Compositions and methods for modifying antibody effector function

Abstract

This invention relates to antibody variants for felines, canines, and equines and their uses therein, as well as compositions and methods for improving therapeutic monoclonal antibodies by modulating immune effector function, and antibody variants using said methods and compositions.

Description

Technical Field

[0001] This application relates to feline, canine, and equine antibody variants and their uses, as well as compositions and methods for modifying therapeutic veterinary monoclonal antibodies by modulating immune effector function, and the use of those methods, compositions, and antibody variants. Background Technology

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

[0003] Feline IgG monoclonal antibodies (mAbs) are being developed as effective therapeutic agents in veterinary medicine. Several years ago, feline IgG subclasses were identified and characterized (Strietzel et al., 2014, Veterinary Immunology and Immunopathology, Vol. 158(3-4), pp. 214-223). Canine IgG monoclonal antibodies (mAbs) are also being developed as effective therapeutic agents in veterinary medicine. Several years ago, four canine IgG subclasses were identified and characterized (Bergeron et al., 2014, Veterinary Immunology and Immunopathology, Vol. 157(1-2), pp. 31-41). The seven IgG subclasses in equines are well known in the field and are well described in the following literature: for example, Wagner et al., 2004, Journal of Immunology, Vol. 173, pp. 3230-3242; Wagner, 2006, Developmental and Comparative Immunology, Vol. 30, pp. 155-164; Sheoran et al., 2000, American Journal of Veterinary Research, Vol. 61, pp. 1099-1105; and Wagner et al., 1998, Immunobiology, Vol. 199(1), pp. 105-118.

[0004] Through a cyclic mechanism, the neonatal Fc receptor (FcRn) prolongs the half-life of IgG in its pH-dependent interaction with its fragmental crystallizable (Fc) region. Specifically, the Fc region, spanning the interface between the CH2 and CH3 domains, interacts with FcRn on the cell surface to regulate IgG homeostasis. Acidic interactions following IgG endocytosis favor this interaction and thereby protect IgG from degradation. The endocytized IgG then cycles back to the cell surface and is released into the bloodstream at a slightly alkaline pH, thus maintaining adequate serum IgG function. Therefore, the pharmacokinetic profile of IgG depends on the structural and functional properties of its Fc region.

[0005] The Fc region is also responsible for antibody immune effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP). These effector functions depend on the interaction between the antibody Fc region and FcγR. Therefore, engineering the Fc region to modulate its interaction with FcγR and C1q has become a promising approach for enhancing the activity of therapeutic antibodies.

[0006] Therefore, novel IgG Fc region mutations are needed to improve the various properties of feline, canine, and equine IgGs so that they can be used as safe therapeutic mAbs, particularly mutations that reduce, eliminate, or enhance the function of immune effectors in cell-based CDC, ADCC, and ADCP assays. Summary of the Invention

[0007] The ability to mediate cytotoxic and phagocytic effector functions is an effective mechanism by which antibodies destroy target cells. The Fc region links the antibody's recognition domain to these effector functions through interactions with Fc receptors and ligands. Manipulating these effector functions by altering the Fc region has important implications for the treatment of many medical conditions, such as cancer, autoimmune diseases, and infectious diseases.

[0008] Fc domains can be modified to achieve beneficial gain-of-function modifications, such as enhancing the immune effector function on therapeutic mAbs targeting cancer cells, bacteria, or viruses to improve the killing / clearance of cell / virus particles. However, in some cases, reducing or eliminating antibody Fc function may be beneficial. These include antibodies used as receptor agonists to crosslink receptors and induce signaling, antibodies used as receptor antagonists to block receptor:ligand interactions and thus prevent signaling, or antibodies used as drug delivery mediators to deliver drugs to antigen-expressing target cells. In these cases, Fc or C1q binding of the receptor on effector cells is unnecessary, as it could lead to unintended killing of biologically important cells expressing the receptor or recruitment of drug-conjugated antibodies to off-target cells. Therefore, IgGs with ineffective effector function are important for many antibody mechanisms across a wide range of disease domains. This is also important in Fc fusion proteins and alternative antibody forms such as bispecific or multispecific antibodies. The following literature describes several strategies for manipulating the binding of human antibodies to FcγR and complement protein C1q (including alterations to the Fc sequence and glycosylation): Saunders, Front Immunology, Article 1296, Vol. 10, June 7, 2019.

[0009] This invention provides modified canine, feline, and equine Fc regions (compared to wt) with advantageous properties. Canine IgG1 and IgG4 subclasses are known to lack effector function, while IgG2 and IgG3 have intact effector function. However, IgG2 is currently the dominant IgG used in therapeutic antibodies due to other properties such as protein A binding capacity and a relatively long half-life compared to other canine IgGs. Therefore, canine IgG2, which produces effector-ineffective function, is highly attractive for certain therapeutic areas. Similarly, feline IgG1 is a preferred IgG subclass for developing therapeutic mAbs, making feline IgG1, which produces effector-ineffective function, also highly attractive for certain therapeutic areas. Seven equine IgG subclasses, namely IgG1–IgG7, have been reported, each with different characteristics in protein A binding, pharmacokinetics, and effector function, but IgG4 and IgG7 are very similar. All seven equine IgG subclasses possess some ability to trigger effector function, with the possible exception of IgG2. Therefore, different subclasses may require different sets of mutations to reduce effector functionality.

[0010] Therefore, this invention relates to modified recombinant feline, canine, and equine IgGs that exhibit desired properties relative to wild-type IgG. Specifically, the inventors of this application have discovered that replacing amino acid residues at positions described herein with another amino acid unexpectedly produces the desired effects. In exemplary embodiments, the unexpected desired effects include, but are not limited to: enhanced affinity for FcRn; reduced complement-dependent cytotoxicity (CDC); reduced antibody-dependent cytotoxicity (ADCC); reduced antibody-dependent phagocytosis (ADCP); reduced or enhanced binding to the Fcγ receptor (bFcγR); or combinations thereof.

[0011] In one respect, using EU indexing as in Kabat, the Fc region of feline IgG1a may contain 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.

[0012] On the other hand, using EU index numbers, the Fc region of feline IgG1a may contain one or more substitutions selected from the following: 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.

