKSN GQPEPETKYS TTPAQLDSDG SYFLYSKLTV ETNRWQQGTT FTCAVMHEAL 360 HNHYTEKSVS KSPGK                                                    375 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-375 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG7 fusion WIN (L234A, S235A, V236G, G237A) SEQ ID NO: 62 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ  60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC GGCPTCPECA AGAPSVFIFP PKPKDVLMIS 180 RTPTVTCVW DVGHDFPDVQ FNWYVDGVET HTATTEPKQE QNNSTYRWS ILAIQHKDWL 240 SGKEFKCKVN NQALPAPVQK TISKPTGQPR EPQVYVLAPH RDELSKNKVS VTCLVKDFYP 300 TDIDIEWKSN GQPEPETKYS TTPAQLDSDG SYFLYSKLTV ETNRWQQGTT FTCAVMHEAL 360 HNHYTEKSVS KSPGK                                                    375 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-375 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG7 fusion WIN_PSS (L234A, S235A, V236G, G237A; A330S, P331S) SEQ ID NO: 63 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC GGCPTCPECA AGAPSVFIFP PKPKDVLMIS 180 RTPTVTCVW DVGHDFPDVQ FNWYVDGVET HTATTEPKQE QNNSTYRWS ILAIQHKDWL 240 SGKEFKCKVN NQALPSSVQK TISKPTGQPR EPQVYVLAPH RDELSKNKVS VTCLVKDFYP 300 TDIDIEWKSN GQPEPETKYS TTPAQLDSDG SYFLYSKLTV ETNRWQQGTT FTCAVMHEAL 360 HNHYTEKSVS KSPGK                                                    375 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-375 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Canine CTLA-4 Equine IgG7 fusion WIN_SAP (L234A, S235A, V236G, G237A; P329S) SEQ ID NO: 64 METDTLLLWV LLLWVPGSTG DMHVAQPAW LASSRGVASF VCEYGSSGNA AEVRVTVLRQ 60 AGSQMTEVCA ATYTVEDELA FLDDSTCTGT SSGNKVNLTI QGLRAMDTGL YICKVELMYP 120 PPYYVGMGNG TQIYVIDPEP CPDSDVIKEC GGCPTCPECA AGAPSVFIFP PKPKDVLMIS 180 RTPTVTCVW DVGHDFPDVQ FNWYVDGVET HTATTEPKQE QNNSTYRWS ILAIQHKDWL 240 SGKEFKCKVN NQALSAPVQK TISKPTGQPR EPQVYVLAPH RDELSKNKVS VTCLVKDFYP 300 TDIDIEWKSN GQPEPETKYS TTPAQLDSDG SYFLYSKLTV ETNRWQQGTT FTCAVMHEAL 360 HNHYTEKSVS KSPGK                                                    375 where amino acids 22-145 comprise a fragment of canine CTLA-4 (Accession XP_038303218)and amino acids 146-375 comprise a fragment of equine IgGl heavy chain containing mutated the constant region. Methods for Making Antibody Molecules of the Invention

[00196] Methods for making antibody molecules are well known in the art and fully described in U.S. Patents 8,394,925; 8,088,376; 8,546,543; 10,336,818; and 9,803,023 and U.S. Patent Application Publication 20060067930, which are incorporated by reference herein in their entirety. Any suitable method, process, or technique, known to one of skilled in the art, can be used. An antibody molecule having a variant Fc region of the invention may be generated according to the methods well known in the art. In some embodiments, the variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or binding domain of a receptor or ligand.

[00197] With the advent of methods of molecular biology and recombinant technology, a person of skilled in the art can produce antibody and antibody-like molecules by recombinant means and thereby generate gene sequences that code for specific amino acid sequences found in the polypeptide structure of the antibodies. Such antibodies can be produced by either cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with assembly of the synthesized chains to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants. This has permitted the ready production of antibodies having sequences characteristic of neutralizing antibodies from different species and sources.

[00198] Regardless of the source of the antibodies, or how they are recombinantly constructed, or how they are synthesized, in vitro or in vivo, using transgenic animals, large cell cultures of laboratory or commercial size, using transgenic plants, or by direct chemical synthesis employing no living organisms at any stage of the process, all antibodies have a similar overall 3 dimensional structure. This structure is often given as H2L2 and refers to the fact that antibodies commonly comprise two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are referred to as “variable” or “V” regions and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity. The variable regions of either H or L chains contain the amino acid sequences capable of specifically binding to antigenic targets.

[00199] As used herein, the term “antigen binding region” refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody binding region includes the “framework” amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the H or L chains that provide for the antigen binding regions are smaller sequences dubbed “hypervariable” because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as “complementarity determining regions” or “CDR” regions. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure.

[00200] The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have three CDR regions, each non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) and variable regions, and, within the latter, there are the CDRs and the so-called “framework regions” made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

[00201] The present invention further provides a vector including at least one of the nucleic acids described above. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which would be capable of encoding the amino acid. The probability that a particular oligonucleotide will, in fact, constitute the actual encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an antibody or portion. Such “codon usage rules” are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using the “codon usage rules” of Lathe, a single nucleotide sequence, or a set of nucleotide sequences that contains a theoretical “most probable” nucleotide sequence capable of encoding feline, equine, or canine IgG sequences can be identified. It is also intended that the antibody coding regions for use in the present invention could also be provided by altering existing antibody genes using standard molecular biological techniques that result in variants of the antibodies and peptides described herein. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the antibodies or peptides.

[00202] For example, one class of substitutions is conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a feline antibody peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Vai, Leu, and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg, replacements among the aromatic residues Phe, Tyr, and the like. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., 247 Science 1306-10 (1990).

[00203] Variant feline antibodies or peptides may be fully functional or may lack function in one or more activities. Fully functional variants typically contain only conservative variations or variations in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[00204] Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham etal., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as epitope binding or in vitro ADCC activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. Mal. Biol. 899-904 (1992); de Vos etal., 255 Science 306-12 (1992).

[00205] Moreover, polypeptides often contain amino acids other than the twenty “naturally occurring” amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP ribosylation, for instance, are described in most basic texts, such as Proteins-Structure and Molecular Properties (2nd ed., T. E. Creighton, W. H. Freeman & Co., N.Y., 1993). Many detailed reviews are available on this subject, such as by Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, N.Y., 1983); Seifter et al. iRZMeth. Enzyniol. 626-46 (1990); and Rattan et al. 663 Arm. NY Acad. Sci. 48-62 (1992).

[00206] In another aspect, the invention provides antibody derivatives. A “derivative” of an antibody contains additional chemical moi eties not normally a part of the protein. Covalent modifications of the protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents, well-known in the art, is useful for cross-linking the antibody or fragment to a water-insoluble support matrix or to other macromolecular carriers.

[00207] Derivatives also include radioactively labeled monoclonal antibodies that are labeled. For example, with radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, tritium (H3) or the like; conjugates of monoclonal antibodies with biotin or avidin, with enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholine esterase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemoluminescent agents (such as acridine esters) or fluorescent agents (such as phycobiliproteins).

[00208] Another derivative bifunctional antibody of the invention is a bispecific antibody, generated by combining parts of two separate antibodies that recognize two different antigenic groups. This may be achieved by crosslinking or recombinant techniques. Additionally, moieties may be added to the antibody or a portion thereof to increase half-life in vivo (e.g., by lengthening the time to clearance from the blood stream. Such techniques include, for example, adding PEG moieties (also termed pegylation), and are well-known in the art. See U.S. Patent. Appl. Pub. No. 20030031671.

[00209] In some embodiments, the nucleic acids encoding a subject antibody are introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. After the subject nucleic acids have been introduced into a cell, the cell is typically incubated, normally at 37° C., sometimes under selection, for a period of about 1-24 hours in order to allow for the expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the media in which the cell is growing. Traditionally, monoclonal antibodies have been produced as native molecules in murine hybridoma lines. In addition to that technology, the present invention provides for recombinant DNA expression of the antibodies. This allows the production of antibodies, as well as a spectrum of antibody derivatives and fusion proteins in a host species of choice.

[00210] A nucleic acid sequence encoding at least one antibody, portion or polypeptide of the invention may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al. 1993 supra, may be used to construct nucleic acid sequences which encode an antibody molecule or antigen binding region thereof.

[00211] A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as peptides or antibody portions in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the analogous art. See, e.g., Sambrook etal., 2001 supra, Ausubel et al., 1993 supra.

[00212] The present invention accordingly encompasses the expression of an antibody or peptide, in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts including bacteria, yeast, insects, fungi, bird and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. The mammalian cell or tissue may be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin. Any other suitable mammalian cell, known in the art, may also be used.

[00213] In one embodiment, the nucleotide sequence of the invention will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. See, e.g., Ausubel et al., 1993 supra. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.

[00214] Example prokaryotic vectors known in the art include plasmids such as those capable of replication in E.coli (such as, for example, pBR322, CoIEl, pSCIOl, pACYC 184, .pi.vX). Such plasmids are, for example, disclosed by Maniatis et al., 1989 supra; Ausubel et al, 1993 supra. Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, in THE MOLEC. BIO. OF THE BACILLI 307329 (Academic Press, NY, 1982).

[00215] Suitable Streptomyces plasmids include plJIOl (Kendall et al., 169 J. Bacterial. 4177-83 (1987), and Streptomyces bacteriophages such as phLC31 (Chater et al., in SIXTH INT'L SYMPOSIUM ON ACTINOMYCET ALES BIO. 45-54 (Akademiai Kaido, Budapest,

[00216] 30 Hungary 1986). Pseudomonas plasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704 (1986); lzak:i, 33 Jpn. J. Bacterial. 729-42 (1978); and Ausubel et al., 1993 supra.

[00217] Alternatively, gene expression elements useful for the expression of cDNA encoding antibodies or peptides include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982), and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985); (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites such as in SV40 (Okayama et al., 1983).

[00218] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51 Gene 21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin genes comprised of part cDNA, part genomic DNA (Whittle et al., I Protein Engin. 499 (1987)), the transcriptional promoter can be human cytomegalovirus, the promoter enhancers can be cytomegalovirus and mouse / human immunoglobulin, and mRNA splicing and polyadenylation regions can be the native chromosomal immunoglobulin sequences.

[00219] In an embodiment, for expression of cDNA genes in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are either or both the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells.

[00220] Each fused gene can be assembled in, or inserted into, an expression vector. Recipient cells capable of expressing the immunoglobulin chain gene product are then transfected singly with a peptide or H or L chain-encoding gene or are co-transfected with H and L chain gene. The transfected recipient cells are cultured under conditions that permit expression of the incorporated genes and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture.

[00221] In one embodiment, the fused genes encoding the peptide or H and L chains, or portions thereof are assembled in separate expression vectors that are then used to cotransfect a recipient cell. Alternatively the fused genes encoding the H and L chains can be assembled on the same expression vector. For transfection of the expression vectors and production of the antibody, the recipient cell line may be a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells such as B lymphocytes of feline or nonfeline origin, hybridoma cells of feline or non-feline origin, or interspecies heterohybridoma cells.

[00222] The expression vector carrying an antibody construct or polypeptide of the invention can be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

[00223] Yeast may provide substantial advantages over bacteria for the production of immunoglobulin H and L chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides). Hitzman et al., 11th IntT Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[00224] Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of peptides, antibodies, fragments and regions thereof. Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A number of approaches can be taken for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK 1985).

[00225] Bacterial strains can also be utilized as hosts for the production of antibody molecules or peptides described by this invention. Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts. The vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells. A number of approaches can be taken for evaluating the expression plasmids for the production of antibodies, fragments and regions or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, 1985 supra; Ausubel, 1993 supra; Sambrook, 2001 supra; Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, N.Y. (1994-2001); Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997-2001).

[00226] Host mammalian cells may be grown in vitro or in vivo. Mammalian cells provide posttranslational modifications to immunoglobulin protein molecules including leader peptide removal, folding and assembly of Hand L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein. Mammalian cells which can be useful as hosts for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vector systems are available for the expression of cloned peptides Hand L chain genes in mammalian cells (see Glover, 1985 supra). Different approaches can be followed to obtain complete H2L2 antibodies. It is possible to co-express Hand L chains in the same cells to achieve intracellular association and linkage of Hand L chains into complete tetrameric H2L2 antibodies and / or peptides. The co-expression can occur by using either the same or different plasmids in the same host. Genes for both Hand L chains and / or peptides can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells can be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker, cell lines producing peptides and / or H2L2 molecules via either route could be transfected with plasmids encoding additional copies of peptides, H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines.

[00227] For long-term, high-yield production of recombinant antibodies, stable expression may be used. For example, cell lines, which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with immunoglobulin expression cassettes and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and grow to form foci which in turn can be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluation of compounds / components that interact directly or indirectly with the antibody molecule.

[00228] Once an antibody of the invention has been produced, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In many embodiments, antibodies are secreted from the cell into culture medium and harvested from the culture medium.

[00229] An antibody having one or more substitutions as described herein can be any suitable antibody known to one of skilled in the art. In one example, the antibody is an anti-IL31 antibody. In another example, the antibody is an anti-NGF antibody.

[00230] Anti-IL31 antibodies, without the substitution described herein, are well known in the art and fully described in, for example, U.S. Patents 10,526,405; 10,421,807; 9,206,253; and 8,790,651. Also, anti-NGF antibodies, without the substitution described herein, are also well known in the art and fully described in, for example, U.S. Patents 10,125,192; 10,093,725; 9,951,128; 9,617,334; and 9,505,829.

[00231] In one embodiment, an anti-IL31 antibody of the invention (i.e., antibody having the substitution) reduces, inhibits, or neutralizes an IL-31-mediated pruritic or allergic condition. In another embodiment, the anti-IL3 1 antibody of the invention reduces, inhibits, or neutralizes IL-31 activity. VL, VH, and CDR sequences of the anti-IL31 antibodies are well known in the art and fully described in, for example, U.S. Patents 11,530,262; 10,526,405; 10,421,807; 9,206,253; and 8,790,651. In one embodiment, an anti-IL31 antibody of the invention may include a variable light chain comprising an amino acid sequence set forth therein. In another embodiment, the anti-IL3 1 antibody of the invention may include a variable heavy chain comprising the amino acid sequence set forth therein.

[00232] In one embodiment, the mutant anti-NGF antibody of the invention (z.e., antibody having the substitution) reduces, inhibits, or neutralizes NGF activity in an animal, and / or enhanced ability to inhibit NGF binding to Trk A and p75, in order to treat an NGF-mediated pain or condition. VL, VH, and CDR sequences of anti-NGF antibodies are also well known in the art and fully described in, for example, U.S. Patents 10,125,192; 10,093,725; 9,951,128; 9,617,334; and 9,505,829. In one example, the anti-NGF antibody of the invention may include at least one of the complementary determining region (CDR) sequences set forth therein. Pharmaceutical and Veterinary Applications

[00233] The invention also provides a pharmaceutical composition comprising molecules of the invention and one or more pharmaceutically acceptable carriers. More specifically, the invention provides for a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antibody or peptide according to the invention.

[00234] “Pharmaceutically acceptable carriers” include any excipient which is nontoxic to the cell or animal being exposed thereto at the dosages and concentrations employed. The pharmaceutical composition may include one or additional therapeutic agents.

[00235] “Pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit / risk ratio.

[00236] Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents and absorption delaying agents.

[00237] Pharmaceutically acceptable carriers include water; saline; phosphate buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphate, citrate and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; EDTA; salt forming counterions such as sodium; and / or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS; isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride; as well as combinations thereof.

[00238] The pharmaceutical compositions of the invention may be formulated in a variety of ways, including for example, liquid, semi-solid, or solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills, or powders. In some embodiments, the compositions are in the form of injectable or infusible solutions. The composition can be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical, or transdermal administration. The composition may be formulated as an immediate, controlled, extended or delayed release composition.

[00239] The compositions of the invention can be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. Administration of the antibodies disclosed herein may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), orally, or by topical administration of the antibodies (typically carried in a pharmaceutical formulation) to an airway surface. Topical administration to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler). Topical administration of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.

[00240] In some desired embodiments, the antibodies are administered by parenteral injection. For parenteral administration, antibodies or molecules can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle may be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as an aqueous carrier such vehicles are water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine and the like. Liposomes and nonaqueous vehicles such as fixed oils can also be used. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjustment agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these formulations can vary widely, for example from less than about 0.5%, usually at or at least about 1% to as much as 15% or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, REMINGTON’S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

[00241] The antibodies or molecules of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins. Any suitable lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss and that use levels may have to be adjusted to compensate. The compositions containing the present antibodies or a cocktail thereof can be administered for prevention of recurrence and / or therapeutic treatments for existing disease. Suitable pharmaceutical carriers are described in the most recent edition of REMINGTON’S PHARMACEUTICAL SCIENCES, a standard reference text in this field of art. In therapeutic application, compositions are administered to a subject already suffering from a disease, in an amount sufficient to cure or at least partially arrest or alleviate the disease and its complications.