[0013] On the other hand, various substitution combinations in the Fc region of feline IgG1a can include one or more of the following, referred to herein by abbreviated names: WIN (M234A, L235A, G237A), GSP (P329G), PSS (P331S), GAP (P329G, S330A), 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, S330A), WIN_DANG (M234A, L235A, G237A, D265A, N297G), WIN_DANG_GAP (M234A, L235A, G237A, D265A, N297G, P329G, S330A), DANG_PSS (D26 5A, N297G, P331S), WIN_PSA (M234A, L235A, G237A, P331A), WIN_DANG_PSS (M234A, L235A, G237A, D265A, N297G, P331S), WIN_PSS (M234A, L 235A, G237A, P331S), WIN_GSA (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] On the other hand, the Fc region of feline IgG1b may also contain one or more substitutions or combinations of substitutions at positions corresponding to those in IgG1a described above. It will also be appreciated that canine and equine IgGs of any subclass may contain one or more corresponding substitutions or combinations of substitutions at positions corresponding to those in feline IgG1a described above, and the short names used above may be used to identify those corresponding substitutions.

[0015] On the other hand, using EU indexing such as in Kabat, the Fc region of canine IgG2 may contain one or more mutations at positions such as 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.

[0016] On the other hand, using EU index numbers such as in Kabat, the Fc region of canine IgG2 may contain one or more substitutions listed in Table 5 and / or substitutions selected from the following: M234A, L235A, L235Y, G236A, G237A, G237W, S239D, D265A, D270G, D270S, N297G, G298A, K322A, P329G, S330A, S330L, P331A, I332E, E333A, R334A.

[0017] On the other hand, the Fc region of canine IgG2 may contain various combinations of substitutions in the Fc region of canine IgG2, which may include one or more of the following, referred to herein by abbreviated names: 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, P331A), K322A), WIN_KAPA (M234A, L235A, G237A, K322A, P331A), WIN_DANG_GAP (M234A, L2 35A, G237A; D265A, N297G; P329G, S330A), AAA (G298A, E333A, R334A), DE (S239D, I 332E), DLE (S239D, S330L, I332E), DAE (G236A, S239D, I332E), YWA (L235Y, G237W, G298A), EFT (D267E, P268F, N324T), Q345R (Q345R), D270G (D270G) and D270S (D270S).

[0018] On the other hand, using EU index numbers such as in Kabat, the Fc region of equine IgG1 may contain substitutions at one or more locations, including 229, 234, 235, 237, 329, 330 and / or 331.

[0019] On the other hand, using EU index numbers such as in Kabat, the Fc region of equine IgG1 can contain one or more substitutions selected from the following: P229S, L234A, L235A, G237A, P329S, Q330A, Q330S, and P331S.

[0020] On the other hand, the Fc region of equine IgG1 can contain various combinations of substitutions in the Fc region, including one or more of the following, referred to herein by abbreviated names: SAP (P329S, Q330A), PAP (Q330A), SQP (P229S), WIN (L234A, L235A, G237A), KA (K322A), PA (P331A), KAPA (K322A, P331A), WIN_PA (M234A, L235A), etc. A, 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] On the other hand, the Fc region of equine IgG4 may contain one or more mutations and / or substitutions selected from the following locations: 234, 235, 237, 239, 330 and / or 331.

[0022] On the other hand, using EU index numbers such as in Kabat, the Fc region of equine IgG4 can contain one or more substitutions selected from the following: L234A, Q235A, G237A, P239S, A330Q, A330S, P331S.

[0023] On the other hand, the Fc region of equine IgG4 may contain various combinations of substitutions in the Fc region of equine IgG4, which may include one or more of the following, referred to herein by abbreviated names: 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] On the other hand, using EU index numbers such as in Kabat, the Fc region of equine IgG7 may contain one or more mutations and / or substitutions selected from the following locations: 234, 235, 236, 237, 239, 330 and / or 331.

[0025] On the other hand, using EU index numbers such as in Kabat, the Fc region of equine IgG7 can contain one or more substitutions selected from the following: L234A, S235A, V236G, G237A, P239S, P329S, A330S, A330Q, P331S.

[0026] On the other hand, the Fc region of equine IgG7 may contain a combination of substitutions in the Fc region of equine IgG7, which may include one or more of the following, referred to herein by abbreviated names: 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 present invention provides a recombinant polypeptide comprising a modified recombinant feline, canine, or equine IgG Fc region as described herein, wherein the Fc region comprises one or more amino acid substitutions as described herein.

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

[0029] In another aspect, the present invention provides a method for generating or manufacturing antibodies or molecules, the method comprising: providing a vector or host cell having a nucleic acid sequence encoding an antibody, wherein the antibody comprises a feline, canine, or equine IgG constant domain, the constant domain comprising one or more amino acid substitutions as described herein.

[0030] Other features and advantages of the invention will become apparent from the following detailed description, examples, and accompanying drawings. However, it should be understood that while the detailed description and specific examples indicate preferred embodiments of the invention, they are given by way of illustration only, as 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. Attached Figure Description

[0031] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a copy of this patent or patent application publication with color drawings.

[0032] Figure 1 Cell-based complement-dependent cytotoxic (CDC) activity of CTLA4 fusion proteins of feline IgG1a, IgG1b and IgG2 Fc subclasses.

[0033] Figure 2A and 2B -4: Cell-based CDC activity of wild-type Fc IgG1a subclass CTLA4 fusion protein in felines and Fc mutants of said subclass. Figure 5-9 Cell-based antibody-dependent phagocytic activity of wild-type IgG1a Fc subclass CTLA4 fusion protein and Fc mutants of the subclass.

[0034] Figure 10-12 Cell-based antibody-dependent cytotoxic (ADCC) activity of wild-type IgG1a Fc subclass CTLA4 fusion protein and Fc mutants of the subclass in felines.

[0035] Figure 13 IgG1a Fc mutation in felines studied by ADCC assay.

[0036] Figures 14A-14C Protein modeling of the Fc regions of IgG1a and IgG1b in felines. Figure 14A An overlay of the protein variation model is shown, with magnified subplots showing the positions of amino acid residues in a ball-and-stick format. Figure 14B The root mean square deviation (RMSD) comparison of wild-type constructs of feline IgG1a and IgG1b is shown. Figure 14C An overlay of protein models with variations of G236, G267, P268, S298, N324, E333, R334, and E345 is shown, with magnified subplots showing the positions of amino acid residues in a ball-and-stick format.

[0037] Figure 15 Cell-based CDC activity of canine IgG1 and IgG2 Fc subclass CTLA4 fusion protein.

[0038] Figure 16 Cell-based CDC activity of wild-type Fc IgG2 subclass CTLA4 fusion protein in canines and Fc mutants of the subclass.

[0039] Figure 17-21 Cell-based ADCP activity of canine wild-type IgG2 Fc subclass CTLA4 fusion protein and Fc mutants of the subclass.

[0040] Figure 22-24Cell-based antibody-dependent cytotoxic (ADCC) activity of the canine wild-type IgG2 Fc subclass CTLA4 fusion protein and the Fc mutant of the subclass.

[0041] Figure 25 IgG2 Fc mutation in canines studied by ADCC assay.

[0042] Figure 26 Cell-based CDC activity of wild-type Fc subclass CTLA4 fusion protein in equines.

[0043] Figure 27 Cell-based CDC activity of wild-type Fc IgG1 subclass CTLA4 fusion protein and Fc mutants of the subclass in equines.

[0044] Figure 28 Cell-based CDC activity of wild-type Fc IgG4 subclass CTLA4 fusion protein and Fc mutants of the subclass in equines.

[0045] Figure 29 Cell-based CDC activity of wild-type Fc IgG7 subclass CTLA4 fusion protein and Fc mutants of the subclass in equines.

[0046] Figure 30 Cell-based ADCP activity of wild-type Fc IgG1 subclass CTLA4 fusion protein and Fc mutants of the subclass in equines.

[0047] Figures 31-32 Cell-based ADCP activity of wild-type Fc IgG4 subclass CTLA4 fusion protein and Fc mutants of the subclass in equines.

[0048] Figures 33-34 Cell-based ADCP activity of wild-type Fc IgG7 subclass CTLA4 fusion protein and Fc mutants of the subclass in equines.

[0049] Figure 35 Cell-based antibody-dependent cell-mediated cytotoxic activity of wild-type Fc subclass CTLA4 fusion protein in equines.

[0050] Figures 36-37 Cell-based antibody-dependent cell-mediated cytotoxicity of wild-type Fc IgG1 subclass CTLA4 fusion protein and Fc mutants of the subclass in equines.

[0051] Figure 38Cell-based antibody-dependent cell-mediated cytotoxic activity of wild-type Fc IgG4 subclass CTLA4 fusion protein and mutant Fc subclasses in equines.

[0052] Figure 39 Cell-based antibody-dependent cell-mediated cytotoxic activity of wild-type Fc IgG7 subclass CTLA4 fusion protein and mutant Fc subclasses in equines.

[0053] Figure 40 Protein modeling of the FcRn and IgG1 Fc regions in equines, with magnified subplots showing the positions of amino acid residues in ball-and-stick format.

[0054] Figure 41 Protein modeling of the FcRn, IgG4a-Fc, and IgG4b-Fc regions in equines, with magnified subplots showing the positions of amino acid residues in ball-and-stick format.

[0055] Figure 42 Protein modeling of the FcRn, IgG7a-Fc, and IgG7b-Fc regions in equines, with magnified subplots showing the positions of amino acid residues in ball-and-stick format.

[0056] Figure 43 Comparison of root mean square deviation (RMSD) of wild-type constructs of IgG1, IgG4a, IgG4b, IgG7a and IgG7b in equines.

[0057] Figure 44 Cell-based CDC activity of wild-type Fc IgG1a subclasses and Fc mutants of said subclasses in felines.

[0058] Figure 45 and 46 Cell-based CDC activity of wild-type Fc IgG1b subclasses and Fc mutants of said subclasses in felines.

[0059] Figure 47 Cell-based CDC activity of wild-type Fc IgG2 subclasses and Fc mutants of said subclasses in felines.

[0060] Figure 48 and 49 Cell-based CDC activity of wild-type Fc IgG3 subclasses and Fc mutants of said subclasses in felines.

[0061] Figure 50 Cell-based CDC activity of feline IgG1a variants.

[0062] Figures 51-61Cell-based CDC activity of wild-type Fc IgG2 subclasses and Fc mutants of said subclasses in canines.

[0063] Figure 62 and 63 Cell-based CDC activity of wild-type Fc IgG3 subclasses and Fc mutants of said subclasses in canines.

[0064] Figure 64 Cell-based ADCP activity of wild-type canine anti-canine CD20 chimeric IgG mAb and Fc mutants of the aforementioned subclass.

[0065] Figure 65-92 Cell-based ADCP activity of the canine wild-type IgG2 Fc subclass CTLA4 fusion protein and the Fc mutant of the subclass.

[0066] Figure 93 Cell-based ADCP activity of wild-type canine anti-canine CD20 chimeric IgG mAb and Fc mutants of the aforementioned subclass.

[0067] Figure 94 This demonstrates the protocol for ADCC measurement.

[0068] Figures 95-98 : Canine wild-type IgG2 Fc subclass antibody and cell-based canine FcRIII of the Fc mutant of the subclass.

[0069] Brief description of the sequence The contents of the electronic sequence list (sequencelisting_ST26.xml; size: 82,432 bytes; and creation date: February 1, 2024) are incorporated herein by reference in their entirety.

[0070] SEQ ID NO: 1 is a sequence of feline IgG1a heavy chain containing the GSP (P329G) mutation in the rituximab variable region and Fc region.

[0071] SEQ ID NO: 2 is a sequence of feline IgG1a heavy chain containing the rituximab variable region and the GSP (P329G) mutation.

[0072] SEQ ID NO: 3 is a sequence of feline IgG1a heavy chain containing GAP (P329G, S330A) mutations in the rituximab variable region and Fc region.

[0073] SEQ ID NO: 4 is a sequence of feline IgG1a heavy chain containing the DANG-GAP (D265A, N297G; P329G, S330A) mutation in the rituximab variable region and Fc region.

[0074] SEQ ID NO: 5 is a sequence of feline IgG1a heavy chain containing the WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) mutation in the rituximab variable region and Fc region.

[0075] SEQ ID NO: 6 is a sequence of feline IgG1a heavy chain containing the DANG (D265A, N297G) mutation in the rituximab variable region and Fc region.

[0076] SEQ ID NO: 7 is a sequence of feline IgG1a heavy chain containing AAA (S298A, E333A, R334A) mutations in the rituximab variable region and Fc region.

[0077] SEQ ID NO: 8 is a sequence of feline IgG1a heavy chain containing DLE (S239D, S330L, I332E) mutations in the rituximab variable region and Fc region.

[0078] SEQ ID NO: 9 is a sequence of feline IgG1a heavy chain containing the DE (S239D, I332E) mutation in the rituximab variable region and Fc region.

[0079] SEQ ID NO: 10 is a sequence of feline IgG1a heavy chain containing DAE (G236A, S239D, I332E) mutations in the rituximab variable region and Fc region.

[0080] SEQ ID NO: 11 is a sequence of feline IgG1a heavy chain containing the YWA (L235Y, G237W, S298A) mutation in the rituximab variable region and Fc region.

[0081] SEQ ID NO: 12 is a sequence of feline IgG1a heavy chain containing EFT (G267E, P268F, N324T) mutations in the rituximab variable region and Fc region.

[0082] SEQ ID NO: 13 is a sequence of feline IgG1a heavy chain containing the E345R (E345R) mutation in the rituximab variable region and Fc region.

[0083] SEQ ID NO: 14 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with a WIN_DANG (M234A, L235A, G237A; D265A, N297G) mutation.