[00242] Effective doses of the compositions of the present invention, for treatment of conditions or diseases as described herein vary depending upon many different factors, including, for example, but not limited to, the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; target site; physiological state of the animal; other medications administered; whether treatment is prophylactic or therapeutic; age, health, and weight of the recipient; nature and extent of symptoms kind of concurrent treatment, frequency of treatment, and the effect desired.

[00243] Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating veterinarian. In any event, the pharmaceutical formulations should provide a quantity of the antibody(ies) of this invention sufficient to effectively treat the subject. In some embodiments, the composition is administered bimonthly, once-in-three months, once-in-four months, once-in-five months, once-in-six months, or once- in-seven months. [00244J Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy. The pharmaceutical compositions of the invention may include a “therapeutically effective amount.” A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.

[00245] In another aspect, the compositions of the invention can be used, for example, in the treatment of various diseases and disorders. As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable. Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented. [00246J The composition having mutant molecule of the invention can be used to treat any suitable disease or disorder. For example, a mutant anti-IL3 1 antibody of the invention can be used to treat an IL-3I-mediated pruritic or allergic condition. The examples of IL-31-mediated pruritic condition include, for example, but not limited to, atopic dermatitis, eczema, psoriasis, scleroderma, and pruritis. The examples of IL-31-mediated allergic condition include, for example, but not limited to, allergic dermatitis, summer eczema, urticaria, heaves, inflammatory airway disease, recurrent airway obstruction, airway hyper-responsiveness, chronic obstruction pulmonary disease, and inflammatory processes resulting from autoimmunity.

[00247] A mutant anti-NGF antibody of the invention can be used to treat an NGF-mediated pain or a condition. The examples of a pain include, for example, but not limited to, a chronic pain, an inflammatory pain, a post-operative incision pain, a neuropathic pain, a fracture pain, an osteoporotic fracture pain, a post-herpetic neuralgia, a cancer pain, a pain resulting from bums, a pain associated with wounds, a pain associated with trauma, a neuropathic pain, a pain associated with a musculoskeletal disorder, a rheumatoid arthritis, an osteoarthritis, an ankylosing spondylitis, a seronegative (non-rheumatoid) an arthropathies, a non-articular rheumatism, a periarticular disorder, or a peripheral neuropathy. In a particular embodiment, the pain is an osteoarthritis pain.

[00248] All patents and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[00249] The following examples are offered to illustrate, but not to limit, the claimed embodiments. It is to be understood that the examples and embodiments described herein are for illustrative purposes only, and persons skilled in the art will recognize various parameters that can be altered without departing from the spirit of the disclosure or the scope of the appended claims. EXAMPLES

[00250] Example 1—CDC Cell-Based Assay of Feline IgG Fc Effector Function

[00251] The cell-based complement-dependent cytotoxicity (CDC) assay was developed and employed to characterize the effectiveness of two CTLA4 feline IgG subclass Fc fusion proteins in mediating CDC and to investigate Fc region mutations in the subclasses. The assay can be used in identifying key residues in the Fc region that determine CDC activity of the feline IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 which binds to CTLA4 on the Fc fusion proteins. These target cells have been used in past canine ADCC assays and were utilized in the CDC assay due to their dependability (Bergeron, McCandless et al. 2014).

[00252] Incubation of the fusion protein-bound target cells with feline complement-preserved serum can result in Fc binding on the fusion proteins to complement component Clq initiating the complement cascade, ultimately forming membrane attack complexes. The pore-forming complexes mediate cell lysis of the target cells measured by loss of cell viability. If there is no Fc binding to Clq there is no resultant cell lysis / death.

[00253] Methods. CD80-expressing CHO cells (target cells) were plated at 40,000 cells / well in CD CHO media in round-bottomed 96-well plates. Titrated fusion proteins in CD CHO media were added to the target cells and allowed to bind for 60 minutes at 37°C. Feline complement preserved serum (25% in CD CHO media) was added to the plates for 60 minutes at 37°C. Cell viability was then measured using CellTiter-Glo and data were expressed as “cell viability % of control” calculated using no fusion protein + complement preserved serum controls.

[00254] Results As shown in Figure 1 and Table 1, Feline IgGla and IgGlb Fc subclasses showed robust and potent CDC activity with EC50 values 66.0 and 68.9 ng / mL respectively. Feline IgG2 Fc subclass showed no CDC effector function activity. Feline IgGla and IgGlb Fc subclasses showed CDC activity. Feline IgG2 Fc subclass showed no CDC activity.

[00255] Table 1 CDC effects induced by feline IgG Fc wildtype subclasses. CTLA4 Feline IgG Fusion Protein CDC EC50 Value CTLA4FcflgGla 66.0 ng / mL CTLA4 FcflgGlb 68.9 ng / mL CTLA4FcflgG2 No CDC

[00256] As shown in Figures 2A and 2B, wild type feline IgGla Fc subclass showed robust and potent CDC activity, but several Fc mutations investigated all showed diminished CDC activity to varying degrees. In Figure 2A, feline IgGla WT Fc CTLA4 fusion protein both showed robust concentration-dependent CDC activity. The Win mutation showed a slight right shift of the concentration response curve. In Figure 2B, all seven of the listed Win plus mutations showed significant (60-75%) knock down of CDC effector function.

[00257] As shown in Figure 3, wild type feline IgGla Fc subclass showed robust and potent CDC activity, while several Fc mutations investigated all showed diminished CDC activity to varying degrees. Again, the Win mutation showed a slight right shift of the concentration response curve. CDC effector function was successively knocked down as the mutations went from DANG < DANG PSS < WinDANG < WinDANGPSS.

[00258] As shown in Figure 4, wild type IgGla Fc subclasses showed robust and potent CDC activity. However, several Fc mutations investigated all showed diminished CDC activity to varying degrees. Again, the Win mutation showed a slight right shift of the concentration response curve. The mutations GSP, GAP, DANG GAP and WinDANG GAP all knocked down CDC effector function 60-70%.

[00259] As shown in Figure 5, wild type IgGla Fc subclass showed robust and potent CDC activity. However, the Fc mutations investigated showed varying effects on CDC effector function ranging from no effect to completely knocking our CDC effector function. The mutations KAPA and WinKAPA both showed dramatic knock down of CDC effector function with WinKAPA showing complete knockout of effector function. However, the mutations D270G and D270S showed little effect on CDC effector function.

[00260] Example 2—ADCP Assay of Feline IgG Effector Function

[00261] The antibody dependent cellular phagocytosis (ADCP) assay utilizes CHO target cells engineered to express canine CD80, which binds to canine CTLA4 on the Fc fusion proteins. The Fc region of the fusion protein may then bridge this complex to Fc gamma receptors on activated macrophage effector cells, which have the capacity to phagocytose the target cell. ADCP is measured by fluorescent intensity and / or fluorescent area of a pH-sensitive fluorescent dye-stained target cells within the population of effector macrophages in the coculture, wherein fluorescent cells are indicative of an effector cell that has successfully internalized a target cell into the acidic lysosome.

[00262] Methods. Canine CD80-expressing CHO cells (CD80 target cells) or wild-type CHO cells which do not express CD80 (parental target cells) were stained with pHrodo red dye for 30 minutes at 37 degrees C. Stained cells were subsequently incubated with feline CTLA4-Fc fusion proteins for 60 minutes to mediate CTLA4:CD80 binding. 30,000 target cells were then added to 7,000 pre-plated Fcwf-4 effector cells (feline macrophage cell line). Co-cultures were maintained for 6-8 hours at 37 C as they were monitored and analyzed by a SX5 Incucyte live cell imaging system. Fluorescent area and / or intensity was quantified using a predefined processing definition in the Incucyte software and expressed as such.

[00263] Results. As shown in Figure 5, wild type IgGla showed concentration dependent ADCP activity. The Fc mutations investigated either showed little effect or a slight knock down of ADCP activity compared to the wild type. The GSP mutation in the Fc showed significant knockdown of ADCP activity.

[00264] As shown in Figure 6, wild type IgGla showed concentration dependent ADCP activity. The Fc mutations investigated showed varying effects on ADCP activity compared to the wild type. The WinDANGPSS mutation in the Fc showed knockdown of ADCP activity. However, the WinPSS mutation in the Fc showed an enhancement of ADCP activity.

[00265] As shown in Figure 7, wild type IgGla showed concentration-dependent ADCP activity. Several Fc mutations investigated either showed little effect or a slight knock down of ADCP activity compared to the wild type. The Win and WinDANG mutations have little or no effect on ADCP activity compared to wild type IgGla, but the WinDANGGAP mutation in the Fc showed knockdown of ADCP activity.

[00266] As shown in Figure 8, wild type IgGla showed concentration-dependent ADCP activity. The Fc mutations investigated either showed little effect or a dramatic knock down of ADCP activity compared to the wild type. The KAPA mutation in the Fc showed a dramatic knockout of ADCP activity. The D270G and D270S mutations showed no effect on ADCP activity.

[00267] As shown in Figure 9, wild type IgGla showed concentration-dependent ADCP activity. The Fc mutations investigated showed moderate knock down of ADCP activity compared to the wild type. The DANGGAP and WinKAPA mutation in the Fc showed a very strong knock down of ADCP activity.

[00268] Example 3—ADCC Assay of Feline IgG Effector Function

[00269] The ADCC cell-based assay was developed and employed to characterize the effectiveness of feline IgGla subclass Fc rituximab chimera IgGs in mediating ADCC and to investigate Fc region mutations in the subclass IgGla. This will help define key residues in the Fc region that determine ADCC activity of the feline IgG subclasses. The assay utilizes specially engineered HiBiT Ramos target cells (Promega) and express human CD20 which binds to our rituximab chimera IgGs. These target cells have been used in past human ADCC assays (Promega).

[00270] Incubation of rituximab chimera-bound target cells with cultured activated PBMCs can result in Fc binding on the IgGs to FcyRIII mediating granzyme and perforin release form the NK cells within the PBMC population. The actions of these proteins will result in cytotoxicity and lysis of the target cells bound by the IgGs. Upon killing of the target cell, the HiBiT fusion protein is released and binds extracellular LgBiT to create a functional NanoBiT® Luciferase enzyme (Promega). Luminescence is measured using a luciferase substrate and the GloMax® Discover System (Promega). If there is no Fc binding to FcyRIII there is no resultant target cell death measured.

[00271] Methods. HiBiT Ramos cells (target cells) were plated at 1,000 cells / well in EMEM media in 384-well plates. Titrated fusion proteins in EMEM media were added to the target cells and allowed to bind for 60 minutes at 37°C. Single donor canine PBMCs (effector cells), cultured overnight in RPMI 1640 medium + IL-2 and IL-15, were added to the plates for 5 hours at 37°C using an effectortarget cell ratio (E:F Ratio) of 25:1. Cells death was then quantified using the Promega Nano-Gio HiBiT Extracellular Detection System. Data were expressed as ’’Fold Induction” normalized to controls (minus effector PBMCs). Figure 13 summarizes the mutations investigated using the ADCC assay.

[00272] Results. As shown in Figure 10, wild type feline IgGla showed concentrationdependent ADCC activity. The Fc mutations investigated either showed a slight knock down or an enhancement of ADCP activity compared to the wild type. The Win mutation in the Fc showed knockdown of ADCC activity. Both the KAPA and DE mutations showed enhancement of ADCC activity, especially the DE mutation.

[00273] As shown in Figure 11, wild type IgGla showed concentration-dependent ADCP activity. The Fc mutations investigated showed varying knockdown to knockout effects on ADCC activity compared to the wild type. The DANG and DANG GAP mutations in the Fc showed knockdown of ADCC activity. The WinDANG GAP mutation in the Fc showed a knockout of ADCC activity.

[00274] As shown in Figure 12, wild type IgGla showed concentration-dependent ADCC activity. The Fc mutations investigated showed knock down of ADCC activity compared to the wild type. The WinKAPA, WinKA and WinPA mutations in the Fc showed knockdown of ADCC activity.

[00275] Example 4—In Silico Modeling of Feline IgG Fc Regions

[00276] The Fc regions of the feline backbone subclasses, IgGla and IgGlb were first designed using their respective CH2 and CH3 regions. The protein modeling feature of Alphafold 2.2 developed by Deepmind was implemented to model the 3D structure of each of wild-type (WT) and mutant constructs of each feline backbone subclass and allotypes.

[00277] Methods. Molecular Operating Environment (MOE) developed by Chemical Computing Group (MOE2019.0102) provides a flexible and automated graphical user interface for protein modeling. To analyze the difference in structures, the sequence-to-profile alignment algorithm uses a scoring algorithm to rank the sequence templates and scores higher than 85% ensure the selection of protein templates with physically realistic structures. The model was then optimized using the same pipeline and the structural stability of the models was verified using Ramachandran Plots, which checks the stereochemical quality of a protein structure.

[00278] This method was performed on wild-type (WT) constructs and mutations in the following positions: M234, L235, G237, S239, D265, D270, N297, K322, P329, S330, P331, and 1332. The models were overlayed, and the mutated residues were displayed in ball and stick form (Fig. 14A). An RMSD plot was generated to calculate the root mean square deviation of the two WT structures, Feline-IgGla WT and Feline-IgGlb WT relative to each other (Fig. 14B). An RMSD value of 2.0A or lower is considered the standard for considering two structures to be alike. Results indicated that Feline IgGla construct was identical in its fold to the feline IgGlb subclass with average RMSD value for the structure being 1.05A. RMSDs at the twelve positions where mutational scanning was performed was also noted in Table 2A with values ranging from 0.2-0.9A.

[00279] Table 2A. Root Mean Square Deviation (RMSD) comparisons of positions where mutations were performed in the protein models of WT Feline IgGla and IgGlb. Residue in IgGla WT Residue in IgGlb WT RMSD (A) M234 P234 0.5 L235 L235 0.9 G237 G237 0.6 S239 S239 0.4 D265 D265 0.4 D270 D270 0.2 N297 N297 0.4 K322 R322 0.3 P329 P329 0.4 S330 S330 0.4 P331 P331 0.3 1332 1332 0.4

[00280] The method described above was performed on the wild-type (WT) constructs and mutations in the following positions: G236, G267, P268, S298, N324, E333, R334 and E345. The models were overlayed, and the mutated residues were displayed in ball and stick form (Fig 14C). The root mean square deviation of the two WT structures, Feline-IgGla WT and Feline-IgGlb WT relative to each other was calculated. RMSDs at the twelve positions where mutational scanning was performed is shown in Table 2B with values ranging from 0.1-0.7A. An RMSD value of 2.0A or lower is considered the standard for considering two structures to be alike. Results indicated that Feline IgGla construct was identical in its fold to the feline IgGlb subclass with average RMSD value for the structure being 1.05A.

[00281] Table 2B. Root Mean Square Deviation (RMSD) comparisons of positions where mutations were performed in the protein models of WT Feline IgGla and IgGlb. Residue in IgGla WT Residue in IgGlb WT RMSD (A) G236 G236 0.7 G267 G267 0.4 P268 P268 0.4 S298 S298 0.4 N324 N324 0.3 E333 E333 0.4 R334 R334 0.3 E345 E345 0.1

[00282] Results. Molecular modeling of all mutations across the two IgGl allotypes, IgGla and IgGlb of feline backbones were performed and validated using MOE2019.0102. The Fc fold and residue conformation of between subclasses were shown to have RMSDs less than 2A, indicating that the protein structures have very high identity and therefore mutations in the positions in IgGla would function in a similar manner when extrapolated to IgGlb.

[00283] Example 5—CDC Cell-Based Assay of Canine IgG Fc Effector Function

[00284] The cell-based complement-dependent cytotoxicity (CDC) assay was developed and employed to characterize the effectiveness of two CTLA4 canine IgG subclass Fc fusion proteins in mediating CDC and to investigate Fc region mutations in the subclasses. The assay can be used in identifying key residues in the Fc region that determine CDC activity of the feline IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 which binds to CTLA4 on the Fc fusion proteins. These target cells have been used in past canine ADCC assays and were utilized in the CDC assay due to their dependability (Bergeron, McCandless et al. 2014).

[00285] Methods. Incubation of the fusion protein-bound target cells with feline complement-preserved serum can result in Fc binding on the fusion proteins to complement component Clq initiating the complement cascade, ultimately forming membrane attack complexes. The pore-forming complexes mediate cell lysis of the target cells measured by loss of cell viability. If there is no Fc binding to Clq there is no resultant cell lysis / death.

[00286] CD80-expressing CHO cells (target cells) were plated at 40,000 cells / well in CD CHO media in round-bottomed 96-well plates. Titrated fusion proteins in CD CHO media were added to the target cells and allowed to bind for 60 minutes at 37°C. Complement preserved serum (25% final concentration in CD CHO media) was added to the plates for 60 minutes at 37°C. Cell viability was then measured using CellTiter-Glo and data were expressed as “cell viability % of control” calculated using no fusion protein + complement preserved serum controls.