[0084] SEQ ID NO: 15 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region having a WIN_DANG_PSS (M234A, L235A, G237A; D265A, N297G; P331S) mutation.

[0085] SEQ ID NO: 16 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to the feline Fc region with a DANG_PSS (D265A, N297G; P331S) mutation.

[0086] SEQ ID NO: 17 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with WIN (M234A, L235A, G237A) mutations.

[0087] SEQ ID NO: 18 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to the feline Fc region with a D270G mutation.

[0088] SEQ ID NO: 19 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused with a feline Fc region having a D270S mutation.

[0089] SEQ ID NO: 20 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with a WIN_PSA (M234A, L235A, G237A; P331A) mutation.

[0090] SEQ ID NO: 21 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with WIN_GSA (M234A, L235A, G237A; P329G, P331A) mutation.

[0091] SEQ ID NO: 22 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with a WIN_GSP (M234A, L235A, G237A; P329G) mutation.

[0092] SEQ ID NO: 23 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with a WIN_PSS (M234A, L235A, G237A; P331S) mutation.

[0093] SEQ ID NO: 24 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to the feline Fc region with a PSS (P331S) mutation.

[0094] SEQ ID NO: 25 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with WIN_GSS (M234A, L235A, G237A; P329G, P331S) mutation.

[0095] SEQ ID NO: 26 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with a WIN_GAP (M234A, L235A, G237A; P329G, S330A) mutation.

[0096] SEQ ID NO: 27 is a sequence of a feline IgG1a heavy chain containing a canine CTLA-4 peptide fused to a feline Fc region with a WIN_GAP (M234A, L235A, G237A; P329G, S330A) mutation.

[0097] SEQ ID NO: 28 is a sequence of canine IgG2 heavy chain containing the GSP (P329G) mutation in the rituximab variable region and Fc region.

[0098] SEQ ID NO: 29 is a sequence of canine IgG2 heavy chain containing GAP (P329G, S330A) mutations in the rituximab variable region and Fc region.

[0099] SEQ ID NO: 30 is a sequence of canine IgG2 heavy chain containing the DANG-GAP (D265A, N297G; P329G, S330A) mutation in the rituximab variable region and Fc region.

[0100] SEQ ID NO: 31 is a sequence of canine IgG2 heavy chain containing KAPA (K322A, P331A) mutations in the rituximab variable region and Fc region.

[0101] SEQ ID NO: 32 is a sequence of canine IgG2 heavy chain containing the WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) mutation in the rituximab variable region and Fc region.

[0102] SEQ ID NO: 33 is a sequence of canine IgG2 heavy chain containing the DANG (D265A, N297G) mutation in the rituximab variable region and Fc region.

[0103] SEQ ID NO: 34 is a sequence of canine IgG2 heavy chain containing AAA (G298A, E333A, R334A) mutations in the rituximab variable region and Fc region.

[0104] SEQ ID NO: 35 is a sequence of canine IgG2 heavy chain containing DLE (S239D, S330L, I332E) mutations in the rituximab variable region and Fc region.

[0105] SEQ ID NO: 36 is a sequence of canine IgG2 heavy chain containing DE (S239D, I332E) mutations in the rituximab variable region and Fc region.

[0106] SEQ ID NO: 37 is a sequence of canine IgG2 heavy chain containing DAE (G236A, S239D, I332E) mutations in the rituximab variable region and Fc region.

[0107] SEQ ID NO: 38 is a sequence of canine IgG2 heavy chain containing YWA (L235Y, G237W, G298A) mutations in the rituximab variable region and Fc region.

[0108] SEQ ID NO: 39 is a sequence of canine IgG2 heavy chain containing EFT (D267E, P268F, N324T) mutations in the rituximab variable region and Fc region.

[0109] SEQ ID NO: 40 is a sequence of canine IgG2 heavy chain containing the Q345R mutation in the rituximab variable region and Fc region.

[0110] SEQ ID NO: 41 is a sequence of canine IgG2 heavy chain containing a canine CTLA-4 peptide fused to the canine Fc region with a D270G mutation.

[0111] SEQ ID NO: 42 is a sequence of canine IgG2 heavy chain containing a canine CTLA-4 peptide fused to the canine Fc region with a D270S mutation.

[0112] SEQ ID NO: 43 is a sequence of canine IgG2 heavy chain containing a canine CTLA-4 peptide fused to the canine Fc region with WIN (M234A, L235A, G237A) mutations.

[0113] SEQ ID NO: 44 is a sequence of canine IgG2 heavy chain containing a canine CTLA-4 peptide fused to the canine Fc region with a WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) mutation.

[0114] SEQ ID NO: 45 is a sequence of equine IgG1 heavy chain containing the rituximab variable region and the wild-type Fc region.

[0115] SEQ ID NO: 46 is a sequence of equine IgG1 heavy chain containing SAP (P329S, Q330A) mutations in the rituximab variable region and Fc region.

[0116] SEQ ID NO: 47 is a sequence of equine IgG1 heavy chain containing WIN (L234A, L235A, G237A) mutations in the rituximab variable region and Fc region.

[0117] SEQ ID NO: 48 is a sequence of equine IgG1 heavy chain containing PSS (Q330S, P331S) mutations in the rituximab variable region and Fc region.

[0118] SEQ ID NO: 49 is a sequence of equine IgG1 heavy chain containing WIN_SAP (L234A, L235A, G237A; P329S, Q330A) mutations in the rituximab variable region and Fc region.

[0119] SEQ ID NO: 50 is a sequence of equine IgG1 heavy chain containing WIN_PSS (L234A, 235A, G237A; Q330S, P331S) mutations in the rituximab variable region and Fc region.

[0120] SEQ ID NO: 51 is a sequence of an equine IgG1 heavy chain containing a canine CTLA-4 peptide fused to the Fc region of an equine with a PAP (Q330A) mutation.

[0121] SEQ ID NO: 52 is a sequence of an equine IgG1 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with an SQP (P229S) mutation.

[0122] SEQ ID NO: 53 is a sequence of an equine IgG4 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with a PSS (A330S, P331S) mutation.

[0123] SEQ ID NO: 54 is a sequence of an equine IgG4 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with an SQP (P239S, A330Q) mutation.

[0124] SEQ ID NO: 55 is a sequence of an equine IgG4 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with WIN_PSS (L234A, Q235A, G237A; A330S, P331S) mutation.

[0125] SEQ ID NO: 56 is a sequence of an equine IgG4 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with a WIN_SAP (L234A, Q235A, G237A; P329S) mutation.

[0126] SEQ ID NO: 57 is a sequence of an equine IgG4 heavy chain containing a canine CTLA-4 peptide fused to the Fc region of an equine with a SAP (P329S) mutation.