[00287] Results. As shown in Figure 15 and Table 3, Canine IgG2 Fc subclass showed robust and potent CDC activity with an EC50 value of 298.7 ng / mL. Canine IgGl Fc subclass showed no CDC effector function activity.

[00288] Table 3. CDC effects induced by canine IgGl and IgG2 Fc wildtype subclasses. CTLA4 Canine IgG Fusion Protein CDC EC50 Value CTLA4 FcflgGl No CDC CTLA4 FcflgG2 298.7 ng / mL

[00289] As shown in Figure 16, wild type canine IgG2 Fc subclass showed robust and potent CDC activity. The Fc mutations investigated showed no effect, diminished, or completely knocked out CDC activity. The D270S mutation of the Fc showed no effect on CDC activity. The Win mutation showed a slight right shift of the concentration response curve. The D70G mutation showed a knockdown of CDC activity. The six mutations GSP, GAP, DANG GAP, WIN DANG GAP, KAPA and WIN KAPA showed complete knockout of CDC effector function.

[00290] Example 6—ADCP Assay of Canine IgG Effector Function

[00291] The antibody dependent cellular phagocytosis (ADCP) assay utilizes CHO target cells engineered to express canine CD80, which binds to canine CTLA4 on the Fc fusion proteins. The Fc region of the fusion protein may then bridge this complex to Fc gamma receptors on activated macrophage effector cells, which have the capacity to phagocytose the target cell. ADCP is measured by fluorescent intensity and / or fluorescent area of a pH-sensitive fluorescent dye-stained target cells within the population of effector macrophages in the coculture, wherein fluorescent cells are indicative of an effector cell that has successfully internalized a target cell into the acidic lysosome.

[00292] Methods. Canine CD80-expressing CHO cells (CD80 target cells) or wild-type CHO cells which do not express CD80 (parental target cells) were stained with pHrodo red dye for 30 minutes at 37 degrees C. Stained cells were subsequently incubated with canine CTLA4-Fc fusion proteins for 60 minutes to mediate CTLA4:CD80 binding. 30,000 target cells were then added to 7,000 pre-plated DH-82 cells (canine macrophage cell line). Co-cultures were maintained for 6-8 hours at 37 °C and they were monitored and analyzed by a SX5 Incucyte live cell imaging system. Fluorescent area and / or intensity was quantified using a predefined processing definition in the Incucyte software and expressed as such.

[00293] Results. As shown in Figure 17, wild type IgG2 showed concentration-dependent ADCP activity. The Fc mutations investigated either showed knockout of ADCP activity compared to the wild type. The Win and GAP mutations in the Fc showed knockout of ADCP activity. [00294J As shown in Figure 18, wild type lgG2 showed concentration-dependent ADCP activity. The Fc mutations investigated showed knockdown effects on ADCP activity compared to the wild type. The DANGGAP and WIN DANG GAP mutations in the Fc showed knockdown of ADCP activity.

[00295] As shown in Figure 19, wild type IgG2 showed concentration-dependent ADCP activity. The Fc mutations investigated either showed knockdown or knockout of ADCP activity compared to the wild type. The KAPA mutation in the Fc showed knockdown of ADCP activity. The GSP mutation showed knockout of ADCP activity.

[00296] As shown in Figure 20, wild type IgG2 showed concentration-dependent ADCP activity. The Fc mutations investigated showed moderate knock down of ADCP activity compared to the wild type. The DANG GAP and WinKAPA mutation in the Fc showed a moderate knockdown to almost complete knockout of ADCP activity, respectively.

[00297] As shown in Figure 21, wild type IgG2 showed concentration dependent ADCP activity. The Fc mutations investigated either showed knockdown of ADCP activity or little effect compared to the wild type. The D270G mutation in the Fc showed knockdown of ADCP activity. The D270S mutation showed no effect on ADCP activity.

[00298] Example 7—ADCC Assay of Canine IgG Effector Function

[00299] The ADCC cell-based assay was developed and employed to characterize the effectiveness of canine subclass IgG2aFc rituximab chimera IgGs in mediating ADCC and to investigate Fc region mutations in the subclasses. This will help define key residues in the Fc region that determine ADCC activity of the feline IgG subclasses. The assay utilizes specially engineered HiBiT Ramos target cells (Promega) and express human CD20 which binds to our rituximab chimera IgGs. These target cells have been used in past human ADCC assays (Promega).

[00300] Incubation of the rituximab chimera-bound target cells with cultured activated PBMCs can result in Fc binding on the IgGs to FcyRIII mediating granzyme and perforin release form the NK cells within the PBMC population. The actions of these proteins will result in cytotoxicity and lysis of the target cells bound by the IgGs. Upon killing of the target cell, the HiBiT fusion protein is released and binds extracellular LgBiT to create a functional NanoBiT® Luciferase enzyme. Luminescence is measured using a luciferase substrate and the GloMax® Discover System. If there is no Fc binding to FcyRIII there is no resultant target cell death measured.

[00301] Methods. HiBiT Ramos cells (target cells) were plated at 1,000 cells / well in EMEM media in 384-well plates. Titrated fusion proteins in EMEM media were added to the target cells and allowed to bind for 60 minutes at 37°C. Single donor canine PBMCs (effector cells), cultured overnight in RPMI 1640 medium + IL-2 and IL-15, were added to the plates for 5 hours at 37°C using an effector:target cell ratio (E:F Ratio) of 25:1. Cells death was then quantified using the Promega Nano-Gio HiBiT Extracellular Detection System. Data were expressed as ’’Fold Induction” normalized to controls (minus effector PBMCs).

[00302] Results. As shown in Figure 22, wild type canine IgG2 showed concentrationdependent ADCC activity. The Fc mutations investigated either showed a slight knock down or knockout of ADCP activity compared to the wild type. The GAP mutation showed a knockdown of ADCC activity. The Win, DANG, DANG_GAP and WinDANG_GAP mutations showed a knockout of ADCC activity.

[00303] As shown in Figure 23, wild type IgG2 showed concentration-dependent ADCC activity. The Fc mutations investigated showed varying knockdown to knockout effects on ADCC activity compared to the wild type. The Win mutation in the Fc showed knockout of ADCC activity. The KAPA, DE, DAE and DLE mutations showed slight enhancement of ADCC activity.

[00304] As shown in Figure 24, wild type IgG2 showed concentration-dependent ADCC activity. The Fc mutations investigated either showed knock down of ADCC activity compared to the wild type. The WinKAPA, WinKA and WinPA mutations in the Fc showed knockdown of ADCC activity with WinKAPA showing the most effective knockout of ADCC activity.

[00305] Example 8—Canine Fey receptor and Clq binding assay.

[00306] Binding of canine WT IgG2 (IgG 65) and mutant Fc were measured by surface plasmon resonance. IgGs were used as the ligand and captured to a sensor chip for analysis of canine Fey receptor and Clq binding. 10 mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% v / v Surfactant P20 pH 7.4 was used as titration and method running buffer. Canine Fey receptors and Human Clq were used as analytes and flowed over captured Fc mutant IgGs. Mutations were made in several locations listed in Table 4.

[00307] Table 4—Listing of mutation locations in canine IgG2 assayed for effect on canine Fey receptor and Clq binding, using EU Index numbering as in Kabat. 231 232 233 234 235 236 237 238 239 240 262 263 264 265 266 267 268 269 270 271 294 295 296 297 298 299 300 301 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 422 423 441

[00308] Table 5- -Listing of mutations in canine IgG2 assayed for effect on canine Fey receptor and Clq binding, using EU Index numbering as in Kabat. A231C A2 31D A231E A231G A2 31H A2 31K A231N A2 31Q A231R A2 31S A231T A2 31V A231Y P232A P232C P232D P232E P232F P232G P232H P232I P232K P232L P232M P232N P232Q P232R P232S P232T P232V P232W P232Y E233A E233C E233D E233F E233G E233H E233I E233K E233L E233M E233N E233P E233Q E233R E233S E233T E233V E233W E233Y M234A M234C M234D M234E M2 34F M234G M234H M2 341 M234K M234L M234N M234P M234Q M234R M2 34S M234T M234W M234Y L235A L235C L235D L235E L235F L235G L235H L235I L235K L235M L235N L235P L235Q L235R L235S L235T L2 35V L235W L235Y G2 3 6A G236C G236D G2 3 6E G236F G236H G236I G236K G236L G2 3 6M G236N G236P G236Q G236R G236S G236T G23 6V G2 3 6W G2 3 6Y G237A G237C G237D G237E G237F G237H G237I G237K G237L G237M G237N G237P G237Q G237R G2 37S G237T G237V G237W G237Y P238A P238C P238D P238E P238F P238G P238H P238I P238K P238L P2 3 8M P238N P238Q P238R P238S P238T P238V P238W P238Y S2 3 9A S239C S239D S239E S239F S239G S239H S239I S239K S239L S239M S239N S239P S239Q S239R S239T S239V S239W S239Y V24 0A V240C V2 4 0D V2 4 0E V24 0F V2 4 0G V240H V2 4 0I V2 4 0K V240L V24 0M V2 4 0N V240P V240Q V2 4 0R V240S V240T V2 4 0W V240Y V2 62A V262C V262D V262E V2 62F V262G V2 62H V2 62K V262L V2 62M V262Q V262R V262S V262T V2 62W V262Y V2 63A V263C V2 63E V263F V263G V263H V263I V263K V263L V2 63M V2 63N V263P V263Q V263S V263T V2 63W V263Y V2 64A V264C V264D V264E V264F V264G V264H V2 64I V264K V264L V2 64M V264N V264P V264Q V264R V264S V264T V264W V264Y D2 65A D265C D2 65E D265F D265G D265H D265I D265K D2 65L D2 65M D265N D265P D2 65Q D265R D265S D265T D2 65V D265W D265Y L2 6 6A L266C L266D L266E L266F L266G L266H L266I L266K L2 6 6M L266N L266P L2 6 6Q L266R L266S L266T L266V L2 6 6W L266Y D2 67A D267C D267E D267F D267G D267H D267I D267K D267L D267M D267N D267P D267Q D267R D267S D267T D267V D267W D267Y P268A P268C P268D P268E P268F P268G P268H P268I P268K P268L P268M P268N P268Q P268R P268S P268T P268V P268W P268Y E269A E269C E269D E269F E269G E269H E269I E269K E269L E269M E269N E269P E269Q E269R E269S E269T E269V E269W E269Y D27 0A D270C D270F D270G D270H D270I D270K D270L D27 0M D270N D270P D270Q D270R D270S D270V D270W D270Y P271C P271F P271G P271H P271I P271K P271L P271M P271N P271Q P271R P271S P271T P271Y E294A E294C E294D E294F E294G E294H E294I E294K E294L E294M E294N E294P E294Q E294R E294S E294T E294V E294W E294Y Q2 95A Q295D Q295E Q295F Q295G Q295K Q295L Q295N Q295P Q295R Q295S Q295T Q295V Q295W Q295Y F2 9 6A F296C F2 9 6D F2 9 6E F296G F2 9 6H F296I F296K F2 9 6L F2 9 6M F2 96N F296P F296Q F296R F296S F296T F2 96V N2 97A N297C N297D N297E N297F N297G N297H N297I N297K N297L N297M N297P N297Q N297R N297S N297T N297V N297W N297Y G298A G298C G298D G298E G298F G298H G298I G298K G298L G298M G298N G298P G298Q G298R G298S G298T G298V G298W G298Y T299A T299C T299D T299E T299F T299G T299H T299I T299K T299L T2 99M T299N T299P T299Q T299R T299S T299V T299W T299Y Y3 0 0A Y300C Y300D Y300E Y300H Y300I Y300K Y300L Y300M Y300N Y300P Y300Q Y300R Y300S Y300T Y3 0 0V Y300W R301A R301C R301D R301E R301F R301G R301H R301I R301L R301P R301Q R301V R301W R301Y V323A V323C V323D V323E V323F V323G V323H V323K V323L V32 3M V323N V323P V323Q V323R V323S V323T V323W V323Y N324A N324C N324D N324E N324F N324G N324H N324I N324K N324L N324M N324P N324Q N324R N324S N324T N324V N324W N324Y N325A N325C N325D N325E N325F N325G N325H N325I N325K N325L N32 5M N325P N325Q N325R N325S N325T N32 5V N325W N325Y K32 6A K326D K326E K326F K326I K326L K32 6N K326R K326S K326T K32 6V K326W A327C A327D A327E A327F A327G A327H A327I A327K A327L A32 7M A327N A327P A327Q A327R A327S A327T A327V A327W A327Y L328A L328C L328D L328E L328F L328G L328H L328I L328K L328M L328N L328P L328Q L328R L328S L328T L328V L328W L328Y K322A K322C K322D K322E K322F K322G K322H K322I K322L K322M K322N K322P K322Q K322R K322S K322T K322V K322W K322Y P329A P329C P329D P329E P329F P329G P329H P329I P329K P329L P329M P329N P329Q P329R P329S P329T P329V P329W P329Y S330A S330C S330D S330E S330F S330G S330H S330I S330K S330L S330M S330N S330P S330Q S330R S330T S330V S330W S330Y P331A I332A I332D I332E I332F I332G I332H I332K I332L I332M I332N I332P I332R I332T I332V I332W I332Y E333A E333D E333H E333I E333K E333N E333R E333S E333V E333Y R334A R334C R334D R334E R334F R334G R334L R334M R334N R334P R334Q R334S R334T R334V R334W R334Y T335C T335H T335I T335K T335L T335P I336D I336E I336G I336K I336N I336Y S337D S337P K338A K338C K338D K338E K338F K338G K338H K33 8I K338L K338M K338N K338P K338Q K338R K338S K338V K338W K338Y A339D A339F A339G A339N A339P A33 9R A339S A339T A3 3 9V A339W A33 9Y R340G T422K T422R F423P L441P

[00309] Kinetics and affinities were determined using Cytiva 8K evaluation software. Blank runs containing buffer only were subtracted out from all runs. Flow cells were regenerated using lOmM Glycine pH 1.5. Runs were performed at room temperature. Mutations (Table 5) made at respective canine mutant Fc IgG positions have a marked effect on the kinetics and / or affinity of the IgG to canine Fey receptors and / or human Clq when compared against the respective WT Fc kinetic and / or affinity values (Table 6).

[00310] Table 6—Binding kinetics and affinities of canine Fey receptors and / or human C Iq to WT IgG2 and mutant canine IgG2 with indicated substitutions in Fc. (LS=Low Signal. NBO=No binding observed.) EU numbering system KD M) ID No. Codon usage mutation AA mutation 1:1 binding Steady State 1:1 binding 1:1 binding canFcgRl canFcgR2b canFcgR3 hClq 1 WT 1 WT1 4.64E-09 2.89E-06 4.81E-07 2.18E-09 2 TGC A231C - 6.51E-07 - 7.09E-09 3 GAC A231D - - - 6.97E-09 4 GAG A231E - - - 1.02E-08 5 GGC A231G - - 1.11E-06 7.61E-09 6 CAC A231H - - - 5.43E-09 7 AAG A231K - 1.24E-05 8.98E-07 5.26E-09 8 AAC A231N - - - 2.03E-08 9 CAG A231Q. - - - 8.55E-09 10 AGG A231R - 8.12E-05 1.45E-06 1.15E-08 11 TCC A231S - - - 7.76E-09 12 ACC A231T - 7.59E-07 - - 13 GTG A231V - - - 1.47E-09 14 TAC A231Y - - 8.90E-07 - 15 GCC P232A - - 8.00E-07 9.63E-09 16 TGC P232C - - - 1.45E-08 17 GAC P232D 2.32E-09 7.96E-07 7.50E-07 6.90E-09 18 GAG P232E - 9.75E-07 - 3.17E-08 19 TTC P232F - - 2.37E-07 4.08E-09 20 GGC P232G - - 1.01E-06 8.20E-07 21 CAC P232H - - 1.23E-06 3.91E-08 22 ATC P232I - 1.00E-06 - 4.25E-09 23 AAG P232K 9.36E-09 LS LS 3.29E-07 24 CTG P232L - 7.74E-07 - - 25 ATG P232M - - - 4.50E-09 26 AAC P232N - - 9.34E-07 5.99E-08 27 CAG P232Q. - - 7.97E-07 7.98E-09 28 AGG P232R 9.84E-09 LS LS 3.10E-07 29 TCC P232S - - 1.26E-06 8.08E-09 30 ACC P232T - - 7.57E-07 8.90E-09 31 GTG P232V - - - 5.42E-09 32 TGG P232W - 7.91E-07 2.45E-07 - 33 TAC P232Y - - - 7.15E-09 34 GCC E233A 8.57E-09 LS 1.65E-06 3.81E-08 35 TGC E233C LS LS - 4.94E-09 36 GAC E233D - - - 5.05E-09 37 TTC E233F 9.87E-09 LS 1.54E-06 1.81E-08 38 GGC E233G 9.48E-09 LS 1.03E-06 LS 39 CAC E233H 1.00E-08 LS 2.86E-06 1.58E-08 40 ATC E233I 8.74E-09 LS LS LS 41 AAG E233K 1.26E-08 LS LS LS 42 CTG E233L 9.20E-09 LS 9.14E-07 1.74E-08 43 ATG E233M 9.07E-09 LS 1.41E-06 3.11E-08 44 AAC E233N 1.03E-08 LS LS LS 45 CCC E233P 9.05E-09 LS 1.09E-06 3.99E-09 46 CAG E233Q. - LS 1.68E-06 1.26E-08 47 AGG E233R 1.18E-08 LS LS LS 48 TCC E233S 9.29E-09 LS LS LS 49 ACC E233T 9.60E-09 LS LS LS 50 GTG E233V 9.78E-09 LS LS 2.82E-08 51 TGG E233W 9.19E-09 LS 6.07E-07 3.99E-09 52 TAC E233Y 1.08E-08 LS LS 1.03E-08 53 GCC M234A - - 1.49E-06 1.34E-08 54 TGC M234C - LS LS LS 55 GAC M234D LS 3.23E-07 LS 3.23E-08 56 GAG M234E 6.26E-09 2.51E-07 LS 1.02E-08 57 TTC M234F - 1.01E-06 8.21E-07 1.03E-08 58 GGC M234G LS LS LS LS 59 CAC M234H 8.01E-09 - 9.05E-07 1.94E-08 60 ATC M234I - - 1.95E-07 - 61 AAG M234K 1.08E-08 LS LS LS 62 CTG M234L - - - 9.20E-09 63 AAC M234N 9.39E-09 - 9.43E-07 - 64 CCC M234P - - - LS 65 CAG M234Q - - - 1.07E-08 66 AGG M234R 9.82E-09 LS LS LS 67 TCC M234S 7.95E-09 - 1.27E-06 3.62E-08 68 ACC M234T - - - 7.14E-09 69 TGG M234W 6.90E-09 8.38E-07 - - 70 TAC M234Y - - LS 2.92E-08 71 GCC L235A NBO - 8.60E-07 LS 72 TGC L235C NBO LS LS LS 73 GAC L235D NBO 3.57E-07 7.79E-07 LS 74 GAG L235E NBO 5.94E-07 - LS 75 TTC L235F LS LS - - 76 GGC L235G NBO LS LS LS 77 CAC L235H LS LS - 3.52E-08 78 ATC L235I 8.78E-09 - - 6.30E-09 79 AAG L235K NBO LS LS LS 80 ATG L235M 7.71E-09 - 8.00E-07 6.44E-09 81 AAC L235N NBO - LS LS 82 CCC L235P NBO - 7.66E-07 - 83 CAG L235Q NBO - 6.05E-07 LS 84 AGG L235R NBO LS LS NBO 85 TCC L235S NBO 8.20E-06 LS LS 86 ACC L235T NBO LS - LS 87 GTG L235V 8.04E-09 - - 4.14E-08 88 WT 2 WT2 6.14E-09 1.65E-04 4.22E-06 1.74E-08 89 TGG L235W LS LS - - 90 TAC L235Y LS - - - 91 GCC G236A 1.23E-08 1.16E-05 LS LS 92 TGC G236C NBO NBO LS NBO 93 GAC G236D 1.42E-08 - LS - 94 GAG G236E 1.15E-08 4.47E-06 LS - 95 TTC G236F 1.42E-08 - LS - 96 CAC G236H LS - LS LS 97 ATC G236I 1.40E-08 LS LS LS 98 AAG G236K NBO NBO LS NBO 99 CTG G236L 1.99E-08 LS LS - 100 ATG G236M 1.83E-08 LS LS 1.05E-06 101 AAC G236N LS LS LS - 102 CCC G236P NBO LS LS LS 103 CAG G236Q 2.12E-08 LS LS LS 104 AGG G236R NBO NBO LS NBO 105 TCC G236S 1.55E-08 - LS LS 106 ACC G236T 1.27E-08 LS LS - 107 GTG G236V 1.50E-08 - LS LS 108 TGG G236W 9.13E-09 1.68E-07 LS 1.18E-09 109 TAC G236Y 1.55E-08 - LS 2.49E-09 110 GCC G237A 1.49E-08 - LS 9.67E-07 111 TGC G237C NBO NBO LS NBO 112 GAC G237D 1.19E-08 LS LS NBO 113 GAG G237E 1.08E-08 LS LS NBO 114 TTC G237F LS LS LS - 115 CAC G237H 2.33E-08 NBO LS NBO 116 ATC G237I NBO LS LS LS 117 AAG G237K NBO NBO LS NBO 118 CTG G237L NBO LS LS NBO 119 ATG G237M 3.39E-08 LS LS NBO 120 AAC G237N 2.67E-08 LS LS NBO 121 CCC G237P NBO LS LS NBO 122 CAG G237Q LS LS LS NBO 123 AGG G237R NBO NBO LS NBO 124 TCC G237S 1.37E-08 LS LS LS 125 ACC G237T 1.30E-08 LS LS NBO 126 GTG G237V LS LS LS LS 127 TGG G237W 1.65E-07 LS LS LS 128 TAC G237Y LS LS LS LS 129 GCC P238A - LS LS NBO 130 TGC P238C NBO LS LS NBO 131 GAC P238D 1.49E-08 LS LS NBO 132 GAG P238E 1.14E-08 LS LS NBO 133 TTC P238F - LS LS NBO 134 GGC P238G 1.99E-08 NBO LS NBO 135 CAC P238H 1.40E-08 NBO LS NBO 136 ATC P238I - LS LS NBO 137 AAG P238K NBO NBO LS NBO 138 CTG P238L - NBO LS NBO 139 ATG P238M - LS LS NBO 140 AAC P238N 1.40E-08 LS LS NBO 141 CAG P238Q 1.42E-08 LS LS NBO 142 AGG P238R NBO LS LS NBO 143 TCC P238S 1.62E-08 LS LS NBO 144 ACC P238T 1.34E-08 LS LS NBO 145 GTG P238V 1.06E-08 LS LS NBO 146 TGG P238W LS LS LS NBO 147 TAC P238Y 1.20E-08 LS LS NBO 148 GCC S239A - - 3.32E-05 - 149 TGC S239C - - LS LS 150 GAC S239D 1.29E-09 5.76E-07 - - 151 GAG S239E 1.80E-09 4.64E-07 - - 152 TTC S239F 1.15E-08 LS LS LS 153 GGC S239G - - LS NBO 154 CAC S239H 1.35E-08 LS LS LS 155 ATC S239I - 6.66E-07 4.21E-05 LS 156 AAG S239K LS LS LS - 157 CTG S239L - 4.78E-07 4.04E-05 LS 158 ATG S239M - 3.27E-06 - - 159 AAC S239N 8.84E-09 - 3.26E-05 - 160 CCC S239P - LS LS NBO 161 CAG S239Q - - 5.88E-05 - 162 AGG S239R 2.09E-08 LS LS - 163 ACC S239T - - 6.33E-05 - 164 GTG S239V - 3.10E-06 LS - 165 TGG S239W 1.00E-08 - LS LS 166 TAC S239Y 1.33E-08 - LS LS 167 GCC V240A - - 6.23E-07 - 168 TGC V240C - - - 1.14E-08 169 GAC V240D LS LS LS LS 170 GAG V240E - - 1.35E-05 1.19E-09 171 TTC V240F - - LS 7.48E-08 172 GGC V240G - - - LS 173 CAC V240H - - - 2.03E-09 174 ATC V240I - - - 1.34E-09 175 AAG V240K NBO LS NBO NBO 176 CTG V240L - - - 7.01E-09 177 ATG V240M - - - 7.63E-09 178 AAC V240N - - - 3.99E-09 179 CCC V240P - LS 2.43E-05 - 180 CAG V240Q - - - 8.62E-10 181 AGG V240R NBO LS NBO NBO 182 WT 3 WT3 5.25E-09 1.17E-06 4.13E-07 8.36E-09 183 TCC V240S - - 1.98E-07 - 184 ACC V240T - - - - 185 TGG V240W 9.66E-09 NBO NBO LS 186 TAC V240Y NBO NBO NBO NBO 187 GCC V262A - LS LS 1.75E-08 188 TGC V262C - 4.26E-05 LS 2.77E-08 189 GAC V262D - 9.25E-05 LS 1.40E-08 190 GAG V262E - NBO NBO 1.13E-07 191 TTC V262F - 1.46E-05 LS 1.71E-07 192 GGC V262G - 7.52E-06 NBO 3.54E-08 193 CAC V262H - 2.48E-05 LS 2.12E-08 194 AAG V262K - 9.55E-05 LS 1.66E-08 195 CTG V262L - - 2.61E-07 - 196 ATG V262M - 2.99E-04 - - 197 CAG V262Q 4.17E-09 - LS 2.54E-08 198 AGG V262R - 1.30E-04 NBO LS 199 TCC V262S - 1.24E-04 LS 1.45E-08 200 ACC V262T - - LS LS 201 TGG V262W - 1.80E-04 LS LS 202 TAC V262Y - 1.82E-04 NBO NBO 203 GCC V263A - - NBO LS 204 TGC V263C - - LS 1.62E-08 205 GAG V263E NBO NBO NBO NBO 206 TTC V263F - 1.78E-04 NBO LS 207 GGC V263G - 9.29E-05 NBO LS 208 CAC V263H NBO NBO NBO NBO 209 ATC V263I - - 2.09E-07 5.58E-09 210 AAG V263K NBO NBO NBO NBO 211 CTG V263L - 1.79E-04 LS LS 212 ATG V263M - 3.78E-04 LS - 213 AAC V263N LS NBO NBO NBO 214 CCC V263P NBO NBO NBO NBO 215 CAG V263Q NBO NBO NBO NBO 216 TCC V263S - 2.22E-05 LS LS 217 ACC V263T - 8.81E-05 - 6.36E-09 218 TGG V263W NBO NBO NBO NBO 219 TAC V263Y NBO NBO NBO NBO 220 GCC V264A - NBO NBO LS 221 TGC V264C - NBO NBO LS 222 GAC V264D LS NBO NBO LS 223 GAG V264E LS NBO NBO LS 224 TTC V264F - NBO NBO - 225 GGC V264G - NBO NBO LS 226 CAC V264H - NBO NBO LS 227 ATC V264I - - 1.78E-07 5.74E-09 228 AAG V264K 4.15E-08 NBO NBO LS 229 CTG V264L - 1.79E-04 NBO - 230 ATG V264M - 1.15E-04 NBO - 231 AAC V264N LS 2.05E-04 NBO - 232 CCC V264P 1.35E-08 NBO NBO LS 233 CAG V264Q - NBO NBO 3.79E-08 234 AGG V264R NBO NBO NBO NBO 235 TCC V264S - NBO NBO 1.54E-08 236 ACC V264T - 2.25E-04 NBO - 237 TGG V264W - NBO NBO 1.85E-08 238 WT 4 WT4 3.48E-09 8.34E-06 2.58E-07 7.66E-09 239 TAC V264Y - LS NBO - 240 GCC D265A LS NBO NBO 3.82E-07 241 TGC D265C 7.29E-09 NBO NBO 2.08E-08 242 GAG D265E 5.07E-09 NBO NBO 3.68E-07 243 TTC D265F NBO NBO NBO NBO 244 GGC D265G NBO NBO NBO LS 245 CAC D265H LS NBO NBO LS 246 ATC D265I NBO NBO NBO NBO 247 AAG D265K NBO NBO NBO LS 248 CTG D265L NBO NBO NBO NBO 249 ATG D265M NBO NBO NBO LS 250 AAC D265N LS NBO NBO - 251 CCC D265P LS NBO NBO NBO 252 CAG D265Q LS NBO NBO LS 253 AGG D265R NBO NBO NBO LS 254 TCC D265S LS NBO NBO 1.45E-07 255 ACC D265T 7.75E-09 NBO NBO NBO 256 GTG D265V NBO NBO NBO NBO 257 TGG D265W NBO NBO NBO NBO 258 TAC D265Y NBO NBO NBO NBO 259 GCC L266A - NBO NBO NBO 260 TGC L266C - NBO NBO NBO 261 GAC L266D 6.90E-09 NBO NBO NBO 262 GAG L266E 6.00E-09 NBO NBO NBO 263 TTC L266F 7.11E-09 NBO NBO NBO 264 GGC L266G 1.56E-09 LS NBO NBO 265 CAC L266H LS NBO NBO NBO 266 ATC L266I 1.69E-09 - - - 267 AAG L266K NBO NBO NBO NBO 268 ATG L266M - - LS NBO 269 AAC L266N - NBO NBO NBO 270 CCC L266P LS NBO NBO NBO 271 CAG L266Q 8.27E-09 NBO NBO NBO 272 AGG L266R NBO NBO NBO NBO 273 TCC L266S 7.84E-09 NBO NBO NBO 274 ACC L266T 7.79E-09 NBO NBO NBO 275 GTG L266V 2.45E-09 - LS - 276 TGG L266W 6.37E-09 NBO NBO NBO Til TAC L266Y 7.00E-09 NBO NBO NBO TT& GCC D267A - 6.31E-05 - NBO Tl^ TGC D267C 9.58E-09 NBO NBO NBO 280 GAG D267E - 5.54E-05 - 2.23E-08 281 TTC D267F 6.75E-09 NBO NBO NBO 282 GGC D267G 6.80E-09 LS NBO NBO 283 CAC D267H 7.19E-09 NBO NBO NBO 284 ATC D267I LS NBO LS NBO 285 AAG D267K NBO NBO NBO NBO 286 CTG D267L - NBO NBO NBO 287 ATG D267M NBO NBO NBO NBO 288 AAC D267N 6.21E-09 NBO LS NBO 289 CCC D267P 6.59E-09 NBO NBO NBO 290 CAG D267Q - NBO NBO NBO 291 AGG D267R NBO NBO NBO NBO 292 TCC D267S - 6.45E-05 - NBO 293 ACC D267T 6.66E-09 NBO LS NBO 294 GTG D267V - NBO LS NBO 295 TGG D267W - NBO LS NBO 296 TAC D267Y - NBO LS NBO 297 GCC P268A 6.53E-09 1.40E-04 LS - 298 TGC P268C - 1.61E-04 LS - 299 GAC P268D - - 1.36E-07 3.33E-09 300 GAG P268E - 6.98E-05 LS 3.37E-09 301 TTC P268F - 5.01E-04 LS 9.93E-10 302 GGC P268G - 1.03E-04 - - 303 CAC P268H - 5.71E-04 LS - 304 ATC P268I - NBO LS - 305 AAG P268K - NBO LS NBO 306 CTG P268L - NBO LS - 307 ATG P268M - NBO - 5.05E-09 308 AAC P268N - - - LS 309 CAG P268Q. - 2.88E-04 - - 310 AGG P268R - NBO LS NBO 311 TCC P268S - 3.09E-05 - - 312 ACC P268T - 2.72E-04 - - 313 GTG P268V - NBO LS - 314 TGG P268W - NBO LS 6.17E-11 315 TAC P268Y - 1.81E-04 LS 5.24E-10 316 GCC E269A - 3.22E-05 - LS 317 TGC E269C - 3.51E-04 - NBO 318 GAC E269D - 1.38E-04 - 3.99E-09 319 TTC E269F - 4.14E-04 LS LS 320 GGC E269G - 1.90E-04 LS LS 321 CAC E269H - 1.37E-04 LS NBO 322 ATC E269I - 1.21E-04 LS NBO 323 AAG E269K 7.73E-09 NBO LS NBO 324 CTG E269L - 7.75E-04 LS LS 325 ATG E269M - 1.17E-04 LS LS 326 AAC E269N - 3.30E-04 LS LS 327 CCC E269P - NBO NBO NBO 328 CAG E269Q - 2.85E-04 LS LS 329 AGG E269R 7.17E-09 NBO NBO NBO 330 TCC E269S - 2.50E-04 LS NBO 331 ACC E269T - 2.30E-04 LS LS 332 WT 5 WT5 2.97E-09 1.41E-06 7.17E-07 9.65E-09 333 GTG E269V - - LS KO 334 TGG E269W - LS LS - 335 TAC E269Y - LS LS LS 336 GCC D270A - 6.49E-06 LS NBO 337 TGC D270C - 4.55E-07 - NBO 338 TTC D270F - - LS - 339 GGC D270G - LS - NBO 340 CAC D270H - - LS LS 341 ATC D270I - - - LS 342 AAG D270K - LS LS NBO 343 CTG D270L - - - 4.90E-07 344 ATG D270M - - - NBO 345 AAC D270N - - - 2.80E-07 346 CCC D270P - - LS NBO 347 CAG D270Q - - - NBO 348 AGG D270R 7.07E-09 NBO NBO NBO 349 TCC D270S - 8.52E-06 - 4.83E-07 350 GTG D270V - - - NBO 351 TGG D270W - - LS NBO 352 TAG D270Y - - LS - 353 TGC P271C - - - NBO 354 TTC P271F - - - LS 355 GGC P271G - - LS - 356 CAC P271H - - - LS 357 ATC P271I - - - LS 358 AAG P271K - - - NBO 359 CTG P271L - - - NBO 360 ATG P271M - - - NBO 361 AAC P271N - - - NBO 362 CAG P271Q. - - - NBO 363 AGG P271R - - LS NBO 364 TCC