[0127] SEQ ID NO: 58 is a sequence of an equine IgG4 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with WIN (L234A, Q235A, G237A) mutations.

[0128] SEQ ID NO: 59 is a sequence of equine IgG7 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with a PSS (A330S, P331S) mutation.

[0129] SEQ ID NO: 60 is a sequence of an equine IgG7 heavy chain containing a canine CTLA-4 peptide fused to the Fc region of an equine with a SAP (P329S) mutation.

[0130] SEQ ID NO: 61 is a sequence of an equine IgG7 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with an SQP (P239S, A330Q) mutation.

[0131] SEQ ID NO: 62 is a sequence of an equine IgG7 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with WIN (L234A, S235A, V236G, G237A) mutation.

[0132] SEQ ID NO: 63 is a sequence of an equine IgG7 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with a WIN_PSS (L234A, S235A, V236G, G237A; A330S, P331S) mutation.

[0133] SEQ ID NO: 64 is a sequence of an equine IgG7 heavy chain containing a canine CTLA-4 peptide fused to the equine Fc region with a WIN_SAP (L234A, S235A, V236G, G237A; P329S) mutation. Detailed Implementation

[0134] The subject matter of the invention can be more readily understood by referring to the following detailed description, which forms a part of this disclosure. It should be understood that the invention is not limited to the specific products, methods, conditions, or parameters described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed invention.

[0135] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms and plural terms shall include singular forms.

[0136] In this disclosure, the singular forms “a,” “an,” and “the” include plural references, and unless the context clearly indicates otherwise, a reference to a particular numerical value includes at least that particular value. Thus, for example, reference to “molecule” or “compound” refers to one or more such molecules or compounds and their equivalents known to those skilled in the art. As used herein, the term “a plurality” means more than one. When expressing a range of values, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another embodiment. All ranges are inclusive and composable.

[0137] In the specification and claims, the amino acid residues in the immunoglobulin heavy chain are numbered according to the Eu index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "The EU index in Kabat" refers to the residue number of the IgG antibody.

[0138] When used in relation to nucleic acids, the term "isolated" refers to nucleic acids that have been identified and isolated from at least one contaminant nucleic acid typically associated with in their natural sources. Isolated nucleic acids exist in a form or environment different from their natural state. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in natural cells. Isolated nucleic acid molecules include those found in cells that typically express polypeptides encoded therein, wherein, for example, the nucleic acid molecules are located in plasmid or chromosomal locations different from those in natural cells. Isolated nucleic acids can exist in single-stranded or double-stranded form. When isolated nucleic acid molecules are used to express proteins, the oligonucleotide or polynucleotide will contain at least a sense strand or coding strand, but may contain both sense and antisense strands (i.e., it may be double-stranded).

[0139] When one nucleic acid molecule is in a functional relationship with another nucleic acid molecule, the nucleic acid molecule is "operably linked" or "operably attached." For example, if a promoter or enhancer influences the transcription of a sequence, it is operably linked to the coding sequence of the nucleic acid; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to the coding sequence of the nucleic acid. If a nucleic acid molecule encoding a variant Fc region is positioned such that the expressed fusion protein contains an upstream or downstream heterologous protein or a functional fragment thereof abutting the variant Fc region polypeptide, the nucleic acid molecule is operably linked to a nucleic acid molecule encoding the heterologous protein (i.e., a protein or a functional fragment thereof that does not contain an Fc region in its natural state); the heterologous protein may be directly adjacent to the variant Fc region polypeptide or may be separated from the polypeptide by a linker sequence of any length and composition. Similarly, when a polypeptide molecule (used herein synonymously with "protein") is in a functional relationship with another polypeptide, it is "operably linked" or "operably attached."

[0140] As used herein, when referring to peptides or proteins (e.g., variant Fc regions or monoclonal antibodies), the term "functional fragment" means a segment of the protein that retains at least one function of the full-length peptide. The size of the fragment can range from six amino acids to the entire amino acid sequence of the full-length peptide minus one amino acid. The functional fragments of the variant Fc region peptides of the present invention retain at least one "amino acid substitution" as defined herein. The functional fragments of the variant Fc region peptides retain at least one function known in the art associated with the Fc region (e.g., ADCC, CDC, Fc receptor binding, C1q binding, downregulation of cell surface receptors), or may, for example, increase the in vivo or in vitro half-life of the peptide to which it is operatively attached.

[0141] The term "purified" or "purified" refers to the substantial removal of at least one contaminant from a sample. For example, antigen-specific antibodies can be purified by completely or substantially removing (at least 90%, 91%, 92%, 93%, 94%, 95%, or more preferably, at least 96%, 97%, 98%, or 99%) at least one contaminating non-immunoglobulin; they can also be purified by removing immunoglobulins that do not bind to the same antigen. Removing non-immunoglobulins and / or removing immunoglobulins that do not bind to a specific antigen increases the percentage of antigen-specific immunoglobulins in the sample. In another example, polypeptides (e.g., immunoglobulins) expressed in bacterial host cells are purified by completely or substantially removing host cell proteins; the percentage of polypeptides in the sample is thus increased.

[0142] The term "natural" as it refers to a polypeptide (e.g., the Fc region) is used herein to mean that the polypeptide has an amino acid sequence consisting of the polypeptide's amino acid sequence (in cases where the polypeptide typically occurs in nature) or its naturally occurring polymorphisms. Natural polypeptides (e.g., natural Fc regions) can be produced by recombinant methods or can be isolated from naturally occurring sources.

[0143] As used herein, the term "expression vector" refers to a recombinant DNA molecule containing the desired coding sequence and the appropriate nucleic acid sequence required to express the operatively linked coding sequence in a particular host organism.

[0144] As used herein, the term “host cell” refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as Escherichia coli, CHO cells, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether it is located in vitro, in situ, or in vivo.

[0145] As used herein, the term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the generally accepted boundaries of the Fc region of the immunoglobulin heavy chain can vary, the IgG heavy chain Fc region is typically defined, for example, as extending from an amino acid residue at approximately position 231 to its carboxyl terminus.

[0146] In some embodiments, variants may contain only a portion of the Fc region and may include or exclude a carboxyl terminus. The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH3. In some embodiments, variants having one or more of these constant domains are contemplated. In other embodiments, variants without such constant domains (or only a portion of such constant domains) are contemplated.

[0147] For example, the "CH2 domain" of the IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The unique feature of the CH2 domain is that it is not tightly paired with another domain. Two N-linked branched carbohydrate chains are interposed between the two CH2 domains of a complete natural IgG molecule.

[0148] The "CH3 domain" of the Fc region of feline IgG is typically an extension from the C-terminus of a residue in the Fc region to the CH2 domain, extending from approximately amino acid residue 341 to approximately amino acid residue 447.

[0149] The “functional Fc region” possesses the “effective function” of the native sequence Fc region. At least one effector function of a polypeptide comprising the variant Fc region of the present invention may be enhanced or weakened relative to the native or parental Fc region comprising said variant. Examples of effector functions include, but are not limited to: C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be operatively linked to a binding domain (e.g., antibody variable domain) and can be assessed using various assays (e.g., Fc binding assay, ADCC assay, CDC assay, target cell depletion in whole blood or graded blood samples, etc.).

[0150] The “natural Fc region” or “wild-type Fc region” refers to the amino acid sequence that is the same as the amino acid sequence of the Fc region that is normally found in nature.

[0151] The “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region (or a fragment thereof) by at least one “amino acid substitution” as defined herein. In a preferred embodiment, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or in the Fc region of the parent peptide, preferably one, two, three, four, or five amino acid substitutions in the native sequence Fc region or the Fc region of the parent peptide. In alternative embodiments, the variant Fc region may be generated according to the methods disclosed herein, and this variant Fc region may be fused with a selected heterologous peptide (such as an antibody variable domain or a non-antibody peptide, such as a receptor or ligand binding domain).

[0152] As used herein, in the context of polypeptides, the term "derivative" refers to a polypeptide comprising an amino acid sequence altered by the introduction of amino acid residue substitutions. As used herein, the term "derivative" also refers to a polypeptide modified by covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, antibodies can be modified, for instance, by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage with cellular ligands or other proteins, etc. Derivative polypeptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, derivative polypeptides have functions similar to or identical to those of the polypeptide from which they are derived. It should be understood that polypeptides comprising the variant Fc region of the present invention can be derivatives as defined herein, preferably with derivatization occurring within the Fc region.

[0153] In this document, the term "substantially feline-derived" as used for reference to polypeptides (e.g., Fc regions or monoclonal antibodies) means that the polypeptide has an amino acid sequence that is 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 the amino acid sequence of a natural feline amino polypeptide. "Substantially canine-derived" and "substantially equine-derived" are similarly defined relative to natural canine or equine polypeptides, respectively.

[0154] The term “Fc receptor” or “FcR” is used to describe a receptor that binds to an Fc region (e.g., the Fc region of an antibody). Preferred FcRs are naturally occurring sequence FcRs. Furthermore, preferred FcRs are those that bind to IgG antibodies (γ receptors) and include receptors of the FcγRI, FcγRII, and FcγRIii subclasses, including allelic variants and alternative splice forms of these receptors. Another preferred FcR includes the neonatal receptor FcRn, responsible for transferring maternal IgG to the fetus (Guyer et al., *Journal of Immunology*, 117:587 (1976); and Kim et al., *Journal of Immunology*, 24:249 (1994)). The term “FcR” as used herein encompasses other FcRs, including those to be identified in the future.

[0155] The phrases “complement-dependent cytotoxicity” and “CDC” refer to the effector functions of IgG and IgM antibodies. When IgG binds to surface antigens on target cells (e.g., cells infected by bacteria or viruses), the classical complement pathway is triggered by the binding of protein C1q to these antibodies, leading to the formation of the membrane attack complex (MAC) and the lysis of the target cell.

[0156] "C1q" is a polypeptide that includes the binding site of the Fc region of immunoglobulins. C1q, together with two serine proteases, C1r and C1s, forms the C1 complex, which is the first component of the CDC pathway.

[0157] The phrases “antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated response in which nonspecific cytotoxic cells expressing FcR (e.g., nonspecific cells such as natural killer (“NK”) cells, neutrophils, and macrophages) recognize antibodies bound to target cells and subsequently cause lysis of the target cells. Primary cells used to mediate ADCC and NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII.

[0158] As used herein, the phrase "effect cell" refers to a leukocyte that expresses one or more FcRs and performs effector functions. Preferably, the cell expresses at least FcγRIii and performs ADCC effector functions. Examples of leukocytes that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood or PBMCs.

[0159] Variant peptides exhibiting "altered" FcRn binding affinity are those that, when measured at pH 6.0, show either increased (i.e., greater, higher) or decreased (i.e., reduced, lower, or less) FcRn binding affinity compared to their parent peptide or peptides containing the native Fc region. Variant peptides showing increased binding affinity to FcRn or increased binding affinity bind FcRn with a greater affinity compared to their parent peptide. Variant peptides showing decreased binding affinity to FcRn or decreased binding affinity bind FcRn with a lower affinity compared to their parent peptide. Such variants showing decreased binding affinity to FcRn may have very little or no binding to FcRn, for example, 0-20% binding to FcRn compared to their parent peptide. When the amounts of the variant peptide and the parent peptide in the binding assay are substantially the same, and all other conditions are identical, a variant peptide that binds FcRn with “enhanced affinity” compared to the parent peptide is a variant peptide that binds FcRn with a higher binding affinity compared to the parent peptide. For example, a variant peptide with enhanced FcRn binding affinity can show an increase in FcRn binding affinity of about 1.10-fold to about 100-fold (more typically about 1.2-fold to about 50-fold) compared to the parent peptide, where the FcRn binding affinity is determined, for example, in an ELISA assay or other methods available to those skilled in the art.

[0160] As used herein, “amino acid substitution” means that at least one existing amino acid residue in a given amino acid sequence is replaced by another different “substitute” amino acid residue. One or more substituted residues can be “naturally occurring amino acid residues” (i.e., encoded by the genetic code) and are selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Substitutions having one or more non-naturally occurring amino acid residues are also covered in the definition of amino acid substitution herein. “Non-naturally occurring amino acid residues” are residues other than those listed above that are naturally occurring amino acid residues, capable of covalently binding to adjacent amino acid residues in a polypeptide chain. Examples of non-naturally occurring amino acid residues include ornithine, ornithine, homoserine, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202: 301-336 (1991).

[0161] The term "assay signal" refers to the output of any method for detecting protein-protein interactions, including but not limited to SPR assays, absorbance measurements from colorimetric assays, fluorescence intensity, or disintegration per minute. Assay formats may include ELISA, facs, SPR, or other methods. Changes in the "assay signal" may reflect changes in cell viability and / or kinetic dissociation rate, kinetic association rate, or both. A "higher assay signal" means that the measured output number is greater than another number (e.g., in an ELISA assay, the measured number for a variant may be higher (larger) compared to the parent peptide). A "lower assay signal" means that the measured output number is less than another number (e.g., in an ELISA assay, the measured number for a variant may be lower (smaller) compared to the parent peptide).