P271S - - - LS 365 ACC P271T - - - NBO 366 TAC P271Y - - - LS 367 GCC E294A - 3.50E-06 - NBO 368 TGC E294C - - - NBO 369 GAC E294D - NBO LS NBO 370 TTC E294F - - - NBO 371 GGC E294G - - LS NBO 372 CAC E294H - - - NBO 373 ATC E294I - - - NBO 374 AAG E294K - - - NBO 375 CTG E294L - - 2.93E-07 LS 376 ATG E294M - - 2.57E-07 NBO 377 AAC E294N - NBO - NBO 378 CCC E294P - - - NBO 379 CAG E294Q. - - - LS 380 AGG E294R - - - NBO 381 TCC E294S - LS LS NBO 382 ACC E294T - LS LS NBO 383 GTG E294V - - - LS 384 TGG E294W - LS - - 385 TAC E294Y - LS - - 386 GCC Q295A - LS LS - 387 GAC Q295D - NBO LS - 388 GAG Q295E - - LS - 389 TTC Q295F - NBO LS LS 390 GGC Q295G - NBO NBO NBO 391 AAG Q295K - LS LS LS 392 CTG Q295L - 8.98E-06 - - 393 AAC Q295N - NBO NBO - 394 CCC Q295P - NBO LS NBO 395 AGG O295R - LS LS LS 396 TCC Q295S - NBO LS - 397 ACC Q295T - NBO LS - 398 GTG Q295V - LS LS - 399 TGG Q295W - NBO NBO NBO 400 TAC Q295Y - NBO LS - 401 GCC F296A - - NBO - 402 TGC F296C - - LS - 403 GAC F296D - - LS - 404 GAG F296E - - LS - 405 GGC F296G - LS LS LS 406 CAC F296H - - LS - 407 ATC F296I - - LS - 408 AAG F296K - - LS - 409 CTG F296L - - LS - 410 ATG F296M - - LS - 411 AAC F296N - - LS - 412 CCC F296P LS NBO NBO LS 413 CAG F296Q - - LS - 414 AGG F296R - - LS - 415 WT 6 WT6 5.31E-09 1.67E-06 4.10E-07 1.06E-08 416 TCC F296S - - NBO NBO 417 ACC F296T - - LS LS 418 GTG F296V - - LS - 419 GCC N297A NBO NBO NBO NBO 420 TGC N297C NBO NBO NBO NBO 421 GAC N297D NBO NBO NBO NBO 422 GAG N297E NBO NBO NBO NBO 423 TTC N297F NBO NBO NBO NBO 424 GGC N297G NBO NBO NBO NBO 425 CAC N297H 1.51E-08 NBO NBO NBO 426 ATC N297I 1.39E-08 NBO NBO NBO 427 AAG N297K NBO NBO NBO NBO 428 CTG N297L NBO NBO NBO NBO 429 ATG N297M NBO NBO NBO NBO 430 CCC N297P NBO NBO NBO NBO 431 CAG N297Q NBO NBO NBO NBO 432 AGG N297R NBO NBO NBO NBO 433 TCC N297S NBO NBO NBO NBO 434 ACC N297T NBO NBO NBO NBO 435 GTG N297V NBO NBO NBO NBO 436 TGG N297W 1.28E-08 NBO NBO NBO 437 TAC N297Y 1.14E-08 NBO NBO NBO 438 GCC G298A 2.90E-09 LS - - 439 TGC G298C 1.02E-09 3.95E-07 - NBO 440 GAC G298D - LS LS LS 441 GAG G298E - LS LS LS 442 TTC G298F - LS LS 1.83E-09 443 CAC G298H - LS LS LS 444 ATC G298I - LS LS 7.06E-10 445 AAG G298K 1.74E-09 - NBO NBO 446 CTG G298L 1.66E-09 3.52E-07 LS 4.49E-09 447 ATG G298M 1.81E-09 8.65E-07 LS - 448 AAC G298N - NBO NBO NBO 449 CCC G298P NBO NBO NBO NBO 450 CAG G298Q 3.46E-09 LS LS LS 451 AGG G298R 2.41E-09 - LS NBO 452 TCC G298S - - LS - 453 ACC G298T 1.79E-09 NBO NBO 5.72E-09 454 GTG G298V - NBO LS 3.02E-09 455 TGG G298W - NBO 1.42E-06 - 456 TAC G298Y - 4.40E-05 - 1.69E-09 457 GCC T299A NBO - NBO NBO 458 TGC T299C NBO NBO NBO NBO 459 GAC T299D NBO NBO NBO NBO 460 GAG T299E NBO NBO NBO NBO 461 TTC T299F NBO NBO NBO LS 462 GGC T299G NBO - NBO NBO 463 CAC T299H NBO NBO LS NBO 464 ATC T299I NBO NBO NBO NBO 465 AAG T299K NBO NBO NBO NBO 466 CTG T299L NBO NBO NBO LS 467 ATG T299M NBO NBO NBO LS 468 AAC T299N NBO NBO NBO LS 469 CCC T299P NBO NBO LS NBO 470 CAG T299Q NBO NBO NBO NBO 471 AGG T299R NBO NBO NBO NBO 472 TCC T299S LS - LS LS 473 GTG T299V NBO NBO LS NBO 474 TGG T299W NBO NBO LS LS 475 TAC T299Y NBO NBO LS - 476 GCC Y300A - LS 2.53E-07 LS 477 TGC Y300C - LS - LS 478 GAC Y300D - - 2.40E-07 6.51E-10 479 GAG Y300E 2.75E-09 - - - 480 CAC Y300H - - - NBO 481 ATC Y300I - - - LS 482 AAG Y300K - LS LS NBO 483 CTG Y300L 2.69E-09 - - LS 484 ATG Y300M - - 1.69E-07 - 485 AAC Y300N - - - LS 486 CCC Y300P LS NBO NBO NBO 487 CAG Y300Q 2.77E-09 - - LS 488 AGG Y300R 3.24E-09 - LS NBO 489 TCC Y300S - LS 3.96E-07 LS 490 ACC Y300T - LS 3.58E-07 LS 491 GTG Y300V - LS - LS 492 TGG Y300W - - LS - 493 GCC R301A - - 1.11E-06 - 494 TGC R301C - LS LS - 495 GAC R301D - LS LS - 496 GAG R301E - LS LS LS 497 TTC R301F - LS LS - 498 GGC R301G - LS LS - 499 CAC R301H - LS LS LS 500 ATC R301I - LS LS - 501 CTG R301L - - LS - 502 WT 7 WT7 5.95E-09 1.41E-06 2.09E-07 1.20E-09 503 CCC R301P 8.46E-09 NBO LS NBO 504 CAG R301Q - LS LS - 505 GTG R301V - - LS - 506 TGG R301W - - LS - 507 TAC R301Y - 2.62E-05 LS - 508 GCC V323A - - - 4.11E-09 509 TGC V323C - - - - 510 GAC V323D 1.13E-08 LS LS NBO 511 GAG V323E - LS LS NBO 512 TTC V323F - - - NBO 513 GGC V323G - - - LS 514 CAC V323H - - 6.08E-08 NBO 515 AAG V323K 1.08E-08 LS NBO NBO 516 CTG V323L - - 1.18E-07 NBO 517 ATG V323M - - - NBO 518 AAC V323N - LS LS NBO 519 CCC V323P 1.00E-08 5.20E-05 LS NBO 520 CAG V323Q - 7.28E-06 LS NBO 521 AGG V323R LS NBO LS NBO 522 TCC V323S - - LS NBO 523 ACC V323T - - - LS 524 TGG V323W - LS LS NBO 525 TAC V323Y - - LS NBO 526 GCC N324A - - - NBO 527 TGC N324C 1.07E-08 - - NBO 528 GAC N324D - - - NBO 529 GAG N324E - - LS NBO 530 TTC N324F - - - NBO 531 GGC N324G - - - LS 532 CAC N324H - - - NBO 533 ATC N324I - - - LS 534 AAG N324K 1.16E-08 LS LS NBO 535 CTG N324L - - - LS 536 ATG N324M - - - NBO 537 CCC N324P NBO NBO NBO NBO 538 CAG N324Q - - - NBO 539 AGG N324R - LS LS NBO 540 TCC N324S - - - NBO 541 ACC N324T - - - NBO 542 GTG N324V - - - NBO 543 TGG N324W - - - LS 544 TAC N324Y - - - NBO 545 GCC N325A - LS - LS 546 TGC N325C - LS LS NBO 547 GAC N325D - - - LS 548 GAG N325E - - LS NBO 549 TTC N325F 9.14E-09 NBO LS NBO 550 GGC N325G - - LS NBO 551 CAC N325H - LS - NBO 552 ATC N325I - NBO LS NBO 553 AAG N325K 1.34E-08 NBO LS NBO 554 CTG N325L - LS LS NBO 555 ATG N325M - LS LS NBO 556 CCC N325P LS NBO LS NBO 557 CAG N325Q - - LS NBO 558 AGG N325R LS NBO LS NBO 559 TCC N325S - - - NBO 560 ACC N325T - - - NBO 561 GTG N325V 1.02E-08 NBO LS NBO 562 TGG N325W 1.05E-08 NBO LS NBO 563 TAC N325Y 1.07E-08 NBO LS NBO 564 GCC K326A - - - 5.69E-10 565 GAC K326D - 9.82E-07 5.78E-06 LS 566 GAG K326E - 9.12E-07 LS NBO 567 TTC K326F - - 4.70E-06 5.73E-10 568 ATC K326I - - - LS 569 CTG K326L - - - LS 570 AAC K326N - 9.58E-07 - 5.72E-10 571 AGG K326R - - - 7.56E-10 572 TCC K326S - - - NBO 573 ACC K326T - - - NBO 574 GTG K326V - - - LS 575 TGG K326W - - - 9.99E-10 576 TGC A327C - - - NBO 577 GAC A327D - - LS LS 578 GAG A327E - 7.48E-07 - LS 579 TTC A327F - - - 5.66E-10 580 GGC A327G - 8.20E-07 - - 581 CAC A327H - - LS LS 582 WT 8 WT8 5.42E-09 5.79E-06 8.54E-07 9.68E-09 583 ATC A327I - - NBO NBO 584 AAG A327K 1.02E-08 NBO NBO NBO 585 CTG A327L - - LS LS 586 ATG A327M - - 2.95E-05 1.34E-06 587 AAC A327N - - LS 2.32E-06 588 CCC A327P - LS NBO NBO 589 CAG A327Q. - - - 9.53E-07 590 AGG A327R 1.26E-08 NBO NBO NBO 591 TCC A327S - - 7.53E-06 1.14E-06 592 ACC A327T 9.27E-09 LS NBO NBO 593 GTG A327V - LS NBO NBO 594 TGG A327W - LS LS 1.03E-06 595 TAC A327Y - 3.22E-05 3.10E-05 - 596 GCC L328A - - LS NBO 597 TGC L328C - LS NBO NBO 598 GAC L328D - LS NBO NBO 599 GAG L328E 9.25E-09 LS NBO NBO 600 TTC L328F 2.50E-09 1.99E-07 NBO NBO 601 GGC L328G 9.21E-09 LS NBO NBO 602 CAC L328H 9.65E-09 NBO NBO NBO 603 ATC L328I - 6.48E-07 - NBO 604 AAG L328K NBO NBO NBO NBO 605 ATG L328M 2.79E-09 6.22E-07 LS NBO 606 AAC L328N - LS LS NBO 607 CCC L328P NBO NBO NBO NBO 608 CAG L328Q - LS NBO NBO 609 AGG L328R NBO NBO NBO NBO 610 TCC L328S - - LS NBO 611 ACC L328T - - 2.05E-05 NBO 612 GTG L328V - 8.81E-07 - 1.71E-06 613 TGG L328W - 1.70E-07 NBO NBO 614 TAC L328Y 2.10E-09 2.46E-07 NBO NBO 615 GCC K322A - - 6.26E-06 NBO 616 TGC K322C - - - NBO 617 GAC K322D - - 5.16E-06 NBO 618 GAG K322E - - - NBO 619 TTC K322F - - 5.48E-06 NBO 620 GGC K322G - - - NBO 621 CAC K322H - - - NBO 622 ATC K322I - - - NBO 623 CTG K322L - - - NBO 624 ATG K322M - - - NBO 625 AAC K322N - - - NBO 626 CCC K322P NBO NBO NBO NBO 627 CAG K322Q - - - NBO 628 AGG K322R - - - NBO 629 TCC K322S - - LS NBO 630 ACC K322T - - - NBO 631 GTG K322V - - - NBO 632 TGG K322W - - - NBO 633 TAC K322Y - - - NBO 634 GCC P329A 9.41E-09 NBO NBO NBO 635 TGC P329C NBO NBO NBO NBO 636 GAC P329D NBO NBO NBO NBO 637 GAG P329E NBO NBO NBO NBO 638 TTC P329F NBO NBO NBO NBO 639 GGC P329G 1.07E-08 NBO NBO NBO 640 CAC P329H NBO NBO NBO NBO 641 ATC P329I NBO NBO NBO NBO 642 AAG P329K NBO NBO NBO NBO 643 CTG P329L NBO NBO NBO NBO 644 ATG P329M NBO NBO NBO NBO 645 AAC P329N NBO NBO NBO NBO 646 CAG P329Q. NBO NBO NBO NBO 647 AGG P329R NBO NBO NBO NBO 648 TCC P329S NBO NBO NBO NBO 649 ACC P329T NBO NBO NBO NBO 650 GTG P329V 7.44E-09 NBO NBO NBO 651 TGG P329W NBO NBO NBO NBO 652 TAG P329Y NBO NBO NBO NBO 653 GCC S330A - - - 1.22E-09 654 TGC S330C 6.54E-10 3.73E-07 - NBO 655 GAC S330D 8.50E-09 LS - NBO 656 GAG S330E - - - NBO 657 TTC S330F - 1.05E-06 - NBO 658 GGC S330G - - - NBO 659 CAC S330H - - - 1.92E-09 660 ATC S330I 1.83E-09 9.18E-07 - NBO 661 AAG S330K 1.58E-09 - 3.86E-07 NBO 662 CTG S330L 1.60E-09 9.57E-07 - NBO 663 ATG S330M 2.52E-09 - 3.29E-07 LS 664 AAC S330N - 9.35E-07 - NBO 665 CCC S330P NBO NBO NBO NBO 666 CAG S330Q 2.77E-09 - - NBO 667 AGG S330R 1.64E-09 - - NBO 668 ACC S330T - - - NBO 669 GTG S330V 1.86E-09 6.28E-07 - NBO 670 TGG S330W - - LS NBO 671 TAC S330Y - 1.09E-06 - NBO 672 GCC P331A - - - LS 673 WT 9 WT9 5.79E-09 2.89E-06 2.08E-07 7.49E-09 674 GCC I332A 8.63E-09 - - - 675 GAC I332D - 7.44E-07 - - 676 GAG I332E - 2.06E-07 - - 677 TTC I332F - LS LS - 678 GGC I332G LS LS LS LS 679 CAC I332H - LS LS LS 680 AAG I332K LS NBO NBO LS 681 CTG I332L - - - 4.27E-09 682 ATG I332M - - - 3.79E-09 683 AAC I332N 9.60E-09 LS LS 2.13E-08 684 CCC I332P 1.06E-08 LS NBO NBO 685 AGG I332R LS NBO NBO LS 686 ACC I332T - - - 1.79E-09 687 GTG 1332V - - - 2.59E-09 688 TGG I332W 8.51E-09 NBO NBO 2.31E-08 689 TAC I332Y 8.27E-09 - - 3.54E-09 690 GCC E333A - - - 4.44E-09 691 GAC E333D - - - 2.70E-09 692 CAC E333H - - - 2.43E-09 693 ATC E333I - - - 1.41E-08 694 AAG E333K - - - 3.83E-09 695 AAC E333N - - - 2.52E-09 696 AGG E333R - - - 1.23E-09 697 TCC E333S - - - 2.73E-09 698 GTG E333V - - - 1.65E-08 699 TAG E333Y - - - 7.48E-10 700 GCC R334A - 6.97E-07 1.05E-07 - 701 TGC R334C - 5.97E-07 9.43E-08 1.07E-08 702 GAC R334D - 9.31E-07 1.10E-07 8.98E-09 703 GAG R334E - 4.69E-07 7.95E-08 - 704 TTC R334F - - - 3.70E-09 705 GGC R334G - 9.47E-07 9.46E-08 - 706 CTG R334L - 6.44E-07 1.13E-07 LS 707 ATG R334M - 5.01E-07 1.02E-07 - 708 AAC R334N - 9.60E-07 1.34E-07 - 709 CCC R334P - - - NBO 710 CAG R334Q. - 5.95E-07 1.13E-07 - 711 TCC R334S - - 1.17E-07 - 712 ACC R334T - 7.47E-07 1.31E-07 3.87E-09 713 GTG R334V - - 1.37E-07 - 714 TGG R334W - - - 1.33E-08 715 TAC R334Y - - - 4.04E-09 716 TGC T335C - - - 1.05E-08 717 CAC T335H - - - 4.93E-09 718 ATC T335I - - - 5.45E-09 719 AAG T335K - - - 6.01E-09 720 CTG T335L - - - 6.55E-09 721 CCC T335P - - - 4.29E-09 722 WT10 WT 10 6.76E-09 9.97E-06 3.51E-07 1.07E-08 723 AAG T422K - - - 2.39E-09 724 AGG T422R - - - 1.45E-09 725 CCC F423P - - - 3.40E-09 726 CCC L441P - - - 2.22E-09 727 WT11 WT 11 5.56E-09 5.60E-07 1.52E-07 5.22E-09 ITS, GAC I336D - - NBO - 729 GAG I336E - - - 1.03E-08 730 GGC I336G - - LS - 731 AAG I336K - - - 9.00E-09 732 AAC I336N - - LS - 733 TAC I336Y 2.36E-09 - - 1.60E-09 734 GAC S337D - - - 2.70E-09 735 CCC S337P - - LS 5.86E-10 736 GCC K338A - - - 3.06E-09 737 TGC K338C - - LS 2.14E-09 738 GAC K338D - - LS 5.24E-10 739 GAG K338E -                -               LS 1.05E-09 740 TTC K338F - - - 1.42E-10 741 GGC K338G - - - 1.10E-09 742 CAC K338H - - - 1.54E-09 743 ATC K338I - - LS 2.07E-09 744 CTG K338L - - LS 2.27E-09 745 ATG K338M - - LS 1.25E-09 746 AAC K338N - - - 4.85E-10 747 CCC K338P - - - 2.51E-09 748 CAG K338Q. - - LS - 749 AGG K338R 1.04E-08 LS LS LS 750 TCC K338S - - - 2.40E-09 751 GTG K338V - LS LS - 752 TGG K338W - - LS 1.23E-10 753 TAC K338Y - - - 7.85E-10 754 GAC A339D - - - 2.18E-09 755 TTC A339F - - - 2.04E-09 756 GGC A339G - - - 1.13E-09 757 AAC A339N - - - 1.45E-09 758 CCC A339P - - - 9.41E-10 759 AGG A339R - - - 2.63E-09 760 TCC A339S - - - 2.71E-09 761 ACC A339T 2.60E-09 - - 1.09E-09 762 GTG A339V 3.07E-09 - - 2.58E-09 763 TGG A339W - - - 7.13E-10 764 TAC A339Y - - - 1.06E-09 765 GGC R340G - - - 2.15E-09

[00311] Example 9—CDC Cell-Based Assay of Equine IgG Fc Effector Function

[00312] The CDC cell-based assay was developed and employed to characterize the effectiveness of the nine CTLA4 equine IgG subclass Fc fusion proteins in mediating CDC and to investigate Fc region mutations in the subclasses. This will help define key residues in the Fc region that determine CDC activity of the equine IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 which binds to CTLA4 on the Fc fusion proteins. These target cells have been used in past canine ADCC assays and were utilized in the CDC assay due to their dependability.

[00313] Incubation of the fusion protein-bound target cells with complement-preserved serum can result in Fc binding on the fusion proteins to complement component Clq initiating the complement cascade, ultimately forming membrane attack complexes. The pore-forming complexes mediate cell lysis of the target cells measured by loss of cell viability. If there is no Fc binding to Clq there is no resultant cell lysis / death.

[00314] Methods. CD80-expressing CHO cells (target cells) were plated at 40,000 cells / well in CD CHO media in round-bottomed 96-well plates. Titrated fusion proteins in CD CHO media were added to the target cells and allowed to bind for 60 minutes at 37°C. Equine complement preserved serum (20% final concentration in CD CHO media) was added to the plates for 45 minutes at 37°C. Cell viability was then measured using CellTiter-Glo and data were expressed as “cell viability % of control” calculated using no fusion protein + complement preserved serum controls.

[00315] Results. As shown in Figure 26 and Table 7, four of the six equine wild type Fc subclasses showed robust and potent CDC activity. The exceptions were IgG2 and IgG5 both of which showed no CDC activity. EC50 values range from 0.19 to 0.29 pg / mL.

[00316] Table 7. CDC effects induced by equine IgG Fc wildtype subclass fusion proteins. CTLA4 Equine IgG Fusion Protein CDC EC50 Value CTLA4 FcelgGl 0.29 [tg / mL CTLA4 Fc elgG2 No CDC CTLA4 FcelgG3 0.21 [tg / mL CTLA4 Fc elgG4 0.19 pg / mL CTLA4 Fc elgG5 No CDC CTLA4 FcelgG7 0.26 pg / mL

[00317] As shown in Figure 27, wild type IgGl Fc subclass showed robust and potent CDC activity. The Fc mutations investigated showed differing effects on CDC activity. The PAP mutation in the Fc showed did not affect CDC activity compared to WT. The PSS mutation showed slightly enhanced CDC activity. The Win and WinPSS mutations showed knockdown of CDC activity. The SQP, SAP and WinSAP mutations most effectively knocked out CDC activity.

[00318] As shown in Figure 28, wild type IgG4 Fc subclass showed robust and potent CDC activity. The Fc mutations investigated showed differing effects on CDC activity. The Win, PSS and WinPSS mutations in the Fc showed slight knockdown of CDC activity compared to WT. The SQP, SAP and WinSAP mutations knocked out CDC activity.

[00319] As shown in Figure 29, wild type IgG7 Fc subclass showed robust and potent CDC activity. The Fc mutations investigated showed differing effects on CDC activity. The Win mutation in the Fc showed no effect on CDC activity compared to WT. The PSS mutation showed moderate knockdown of CDC activity. The SQP, SAP, WinSAP and WinPSS mutations knocked out CDC activity.

[00320] Example 10—ADCP Assay of Equine IgG Effector Function

[00321] The antibody dependent cellular phagocytosis (ADCP) assay utilizes CHO target cells engineered to express canine CD80, which binds to canine CTLA4 on the Fc fusion proteins. The Fc region of the fusion protein may then bridge this complex to Fc gamma receptors on activated macrophage effector cells, which have the capacity to phagocytose the target cell. ADCP is measured by fluorescent intensity and / or fluorescent area of a pH-sensitive fluorescent dye within the population of effector macrophages in the co-culture, wherein fluorescent cells are indicative of an effector that has successfully internalized a fluorescent labeled target cell into the acidic lysosome.

[00322] Methods. Canine CD80-expressing CHO cells (CD80 target cells) or wild-type CHO cells which do not express CD80 (parental target cells) were stained with pHrodo red dye for 30 minutes at 37 degrees C. Stained cells were subsequently incubated with equine CTLA4-Fc fusion proteins for 60 minutes to mediate CTLA4:CD80 binding. 40,000 target cells were then added to 8,000 pre-plated equine activated macrophages, isolated and differentiated from PMBCs. Co-cultures were maintained for 6-8 hours at 37 C as they were monitored and analyzed by a SX5 Incucyte live cell imaging system. Fluorescent area and / or intensity was quantified using a predefined processing definition in the Incucyte software and expressed as such.

[00323] Results As shown in Figure 30, wild type equine IgGl Fc subclass showed robust and potent ADCP activity. The Fc mutations investigated showed diminished ADCP activity to varying degrees. The Win and SAP mutations in the Fc showed knockdown of ADCP activity.

[00324] As shown in Figure 31, wild type IgG4 Fc subclass showed robust and potent ADCP activity. The Fc mutations investigated showed diminished ADCP activity to varying degrees. . The Win and SAP mutations in the Fc showed knockdown of ADCP activity.

[00325] As shown in Figure 32, wild type IgG4 Fc subclass showed robust and potent ADCP activity. The Fc mutations investigated showed either no effect or knockout of ADCP activity. The WinSAP mutation in the Fc showed a knockout of ADCP activity.

[00326] As shown in Figure 33, wild type IgG7 Fc subclass showed robust and potent ADCP activity. The Fc mutations investigated showed diminished ADCP activity to varying degrees. The Win and SAP mutations in the Fc showed knockdown of ADCP activity.

[00327] As shown in Figure 34, wild type IgG7 Fc subclass showed robust and potent ADCP activity. The Fc mutations investigated showed diminished ADCP activity. The WinSAP and SQP mutations in the Fc showed knockdown of ADCP activity.

[00328] Example 11—ADCC Assay of Equine IgG Effector Function.

[00329] The ADCC cell-based assay was developed and employed to characterize the effectiveness of the nine CTLA4 equine IgG subclass Fc fusion proteins in mediating ADCC and to investigate Fc region mutations in the subclasses. This will help define key residues in the Fc region that determine ADCC activity of the equine IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 which binds to CTLA4 on the Fc fusion proteins. These target cells have been used in past canine ADCC assays and were utilized in the CDC assay due to their dependability.

[00330] Methods. CD80-expressing CHO cells (target cells) were plated at 20,000 cells / well in CD CHO media in round-bottomed 96-well plates. Titrated fusion proteins in CD CHO media were added to the target cells and allowed to bind for 60 minutes at 37°C. Single donor equine PBMCs (effector cells), cultured overnight in RPMI 1640 medium + IL-2 and IL-15, were added to the plates for 18-20 hours at 37°C using an effector:target cell ratio (E:F Ratio) of 40-50:1. Cells were then stained fixed and analyzed via flow cytometry quantifying the live / dead stain of the target cells. Data were expressed as “% Dead Target cells” normalized to fusion protein alone (minus effector PBMCs).

[00331] Results. As shown in Figure 35 and Table 8, four of the six equine wild type Fc subclasses showed robust and potent ADCC activity. The exceptions were of IgG2 and IgG5 which showed no ADCC activity or greatly diminished ADCC activity, respectively. EC50 values ranged from 16.1 to 108.6 ng / mL. Equine IgG 1, 3, 4, and 7 Fc CTLA4 fusion proteins all showed ADCC activity with varying potency. IgG2 and 5 showed no ADCC activity.

[00332] Table 8. ADCC effects induced by equine IgG Fc wildtype subclass CTLA4 fusion proteins. CTLA4 Equine IgG Fusion Protein ADCC EC5O Value CTLA4 FcelgGl 108.6 ng / mL CTLA4 FcelgG2 No ADCC CTLA4 FcelgG3 16.1 ng / mL CTLA4 FcelgG4 53.8 ng / mL CTLA4 FcelgG5 No ADCC CTLA4 FcelgG7 22.5 ng / mL [00333J As shown in Figures 36 and 37, wild type IgGl subclass showed robust and potent ADCC activity. The Fc mutations investigated showed differing effects on ADCC activity. The Win, PSS, WinPSS and SQP mutations of the Fc knocked down ADCC effector function activity (Figure 36). The SAP and WinSAP mutations knocked out ADCC effector function activity (Figure 37). The PAP mutation appeared to have little effect on this ADDC effector function activity compared to IgGl wild type Fc (Figure 37).

[00334] As shown in Figure 38, wild type IgG4 subclass showed robust and potent ADCC activity. The Fc mutations investigated showed differing effects on ADCC activity. The Win, PSS, WinPSS and SQP mutations of the Fc showed no effect on ADCC effector function activity compared to IgG4 wild type Fc. The SAP mutation knocked down ADCC effector function activity and the WinSAP mutation knocked out ADCC effector function activity.

[00335] As shown in Figure 39, wild type IgG7 subclass showed robust and potent ADCC activity. The Fc mutations investigated showed differing effects on ADCC activity. The Win, SAP and PSS mutations of the Fc knocked down ADCC effector function activity compared to IgG7 wild type Fc. The WinSAP and Win PSS showed greater knockdown of ADCC activity. The SQP mutation knocked out ADCC effector function activity most effectively. Example 12—In Silico Modeling of Equine IgG Fc Regions

[00336] The Fc regions of the four equine allotypes, IgGl, IgG4a, IgG4b, IgG7a and IgG7b were first designed using their respective CH2 and CH3 regions. The protein modeling feature of Alphafold 2.2 developed by Deepmind was implemented to model the 3D structure of Equine FcRn and each of the wild-type (WT) and mutant constructs of each equine allotype. [00337J Molecular Operating Environment (MOE) developed by Chemical Computing Group (MOE2019.0102) provides a flexible and automated graphical user interface for protein modeling. To analyze the difference in structures, the sequence-to-profile alignment algorithm uses a scoring algorithm to rank the sequence templates and scores higher than 85% ensure the selection of protein templates with physically realistic structures. The model was then optimized using the same pipeline and the structural stability of the models was verified using Ramachandran Plots, which checks the stereochemical quality of a protein structure.

[00338] The method described above was performed on the Equine wild-type (WT) constructs and the following residues were mutated in IgGl: L234, L235, G237, P329, Q330, P331. The positions for the mutational library are represented in a ball-and-stick form in Fig. 40. The same procedure was also followed to investigate the following residue positions in equine subclasses, IgG4 (IgG4a and IgG4b) and IgG7 (IgG7a and IgG7b): L234, L235, G237, P329, A33O, P331 . The positions for the mutational library are represented in a ball-and-stick form in Fig. 41 and Fig.42.

[00339] An RMSD plot was generated to calculate the root mean square deviation of the WT structures, Equine-IgG4a, Equine-IgG4b, Equine-IgG7a and Equine IgG7b relative to each other (Figure 43) . An RMSD value of 2.0A or lower is considered the standard for considering two structures to be alike. Results indicated that equine IgG4a construct was identical to the equine IgG4b allotype with average RMSD value for the structure being 0.44A (individual position RMSD in Table 9). Equine IgG7a construct was identical to the equine IgG7b allotype with an average RMSD value of 0.9 A (individual position RMSD in Table 10).

[00340] Table 9. Root Mean Square Deviation (RMSD) comparisons of residues in the protein models of WT Equine lgG4a and IgG4b. Residue in Equine-lgG4a WT Residue in Equine-lgG4b WT RMSD (A) L234 L234 1.6 L235 L235 1.5 G237 G237 0.7 P329 P329 0.6 A330 A330 0.5 P331 P331 0.5

[00341] Table 10. Root Mean Square Deviation (RMSD) comparisons of residues in the protein models of WT Equine IgG7a and IgG7b. Residue in Equine-lgG7a WT Residue in Equine-lgG7b WT RMSD (A) L234 L234 1.6 L235 L235 1.5 G237 G237 0.7 P329 P329 0.9 A330 A330 0.88 P331 P331 0.88 Example 13—CDC Cell-Based Assay of Feline IgGla, IgGlb, IgG2 and IgG3 Subclasses

[00342] The CDC cell-based assay was developed and employed to characterize the effectiveness of feline IgG subclasses IgGla, IgGlb, IgG2 and IgG3 Fc mAbs in mediating CDC and to investigate Fc region mutations in the subclasses. This will help identify residues in the Fc region that determine CDC activity of the feline IgG subclasses. The assay utilizes CLBL-1 target cells that express canine CD20 which binds to the anti-canine CD20 test mAbs. These target cells have been used in past canine CDC assays and have been shown to be dependable.

[00343] Incubation of the mAb-bound target cells with complement-preserved serum can result in Fc binding on the mAbs to complement component Clq initiating the complement cascade, ultimately forming membrane attack complexes. The pore-forming complexes mediate cell lysis of the target cells measured by loss of cell viability. If there is no Fc binding to Clq there is no resultant cell lysis / death.

[00344] Briefly, canine CD20-expressing CLBL-1 (target cells) were plated at 40,000 cells / well in complete RPMI 1640 media in round-bottomed 96-well plates. Titrated fusion proteins in complete RPMI 1640 media were added to the target cells and allowed to bind for 60 minutes at 37°C. Feline complement preserved serum (25% in complete RPMI 1640 media) was added to the plates for 60 minutes at 37°C. Cell viability was then measured using CellTiter-Glo and data were expressed as “cell viability % of control” calculated using no fusion protein + complement preserved serum controls.

[00345] Results. As shown in Fig. 44, wild type feline IgGla Fc subclass showed robust and potent CDC activity. The Fc mutations investigated showed very good to complete knock out CDC activity. All of the mutations of the Fc tested in this figure showed knockout of CDC activity, with the WinKAPA and WinPA mutations showing the greatest knockout.

[00346] As shown in Fig. 45, wild type IgGlb Fc subclass showed robust and potent CDC activity. All of the Fc mutations investigated showed little to no effect on CDC activity. Four of the mutations showed slight knockdown of CDC activity with the M234A, L235A, G237A mutation showing the most knockdown. The S239D, I332E mutation showed slight enhancement of CDC activity. Feline IgGlb WT showed robust concentration-dependent CDC activity. All of the Fc mutations tested showed very little effect on CDC activity. Four of the mutations showed slight knockdown of CDC activity with the M234A, L235A, G237A mutation showing the most knockdown. The S239D, I332E mutation showed slight enhancement of CDC activity.