[0162] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in concentration) associated with each Fc receptor-Fc binding interaction. Binding affinity is correlated with the kinetic dissociation rate (usually reported in reverse time, e.g., seconds). 1 The ratio of the concentration to the kinetic association rate (usually reported as concentration per unit time, e.g., moles / second) is directly related. Generally, it is impossible to definitively say whether a change in the equilibrium dissociation constant is due to a difference in the association rate, dissociation rate, or both, unless each of these parameters is experimentally determined (e.g., measured by BIACORE or SAPIDYNE).

[0163] As used herein, the term "hinge region" refers to an extension of an amino acid in, for example, feline IgG1a (e.g., from position 216 to position 230 in feline IgG1a). Hinge regions of other IgG isotypes can be aligned with the IgG sequence by placing the first and last cysteine ​​residues that form the inter-heavy-chain disulfide (SS) bond in the same position.

[0164] As used herein, the term "antibody" is used interchangeably with "immunoglobulin" or "Ig," and is used in the broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological or functional activity. Single-chain antibodies and chimeric, feline or feline-derived, canine or canine-derived, or equine or equine-derived antibodies comprising portions derived from different species, as well as chimeric or CDR-grafted single-chain antibodies, are also covered in this invention and the term "antibody." The various portions of these antibodies can be synthetically and chemically linked together using conventional techniques, or can be prepared as a continuous protein using genetic engineering techniques. For example, nucleic acids encoding chimeric or feline-derived chains can be expressed to produce continuous proteins. See, for example, U.S. Patent Nos. 4,816,567; 4,816,397; WO 86 / 01533; 5,225,539; and 5,585,089 and 5,698,762. For primate-derived antibodies, see also Newman, R. et al., *BioTechnology*, 10: 1455-1460, 1993, and for single-chain antibodies, see Ladner et al., U.S. Patent No. 4,946,778, and Bird, RE et al., *Science*, 242:423-426, 1988. It should be understood that all forms of antibodies containing the Fc region (or a portion thereof) are covered herein within the term "antibody". Furthermore, antibodies can be labeled with detectable markers, immobilized on a solid phase and / or conjugated with heterologous compounds (e.g., enzymes or toxins) according to methods known in the art.

[0165] As used herein, the term "antibody fragment" refers to a portion of a complete 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. Antibody fragments preferably retain at least a portion of the hinge of the IgG heavy chain and optionally the CHI region. In other preferred embodiments, the antibody fragment comprises at least a portion or the entire CH2 region.

[0166] As used herein, the term "functional fragment" in reference to monoclonal antibodies is intended to refer to a portion of the monoclonal antibody that retains its functional activity. Functional activity can be, for example, antigen-binding activity or specificity, receptor-binding activity or specificity, effector functional activity, etc. Monoclonal antibody functional fragments include, for example, individual heavy or light chains and their fragments, 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, for example, in Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Enzymological Methods, 178:497-515 (1989); and Day, ED, Advanced Immunochemistry, 2nd ed., Wiley-Liss, Inc., New York, NY (1990). The term "functional fragment" is intended to include, for example, fragments produced by protease digestion or reduction by monoclonal antibodies, as well as fragments produced by recombinant DNA methods known to those skilled in the art.

[0167] 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 consecutive amino acid residues containing 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.

[0168] As used herein, the term "chimeric antibody" includes monovalent, bivalent, or multivalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by the association of a chimeric heavy chain with a chimeric light chain via a disulfide bridge. A bivalent chimeric antibody is a tetramer formed by the association of two heavy-light chain dimers via at least one disulfide bridge. The chimeric feline heavy chain of an antibody includes an antigen-binding region derived from a non-feline antibody heavy chain, said antigen-binding region being linked to at least a portion of a feline heavy chain constant region (such as CHI or CH2). The chimeric light chain of a feline antibody includes an antigen-binding region derived from a non-feline antibody light chain, said antigen-binding region being linked to at least a portion of a feline light chain constant region (CL). Chimeric canine and equine antibodies are similarly defined.

[0169] Antibodies, fragments, or derivatives of chimeric heavy and light chains with the same or different variable region binding specificities can also be prepared by appropriate association of individual polypeptide chains according to known method steps. Using this method, the host expressing the chimeric heavy chain and the host expressing the chimeric light chain are cultured separately, and the immunoglobulin chains are recovered separately and then associated. Alternatively, the hosts can be co-cultured, allowing the chains to spontaneously associate in the culture medium, after which the assembled immunoglobulin or fragment, or both the heavy and light chains can be expressed in the same host cell. Methods for generating chimeric antibodies are well known in the art (see, for example, U.S. Patent Nos. 6,284,471; 5,807,715; 4,816,567; and 4,816,397).

[0170] As used herein, a “felicized” form of a non-feline (e.g., mouse) antibody (i.e., a felicized antibody) is an antibody containing a minimal sequence derived from a non-feline immunoglobulin or lacking said sequence. In most cases, a felicized antibody is a feline immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-feline species (donor antibody) with the desired specificity, affinity, and capability. In some cases, frame region (FR) residues of the feline immunoglobulin are replaced by corresponding non-feline residues. Furthermore, felicized antibodies may contain residues not present in either the recipient or donor antibody. These modifications are typically made to further optimize antibody performance. Typically, a felicized antibody will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops (CDRs) correspond to hypervariable loops of a non-feline immunoglobulin, and all or substantially all FR residues are hypervariable loops of the feline immunoglobulin sequence. Feline antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of feline immunoglobulins.

[0171] As used herein, a “canidified” form of a non-canine (e.g., mouse) antibody (i.e., a canidified antibody) is an antibody containing a minimal sequence derived from a non-canine immunoglobulin or lacking said sequence. In most cases, a canidified antibody is a canine immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-canine species (donor antibody) with the desired specificity, affinity, and capability. In some cases, frame region (FR) residues of the canine immunoglobulin are replaced by corresponding non-canine residues. Furthermore, canidified antibodies may contain residues not present in either the recipient or donor antibody. These modifications are typically made to further optimize antibody performance. Generally, canidified antibodies will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops (CDRs) correspond to hypervariable loops of a non-canine immunoglobulin, and all or substantially all FR residues are hypervariable loops of the canine immunoglobulin sequence. Canine-derived antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of canine immunoglobulins.

[0172] As used herein, the “equinologized” form of a non-equine (e.g., mouse) antibody (i.e., an equinologized antibody) is an antibody containing a minimal sequence derived from a non-equine immunoglobulin or lacking said sequence. In most cases, an equinologized antibody is an equine immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-equine species (donor antibody) with the desired specificity, affinity, and capability. In some cases, frame region (FR) residues of the equine immunoglobulin are replaced by corresponding non-equine residues. Furthermore, equinologized antibodies may contain residues not present in either the recipient or donor antibody. These modifications are typically made to further optimize antibody performance. Generally, equinologized antibodies will contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops (CDRs) correspond to hypervariable loops of the non-equine immunoglobulin, and all or substantially all FR residues are hypervariable loops of the equine immunoglobulin sequence. Equine-derived antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of equine immunoglobulins.