[00347] As shown in Fig. 46, wild type feline IgGlb Fc subclass showed robust and potent CDC activity. All of the Fc mutations investigated showed knockout of CDC activity to varying degrees. Feline IgGlb WT showed robust concentration-dependent CDC activity. Eight of the Fc mutations tested showed knockout of CDC activity. All five of the mutations tested showed knockdown of CDC activity with the M234A, L235A, G237A, K332A, P331S mutation showing the greatest knockdown of CDC activity.

[00348] As shown in Fig. 47, wild type feline IgG2 Fc subclass Wild Type (WT) and all eight of the Fc mutations investigated showed no CDC activity.

[00349] As shown in Fig. 48, wild type feline IgG3 Fc subclass showed robust and potent CDC activity. All five of the Fc mutations investigated showed little to no effect on CDC activity.

[00350] As shown in Fig. 49, four additional feline IgG3 Fc subclass mutations investigated showed little to no effect on CDC activity and one showed moderate knock down of CDC activity. The D265A_N297G mutation showed moderate knock down of CDC activity.

[00351] Conclusions. Among the Fc mutations assayed above, the Fc mutation with the strongest knockout of feline CDC mediated by the feline IgGla subclass are: WinKAPA and WinKA, which produced greater knockout than WinPA. The Fc mutation that best knocks out feline CDC mediated by the feline IgGlb subclass is M234A_L235A_G237A_K332A_P331S. The Fc mutation that best knocks out feline CDC mediated by the IgG3 subclass is D265A_N297G.

[00352] Example 14—ADCP Assay of Feline IgG Effector Function

[00353] The antibody dependent cellular phagocytosis (ADCP) assay was performed as described in Example 2 on feline IgGla variants.

[00354] Results. As shown in Fig. 50, wild type feline IgGla showed concentrationdependent ADCP activity. The Fc mutations WinKAPA, WinKA, and WinPA showed moderate to strong knock down of ADCP activity compared to the wild type, with the WinPA mutation showing the greatest knockdown.

[00355] Example 15—CDC Cell-Based Assay of Canine IgG Fc Effector Function

[00356] The CDC cell-based assay was developed and employed to characterize the effectiveness of canine IgG subclasses IgG2 and IgG3 Fc mAbs in mediating CDC and to investigate Fc region mutations in the subclass. This will help define key residues in the Fc region that determine CDC activity of the canine IgG subclasses. The assay utilizes CLBL-1 target cells that express canine CD20 which binds to the anti-canine CD20 test mAbs. These target cells have been used in past canine CDC assays and have been shown to be dependable.

[00357] Incubation of the mAb-bound target cells with canine complement-preserved serum can result in Fc binding on the mAbs to complement component Clq initiating the complement cascade, ultimately forming membrane attack complexes. The pore-forming complexes mediate cell lysis of the target cells measured by loss of cell viability. If there is no Fc binding to Clq there is no resultant cell lysis / death. [00358J Methods. Briefly, canine CD20-expressing CLBL-1 (target cells) were plated at 40,000 cells / well in complete RPMI 1640 media in round-bottomed 96-well plates. Titrated mAbs in complete RPMI 1640 media were added to the target cells and allowed to bind for 60 minutes at 37°C. Canine complement preserved serum (25% in complete RPMI 1640 media) was added to the plates for 60 minutes at 37°C. Cell viability was then measured using CellTiter-Glo and data were expressed as “cell viability % of control” calculated using no fusion protein + complement preserved serum controls.

[00359] Results. As shown in Fig. 51, wild type canine IgG2 Fc subclass showed robust and potent CDC activity. The Win, KAPA, WinKAPA, WinKA, and WinPA mutations of the Fc showed knockout of CDC activity. This differs slightly from the previous canine CDC assay using CD-80 expressing CHO target cells, CTLA4 fusion protein test articles and feline complement preserved serum. In that assay the Win mutation showed only a slight right shift of the response curve and only partial knockdown of CDC activity, whereas here the Win mutation showed an almost complete knockout of CDC activity.

[00360] As shown in Fig. 52, the D270S variation of canine IgG2 did not knockout CDC activity, but D270G did. GSP, GAP, DANG, Win DANG GAP, and DANG GAP mutations all showed knockout of CDC activity. As shown in Fig. 53, of the mutations tested in this set, two showed enhanced CDC activity, four showed little to no effect on CDC activity and one (DLE) showed knockout of CDC activity. The Q345R mutation showed the greatest enhancement of about a half logarithm increase in CDC potency. As shown in Fig. 54, the mutations tested in this set showed varying degrees of knockdown of CDC activity with the triple mutant, D276K T299E N324P, showing the greatest knock down in this set. [00361J As shown in Fig. 55, the mutations of IgG2 tested in this set showed little effect to slight knock down of CDC activity, including the triple mutants. This may be contrasted with Fig. 56, in which the mutations tested in this set all showed knockout of CDC activity, whether a single mutation site, around residues 263, 264, 265 266 or 267, or a combination of mutation sites added to V263W. As shown in Fig. 57, the mutations tested in this set showed knockout of CDC activity whether a single mutation site, around aa 299 or 328, or a combination of mutation sites added to T299E. As shown in Fig. 58, the mutations tested in this set showed knockout of CDC activity whether a single mutation site, or a combination of mutation sites. As shown in Fig. 59, the mutations tested in this set on residue N297 all showed knockdown of CDC activity with the N297A, N297D and N297K mutations showing the greatest knock down. As shown in Fig. 60, of the mutations tested in this set, the Q295G mutation showed little effect on CDC activity. All of the other tested mutations showed enhanced CDC activity with the P268W mutation by far showing the greatest enhancement of about one log unit increase in potency.

[00362] As shown in Fig. 61, of the mutations of IgG2 tested in this set, most of the combination mutations tested that included the V262, V264, P268 sites, showed enhanced CDC activity. The V262L_Q295N_N324P_Q345R and the V264F_F269A_N324P_Q345R combination mutations showed slight knock down of CDC activity. The P268WQ345R double mutation by far showed the greatest enhancement of about one log unit.

[00363] As shown in Fig. 62 the mutations of IgG3 tested in this set showed either no effect or great enhancement of CDC activity. As shown in Fig. 63, of the mutations tested in this set, most of the mutations tested showed no effect on CDC activity. The P268W mutation showed great enhancement of CDC activity approaching one log unit increase in potency.

[00364] From these assays, it can be observed that the Fc mutations that best knockout canine CDC mediated by the IgG2 subclass are: Win, WinKAPA, WinKA, Win PA, D270G, GSP, GAP, DLE, and the mutations around positions 238, 263, 265, 266,267, 294, 299 and 328.

[00365] The Fc mutations that best knockout canine CDC mediated by the IgG3 subclass are: N324P, K322A P331A, S239D I332E, G236A_S239D_I332E, L234A_L235A_G237A, L234A_L235A_G237A_K322A, and L234A_L235A_G237A_K322A_P331A. The Fc mutations that best enhance canine CDC are: P268W and P268W Q345R for IgG2 subclass and P268W for IgG3 subclass.

[00366] Example 16—ADCP Assay of Canine IgG Effector Function

[00367] The antibody dependent cellular phagocytosis (ADCP) assay utilizes CHO target cells engineered to express canine CD80 or CLBL-1 target cells that express canine CD20. These cells are stained with a pH-sensitive fluorescent dye which can be imaged as the cells are phagocytosed. Our test articles are either canine CTLA4 IgG2 Fc fusion proteins that bind CD80 or anti-canine CD20 chimeric monoclonal IgG2 antibodies (mAbs). The Fc region of the fusion protein or mAb may then bridge the target cell / test article complex to Fc gamma receptors on DH82 effector cells (a canine macrophage cell line), which have the capacity to phagocytose the stained target cells. ADCP is measured by fluorescent intensity and / or fluorescent area of the pH-sensitive fluorescent dye within the population of effector DH82 cells in the co-culture, wherein fluorescent cells are indicative of an effector cell that has successfully internalized a fluorescent labeled target cell into the acidic lysosome.

[00368] Methods. Briefly, target cells were stained with pHrodo red dye for 30 minutes at 37°C and were subsequently incubated with our test articles for 60 minutes to mediate test article:target cell binding. The complex was then added, 30,000 cells / well, to pre-plated DH-82 cells, 7,000 / well. The plates were maintained for 24 hours at 37°C and were monitored and analyzed by a SX5 Incucyte live cell imaging system. Fluorescent area and / or intensity was quantified using a predefined processing definition in the Incucyte software and expressed as such.

[00369] As shown in Fig. 64, wild type canine IgG2 showed concentration-dependent ADCP activity. The Fc mutations investigated in this set either showed knockdown of ADCP activity or little effect compared to the IgG2 WT. The Win, WinKAPA, WinKA and WinPA mutations in the Fc showed very good knockdown of ADCP activity. The KAPA mutation showed no effect on ADCP activity. As shown in Fig. 65, among the mutations tested in this set, the N297P, N297A, N297D mutations in the Fc showed knockdown of ADCP activity. As shown in Fig. 66, among the mutations tested in this set, the N297F and N297K mutations in the Fc showed knockout of ADCP activity. The N297G mutation showed knockdown of ADCP activity. As shown in Fig. 67, among the mutations tested in this set, the T299I, T299E, T299Q mutations in the Fc showed knockdown of ADCP activity with the T299I showing the least knockdown.

[00370] As shown in Fig. 68, among the mutations of canine IgG2 tested in this set, the T299R mutation in the Fc showed complete knockout of ADCP activity. The N324P mutation robust enhancement of ADCP activity. The L328K mutation showed no effect on ADCP activity. As shown in Fig. 69, among the mutations tested in this set, the P238L and V264F mutations in the Fc showed knockdown of ADCP activity. The L328P mutation showed no effect on ADCP activity. As shown in Fig. 70, among the mutations tested in this set, the L266P mutation, E269P mutation, and the E294H mutation in the Fc showed knockdown of ADCP activity. As shown in Fig. 71, among the mutations tested in this set, the Q295G and Q295N mutations in the Fc showed knockdown of ADCP activity. The E294N mutation showed no effect on ADCP activity.

[00371] As shown in Fig. 72, among the mutations of canine IgG2 tested in this set, the F296A and F296S mutations in the Fc showed knockout of ADCP activity. The Q295W mutation showed no effect on ADCP activity. As shown in Fig. 73, among the mutations tested in this set, G298M mutation in the Fc showed slight knockdown of ADCP activity. The M234D, G236E, S239L mutation showed knockout of ADCP activity. The L266G, G298L, Y300Q mutation showed no effect on ADCP activity. As shown in Fig. 74, among the mutations tested in this set, the M234E and V262L mutations in the Fc showed knockdown of ADCP activity. The M234E, L325D mutation showed knockout of ADCP activity. As shown in Fig. 75, among the mutations tested in this set, the D267K, T299E mutation in the Fc showed almost complete knockout of ADCP activity. The D267K, N324P mutation and the T299E, N324P mutation showed complete knockout of ADCP activity.

[00372] As shown in Fig. 76, among the mutations of canine IgG2 tested in this set, the V240S, V262L, E294M mutation in the Fc showed knockdown of ADCP activity. The P268W mutation showed no effect on ADCP activity. The V240Q, P268W, G298I mutation showed knockout of ADCP activity. As shown in Fig. 77, among the mutations tested in this set, the D2267K, T299E, N324P and the V263W, T299Q mutations in the Fc showed knockdown of ADCP activity with he latter showing better knockdown. The V263W, N297A mutation showed no effect on ADCP activity. As shown in Fig. 78, among the mutations tested in this set, the V263H mutation in the Fc showed slight knockdown of ADCP activity. The other mutations showed no effect on ADCP activity. As shown in Fig. 79, among the mutations tested in this set, the D265F mutation in the Fc showed knockdown of ADCP activity. The V263W and V264R mutations showed little to no effect on ADCP activity compared to WT.

[00373] As shown in Fig. 80, among the mutations of canine IgG2 tested in this set, the D265V, L266K, and D267K mutations in the Fc showed moderate knockdown of ADCP activity. As shown in Fig. 81, among the mutations tested in this set, the Win mutation, and the M234D, I332E mutation in the Fc showed very good knockdown of ADCP activity. The M234D, L328F mutation in the Fc showed moderate knockdown of ADCP activity. As shown in Fig. 82, among the mutations tested in this set, the S239L, I332E mutation, the L328F, I332E mutation, and the M234D, S239L, I332E mutation in the Fc showed moderate to very good knockdown of ADCP activity. As shown in Fig. 83, among the mutations tested in this set, the M234D, L328F, I332E mutation and the M234D, S239L, L328F, I332E mutation in the Fc showed knockdown of ADCP activity. The S239L, L328F, I332E mutation showed no effect on ADCP activity. As shown in Fig. 84, among the mutations tested in this set, the L325D, I332E mutation showed moderate knockdown in ADCP activity. The L325D, S239L mutation, and the L235D, S239L, I332E mutation in the Fc showed very good knockdown to almost knockout of ADCP activity. As shown in Fig. 85, among the mutations tested in this set, the L235D, L328F, I332E mutation, the L235D, S239L, L328F, I332E mutation, and the G236W, S239L mutation in the Fc showed slight to moderate knockdown of ADCP activity.

[00374] As shown in Fig. 86, among the mutations of canine IgG2 tested in this set, the G236W, I332E mutation, the G236W, S239L, I332E mutation and the G236W, L328F, I332E mutation in the Fc showed moderate knockdown of ADCP activity. As shown in Fig. 87, among the mutations tested in this set, the G236W, S239L, L328F, I332E mutation, the M234D, S239L mutation, and the G236E, S239L mutation in the Fc showed slight to moderate knockdown of ADCP activity. As shown in Fig. 88, among the mutations tested in this set, the L235D, G236W mutation, and the G236W, L328F mutation in the Fc showed moderate knockdown of ADCP activity. The L235D, G236W, L328F showed slight knockdown of ADCP activity. As shown in Fig. 89, among the mutations tested in this set, the L266G, G298L mutation and the L266G, S330K mutation in the Fc showed little effect on ADCP activity. The G298L,Y300Q mutation showed moderate knockdown of ADCP activity. As shown in Fig. 90, among the mutations tested in this set, the G298L, S330K mutation and the L266G, G298L, S330K mutation in the Fc showed knockdown of ADCP activity. The Y300Q, S330K mutation showed no effect on ADCP activity.

[00375] As shown in Fig. 91, among the mutations of canine IgG2 tested in this set, the L266G, Y300Q, S33OK mutation and the L266G, G298L, Y300Q, S330K mutation in the Fc showed knockdown of ADCP activity. The G298L, Y300Q, S33OK mutation showed little to no effect on ADCP activity. As shown in Fig. 92, among the mutations tested in this set, the L266V, G298K mutation, the G298K, S33OL mutation, the L266V, S33OL mutation and the L266V, G298K, S33OL in the Fc showed knockdown of ADCP activity with the L266V, G298K, S330L mutation showing the greatest knockdown.

[00376] As shown in Fig. 91, wild type IgG3 showed concentration-dependent ADCP activity. Among the mutations of canine IgG3 tested in this set, the N324P mutation in the Fc showed no effect on ADCP activity. The L234A, L235A, G237A, the L234A, L235A, G237A, K322A, P331A, theL234A, L235A, G237A, K322A, P331A mutation, the L234A, L235A, G237A, P331A mutation, and the T299R mutations in the Fc all showed knockout of ADCP activity.

[00377] From these assays, it can be observed that the Fc mutations that best knockout canine ADCP mediated by the IgG2 subclass are: Win, WinKAPA, WinKA, Win PA, N297F, N297K, T299E, T299Q, T299R, FF296A, F296S, M234D_G236E_S239L, M234EL325D, D267K_N324P, T299E_N324P and V240Q_P268W_G298I. The Fc mutations that best knockout canine ADCP mediated by the IgG3 subclass are: L234A L235A G237A, L234A_L235A_G237A_K322A_P331 A, L234A_L235A_G237A_K322A_P331 A, L234A_L235A_G237A_P331 A, and T299R. The Fc mutation that best enhanced canine ADCP is N324P for the IgG2 subclass.

[00378] Example 17—ADCC Assay of Canine IgG Effector Function

[00379] The canine FcyRIII binding (ADCC) cell-based assay was developed and employed to characterize the effectiveness of canine subclass IgG2 Fc anti-canine CD20 chimera IgGs in mediating ADCC and to investigate Fc region mutations in the subclass. This will help define key residues in the Fc region that determine ADCC activity of the canine IgG2 subclass. A schema for an ADCC assay is illustrated in Fig. 94. The assay utilizes specially engineered Jurkat “effector cells” (Promega) in which express canine FcRylll extracellularly and express a NF AT-Response Element linked to a luciferase reporter intracellularly. The “target cells” were CLBL-1 cells that express canine CD20. Our test articles were anti-canine CD20 chimera constructs of canine IgG2 wild type (WT) Fc and mutations of the canine IgG2 WT Fc.

[00380] Following engagement with the Fc region of our anti-canine CD20 canine IgG2 test articles that are bound to CLBL-1 “target cells”, the ADCC Bioassay Effector Cells expressing canine FcyRIII will transduce intracellular signals resulting in NF AT-mediated luciferase activity that can be easily quantified using BioGio™ Luciferase Assay Reagent (Promega) and a luminometer plate reader. The more binding of the Fc of our test articles to the FcyRIII on the “effector cells, the higher the luminescence. If there is no Fc binding to FcyRIII there is no resultant luminescence measured.

[00381] Methods. Briefly, CLBL-1 cells (target cells) were plated at -6,000 cells / well in RPMI1640 media in 96-well plates. Titrated test canine mAbs in RPMI 1640 media were added to the target cells and allowed to bind for 60 minutes at 37°C. The engineered “effector cells”, were counted and added to the plates for 6 hours at 37°C using an effectortarget cell ratio (E:F Ratio) of 10:1. FcyRIII binding was then quantified using the Promega Bio-Gio Luciferase Assay Reagent Luminescent Detection. Data were expressed as ’’Fold Increase” normalized to controls (minus effector cells).

[00382] Results. As shown in Fig. 95, wild type canine IgG2 showed concentration-dependent and robust canine FcyRIII binding translating to ADCC activity. The Fc mutations investigated either showed complete knockout of canine FcyRIII binding suggesting translating to ADCC activity. Canine IgG2 Fc showed ADCC activity via strong canine FcRylll binding. All of the mutations tested in this figure showed complete knockout of ADCC activity.

[00383] As shown in Fig. 96, the Fc mutations investigated in this set either showed complete knockout, knock down or enhancement of canine FcyRIII binding suggesting translating to ADCC activity. The WinKAPA, WinKA and WinPA mutations all showed complete knockout of FcyRIII binding. The KAPA and DAE mutations showed knock down FcyRIII binding. The DLE and DE mutations showed enhancement of FcyRIII binding.

[00384] As shown in Fig. 97, the Fc mutations investigated in this set either showed complete knockout, knock down, no effect or slight enhancement of canine FcyRIII binding suggesting translating to ADCC activity. The D270G, D270S and EFT mutations in the Fc showed knockdown of FcyRIII binding. The YWA mutation showed complete knockout of FcyRIII binding. The Q345R mutation showed no effect on FcyRIII binding compared to WT and the AAA mutation showed enhancement of FcyRIII binding.

[00385] As shown in Fig. 98, the Fc mutations investigated in this set showed complete knockout or enhancement of canine FcyRIII binding suggesting translating to ADCC activity. The Win and L266K mutations in the Fc completely knocked out FcyRIII binding and the V240S V262L E294M triple mutation showed enhancement of FcyRIII binding.

[00386] Conclusions. From these assays, it can be observed that, the Fc mutations that best knockout FcyRIII binding for ADCC mediated by the canine IgG2 subclass are: Win, WinKAPA, WinKA, Win PA, GSP, GAP, YWA, and L266K. From what is reported here, the Fc mutations that best enhance canine FcyRIII binding for ADCC mediated by the canine IgG2 subclass are: DLE, DE, AAA and V240S_V262L_E294M.

Claims

1. An Fc region of feline IgGla comprising one or more substitutions at positions 234, 235, 235, 236, 237, 237, 239, 329, 330, 331, 345, 265, 267, 268, 270, 270, 297, 298, 322, 324, 329, 330, 331, 332, 333, and 334 using EU Index numbering as in Kabat.

2. The Fc region of feline IgGla of claim 1, comprising one or more substitutions selected from among M234A, L235A, L235Y, G236A, G237A, G237W, S239D, P329G, P329S, S33OA, P331 A, P331S, E345R, D265A, G267E, P268F, D270G, D270S, N297G, S298A, K322A, N324T, P329G, S330A, S330L, P331 A, P331S, I332E, E333A, and R334A using EU Index numbering as in Kabat.

3. The Fc region of feline IgGla of claim 1, comprising one or more of the following combinations of substitutions: (M234A, L235A, G237A), (P329G), (P331S), (P329G, S330A), (D265A, N297G), (K322A, P331 A), (M234A, L235A, G237A, P329G, S330A), (D265A, N297G, P329G, S330A), (M234A, L235A, G237A, D265A, N297G), (M234A, L235A, G237A, D265A, N297G, P329G, S330A), (D265A,N297G,P331S), (M234A, L235A, G237A, P331A), (M234A, L235A, G237A, D265A, N297G, P331S), (M234A, L235A, G237A, P331S), WIN GSA (M234A, L235A, G237A, P329G, P331A), (M234A, L235A, G237A, P329G), WIN_GSS (M234A, L235A, G237A, P329G, P331S), (M234A, L235A, G237A, P329S, S330A, P331S), (S298A, E333A, R334A), (S239D, S33OL, I332E), DE (S239D, I332E), (G236A, S239D, I332E), (L235Y, G237W, S298A), (G267E, P268F, N324T), (D270G), (D270S), and (E345R) using EU Index numbering as in Kabat.

4. An Fc region of feline IgGlb comprising one or more substitutions or combinations of substitutions in positions corresponding to the positions in feline IgGla in any of claims 1-3.

5. An Fc region of feline IgG3 comprising one or more substitutions or combinations of substitutions in positions corresponding to the positions in feline IgGla in any of claims 1-3.

6. An Fc region of feline IgG3 comprising substitutions in positions D265A and N297G.

7. An Fc region of canine IgG2 comprising one or more mutations at locations selected fromamong 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 294, 295, 296, 297, 298, 299, 300, 301, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 422 423, and 441, using EU Index numbering as in Kabat.

8. An Fc region of canine IgG2 of claim 7 comprising one or more substitutions selected fromamongA231C, A231D, A231E, A231G, A231H, A231K, A231N, A231Q, A231R,A231S, A231T, A231V, A231Y, P232A, P232C, P232D, P232E, P232F, P232G, P232H, P232I, P232K, P232L, P232M, P232N, P232Q, P232R, P232S, P232T, P232V, P232W, P232Y, E233A, E233C, E233D, E233F, E233G, E233H, E233I, E233K, E233L, E233M, E233N, E233P, E233Q, E233R, E233S, E233T, E233V, E233W, E233Y, M2 34A, M234C, M234D, M234E, M234F, M234G, M234H, M2 341, M234K, M234L, M234N, M234P, M234Q, M234R, M234S, M234T, M234W, M234Y, L235A, L235C, L235D, L235E, L235F, L235G, L235H, L235I, L235K, L235M, L235N, L235P, L235Q, L235R, L235S, L235T, L235V, L235W, L235Y, G236A, G236C, G236D, G236E, G236F, G236H, G236I, G236K, G236L, G236M, G236N,G236P, G236Q, G236R, G236S, G236T, G236V, G236W, G236Y, G237A G237C, G237D, G237E, G237F, G237H, G237I, G237K, G237L, G237M G237N, G237P, G237Q, G237R, G237S, G237T, G237V, G237W, G237Y P238A, P238C, P238D, P238E, P238F, P238G, P238H, P238I, P238K P238L, P238M, P238N, P238Q, P238R, P238S, P238T, P238V, P238W P238Y, S2 3 9A, S239C, S239D, S239E, S239F, S239G, S239H, S239I S239K, S239L, S239M, S239N, S239P, S239Q, S239R, S239T, S239V S239W, S239Y, V2 4 0A, V240C, V240D, V240E, V240F, V240G, V240H V240I, V240K, V240L, V240M, V240N, V240P, V240Q, V240R, V240S V240T, V240W, V240Y, V262A, V262C, V262D, V262E, V262F, V262G V262H, V262K, V262L, V262M, V262Q, V262R, V262S, V262T, V262W V262Y, V2 63A, V263C, V263E, V263F, V263G, V263H, V263I, V263K V263L, V263M, V263N, V263P, V263Q, V263S, V263T, V263W, V263Y V264A, V264C, V264D, V264E, V264F, V264G, V264H, V264I, V264K V264L, V264M, V264N, V264P, V264Q, V264R, V264S, V264T, V264W V264Y, D2 65A, D265C, D265E, D265F, D265G, D265H, D265I, D265K D2 65L, D265M, D265N, D265P, D2 65Q, D265R, D265S, D265T, D265V D265W, D265Y, L2 6 6A, L266C, L266D, L266E, L266F, L266G, L266H L266I, L266K, L266M, L266N, L2 6 6P, L2 6 6Q, L266R, L266S, L266T L266V, L266W, L266Y, D267A, D267C, D267E, D267F, D267G, D267H D267I, D267K, D267L, D267M, D267N, D267P, D267Q, D267R, D267S D267T, D267V, D267W, D267Y, P268A, P268C, P268D, P268E, P268F P268G, P268H, P268I, P268K, P268L, P268M, P268N, P268Q, P268R P268S, P268T, P268V, P268W, P268Y, E2 69A, E269C, E269D, E269FE269G, E269H, E269I, E269K, E269L, E269M, E269N, E269P, E269Q E269R, E269S, E269T, E269V, E269W, E269Y, D270A, D270C, D270F D270G, D270H, D270I, D270K, D270L, D270M, D270N, D270P, D270Q D270R, D270S, D270V, D270W, D270Y, P271C, P271F, P271G, P271H P271I, P271K, P271L, P271M, P271N, P271Q, P271R, P271S, P271T P271Y, E294A, E294C, E294D, E294F, E294G, E294H, E294I, E294K E294L, E294M, E294N, E294P, E294Q, E294R, E294S, E294T, E294V E294W, E294Y, Q295A, Q295D, Q295E, Q295F, Q295G, Q295K, Q295L Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, F2 9 6A F296C, F296D, F296E, F296G, F296H, F296I, F296K, F296L, F2 9 6M F296N, F296P, F296Q, F296R, F296S, F296T, F296V, N297A, N297C N297D, N297E, N297F, N297G, N297H, N297I, N297K, N297L, N297M N297P, N297Q, N297R, N297S, N297T, N297V, N297W, N297Y, G298A G298C, G298D, G298E, G298F, G298H, G298I, G298K, G298L, G298M G298N, G298P, G298Q, G298R, G298S, G298T, G298V, G298W, G298Y T299A, T299C, T299D, T299E, T299F, T299G, T299H, T299I, T299K T299L, T299M, T299N, T299P, T299Q, T299R, T299S, T299V, T299W T299Y, Y3 0 0A, Y300C, Y300D, Y300E, Y300H, Y300I, Y300K, Y300L Y300M, Y300N, Y300P, Y300Q, Y300R, Y300S, Y300T, Y300V, Y300W R301A, R301C, R301D, R301E, R301F, R301G, R301H, R301I, R301L R301P, R301Q, R301V, R301W, R301Y, V323A, V323C, V323D, V323E V323F, V323G, V323H, V323K, V323L, V323M, V323N, V323P, V323Q V323R, V323S, V323T, V323W, V323Y, N324A, N324C, N324D, N324E N324F, N324G, N324H, N324I, N324K, N324L, N324M, N324P, N324QN324R, N324S, N324T, N324V, N324W, N324Y, N325A, N325C, N325D N325E, N325F, N325G, N325H, N325I, N325K, N325L, N325M, N325P N325Q, N325R, N325S, N325T, N325V, N325W, N325Y, K326A, K326D K326E, K326F, K326I, K326L, K326N, K326R, K326S, K326T, K32 6V K326W, A327C, A327D, A327E, A327F, A327G, A327H, A327I, A327K A327L, A327M, A327N, A327P, A327Q, A327R, A327S, A327T, A327V A327W, A327Y, L328A, L328C, L328D, L328E, L328F, L328G, L328H L328I, L328K, L328M, L328N, L328P, L328Q, L328R, L328S, L328T L328V, L328W, L328Y, K322A, K322C, K322D, K322E, K322F, K322G K322H, K322I, K322L, K322M, K322N, K322P, K322Q, K322R, K322S K322T, K322V, K322W, K322Y, P329A, P329C, P329D, P329E, P329F P329G, P329H, P329I, P329K, P329L, P329M, P329N, P329Q, P329R P329S, P329T, P329V, P329W, P329Y, S330A, S330C, S330D, S330E S330F, S330G, S330H, S330I, S330K, S330L, S330M, S330N, S330P S330Q, S330R, S330T, S330V, S330W, S330Y, P331A, I332A, I332D I332E, I332F, I332G, I332H, I332K, I332L, I332M, I332N, I332P I332R, I332T, I332V, I332W, I332Y, E333A, E333D, E333H, E333I E333K, E333N, E333R, E333S, E333V, E333Y, R334A, R334C, R334D R334E, R334F, R334G, R334L, R334M, R334N, R334P, R334Q, R334S R334T, R334V, R334W, R334Y, T335C, T335H, T335I, T335K, T335L T335P, I336D, I336E, I336G, I336K, I336N, I336Y, S337D, S337P K338A, K338C, K338D, K338E, K338F, K338G, K338H, K338I, K338L K338M, K338N, K338P, K338Q, K338R, K338S, K338V, K338W, K338Y A339D, A339F, A339G, A339N, A339P, A339R, A339S, A339T, A3 3 9VA339W, A339Y, R340G, T422K, T422R, F423P, and L441P using EU Index numbering as in Kabat.

9. An Fc region of canine IgG2 of claim 7 comprising one or more substitutions or combinations of substitutions selected from among (P329G), (P329G, S330A), (D265A, N297G), (D265A, N297G; P329G, S330A), (M234A, L235A, G237A), (M234A, L235A, G237A; D265A, N297G; P329G, S330A), (G298A, E333A, R334A), (S239D, I332E), (S239D, S330L, I332E), (G236A, S239D, I332E), (L235Y, G237W, G298A), (D267E, P268F, N324T), (Q345R), (D270G), and (D270S), using EU Index numbering as in Kabat.

10. An Fc region of canine IgG3 comprising one or more substitutions or combinations of substitutions in positions corresponding to the positions in feline IgG2 in any of claims 7-9, using EU Index numbering as in Kabat.

11. An Fc region of canine IgG3 comprising one or more substitutions or combinations of substitutions in positions selected from among N324P, (K322A, P331A), (S239D, I332E), (G236A, S239D, I332E), (L234A, L235A ,G237A), (L234A, L235A, G237A, K322A), (L234A, L235A, G237A, K322A, P331A), (L234A, L235A, G237A, P331A), and T299R, using EU Index numbering as in Kabat.

12. An Fc region of equine IgGl comprising mutations or substitutions at one or more positions selected from among 229, 234, 235, 237, 329, 330, and 331, using EU Index numbering as in Kabat.

13. An Fc region of equine IgGl of claim 12 comprising one or more substitutions selected from among P229S, L234A, L235A, G237A, P329S, Q330A, Q330S, and P331S, using EU Index numbering as in Kabat.

14. An Fc region of equine IgGl of claim 12 comprising one or more combinations of substitutions in a Fc region selected from among (P329S, Q330A), (Q330A), (P229S), (L234A, L235A, G237A), (Q330S, P331S), (L234A, L235A, G237A; P329S, Q330A), and (L234A, L235A, G237A, Q330S, P331S), using EU Index numbering as in Kabat.

15. An Fc region of equine IgG4 comprising one or more mutations and / or substitutions at positions selected from among 234, 235, 237, 239, 330, and 331, using EU Index numbering as in Kabat.

16. An Fc region of equine IgG4 of calim 15, comprising one or more substitutions selected from among L234A, Q235A, G237A, P239S, A330Q, A330S, P331S, using EU Index numbering as in Kabat.

17. An Fc region of equine IgG4 of calim 15, comprising one or more substitutions or combinations of substitutions in a Fc region of equine IgG4 selected from among (A330S, P331S), (P239S, A330Q), (L234A, Q235A, G237A), (L234A, Q235A, G237A; A330S, P331S), (P329S), and (L234A, Q235A, G237A; P329S), using EU Index numbering as in Kabat.

18. An Fc region of equine IgG7 comprising one or more mutations and / or substitutions at positions selected from among 234, 235, 236, 237, 239, 330, and 331, using EU Index numbering as in Kabat.

19. An Fc region of equine IgG7 of claim 18, comprising one or more substitutions selected from among L234A, S235A, V236G, G237A, P239S, P329S, A330S, A33OQ, P331S, using EU Index numbering as in Kabat.

20. An Fc region of equine IgG7 of claim 18, comprising substitions or combinations of substitutions in a Fc region of equine IgG7 selected from among (A330S, P331S), (P329S), (P239S, A330Q), (L234A, S235A, V236G, G237A), (L234A, S235A, V236G, G237A; A330S, P331S), and (L234A, S235A, V236G, G237A; P329S).

21. A recombinant polypeptide comprising a modified recombinant feline, canine, or equine IgGFc region of any of claims 1-20.

22. A recombinant antibody or a molecule comprising a feline, canine, or equine IgGFc region of any of claims 1-20.

23. A method for producing or manufacturing an antibody or a molecule, the method comprising: providing a vector or a host cell having a nucleic acid sequence that encodes an antibody or molecule of Claim 22.

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