[0173] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding domain of a heterologous "adhesin" protein (e.g., a receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, an immunoadhesin comprises an adhesin amino acid sequence that is not an antibody's antigen recognition and binding site (antigen combination site) (i.e., "heterologous") with an immunoglobulin constant domain sequence at the desired binding specificity.

[0174] As used herein, the term "ligand-binding domain" refers to any native receptor or any region or derivative thereof that retains at least the qualitative ligand-binding ability of the corresponding native receptor. In some embodiments, the receptor is derived from a cell surface polypeptide having an extracellular domain homologous to a member of the immunoglobulin superfamily. Other receptors that are not members of the immunoglobulin superfamily but are still specifically covered within this definition are receptors for cytokines, and specifically receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hemopoietin and nerve growth factor receptor superfamily, and cell adhesion molecules (e.g., E-, L-, and P-selectins).

[0175] As used herein, the term “receptor-binding domain” refers to any natural ligand of a receptor, including, for example, cell adhesion molecules, or any region or derivative of such natural ligands that at least retains the qualitative receptor-binding ability of the corresponding natural ligand.

[0176] As used herein, an “isolated” polypeptide is a polypeptide that has been identified, isolated, and / or recovered from components of its natural environment. Contaminant components in its natural environment are materials that would interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the isolated polypeptide is purified to (1) greater than 95% by weight of the polypeptide, as determined by the Lowry method, and preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotary cup sequencer, or (3) homogeneity achieved by SDS-page under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated polypeptides include recombinant intracellular in situ polypeptides, since at least one component of the polypeptide’s natural environment will be absent. However, typically, isolated polypeptides are prepared by at least one purification step.

[0177] As used herein, the terms “symptom” and “disease” are used interchangeably to refer to any symptom that would benefit from treatment with a variant polypeptide (a polypeptide comprising the variant Fc region of the present invention), including chronic and acute symptoms or diseases (e.g., pathological symptoms that make a patient susceptible to a specific symptom).

[0178] As used herein, the term "receptor" refers to a polypeptide capable of binding to at least one ligand. Preferred receptors are cell surface receptors or soluble receptors having an extracellular ligand-binding domain and optionally other domains (e.g., transmembrane domains, intracellular domains, and / or membrane anchors). The receptor to be evaluated in the assays described herein can be a complete receptor or a fragment or derivative thereof (e.g., a fusion protein containing the binding domain of a receptor fused to one or more heterologous polypeptides). Furthermore, receptors to be evaluated for their binding properties can be present in cells or isolated, and can optionally be coated onto an assay plate or some other solid phase, or directly labeled and used as probes.

[0179] Feline IgG The inventors of this application have discovered that by substituting one or more amino acid residues in the Fc region of feline IgG1, advantageous properties can be introduced into feline IgG, such as reducing or completely knocking out CDC, ADCC, and ADCP activities.

[0180] Therefore, referring to the positions encoded according to the EU index as in Kabat, the Fc region of feline IgG1a may contain one or more mutations or substitutions at positions including amino acids 234, 235, 236, 237, 239, 329, 265, 267, 268, 270, 297, 298, 330, 331, 332, 333, 334 and / or 345 (Kabat et al., Sequences of Immunologically Significant Proteins, 5th Edition, Department of Public Health Services, National Institutes of Health, Bethesda, Maryland (1991)).

[0181] In various embodiments, using EU index numbering, the Fc region of feline IgG1a may contain one or more substitutions selected from the following: 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.For example, various substitution combinations in the Fc region of feline IgG1a can include one or more of the following, referred to herein by abbreviated names: 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, P322A), P234A, L235A, G237A, P322A, P322A, GAP (P329G, S330A), DANG (D265A, N297G), KA (K322A), PA (P331A), KAPA (K322A, P331A), WIN_KA (M234A, L235A, G237A, P322A), WIN_PA (M234A, L235A, G237A, P322A), P322A, P322A, GAP (P329G, S330 ... 331A), WIN_KAPA (M234A, L235A, G237A, K322A, P331A), WIN_GAP (M234A, L235A, G237A, P329G, S330A), DANG-GAP (D265A, N297G, P329G, S3 30A), WIN_DANG (M234A, L235A, G237A, D265A, N297G), WIN_DANG_GAP (M234A, L235A, G237A, D265A, N297G, P329G, S330A), DANG_PSS (D265 A, N297G, P331S), WIN_PSA (M234A, L235A, G237A, P331A), WIN_DANG_PSS (M234A, L235A, G237A, D265A, N297G, P331S), WIN_PSS (M234A, L2 35A, G237A, P331S), WIN_GSA (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). Due to significant sequence and structural similarity, the Fc region of feline IgG1b may also contain one or more corresponding substitutions or combinations of substitutions at positions corresponding to those in IgG1a described above.

[0182] As another example, feline IgG1a Fc can be contained within a recombinantly modified protein, such as a feline IgG heavy chain containing the rituximab variable region and mutation invariant region, or a feline IgG1a Fc fused to another functional peptide, such as a CTLA-4 peptide fragment. Examples of such proteins include the following sequences: Rituximab feline IgG1a GSP (P329G) SEQ ID NO: 1 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0183] Rituximab feline IgG1aGAP (P329G, S330A) SEQ ID NO: 2 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0184] Rituximab feline IgG1a DANG-GAP (D265A, N297G; P329G, S330A) SEQ ID NO: 3 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0185] Rituximab feline IgG1a KAPA (K322A, P331A) SEQ ID NO: 4 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0186] Rituximab feline IgG1a WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) SEQ ID NO: 5 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0187] Rituximab feline IgG1aDANG (D265A, N297G) SEQ ID NO: 6 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0188] Rituximab feline IgG1aAAA (S298A, E333A, R334A) SEQ ID NO: 7 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0189] Rituximab feline IgG1aDLE (S239D, S330L, I332E) SEQ ID NO: 8 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0190] Rituximab feline IgG1aDE (S239D, I332E) SEQ ID NO: 9 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0191] Rituximab feline IgG1aDAE (G236A, S239D, I332E) SEQ ID NO: 10 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0192] Rituximab feline IgG1a YWA (L235Y, G237W, S298A) SEQ ID NO: 11 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0193] Rituximab feline IgG1a EFT (G267E, P268F, N324T) SEQ ID NO: 12 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0194] Rituximab feline IgG1a E345R (E345R) SEQ ID NO: 13 Amino acids 1-116 of the sequence contain a rituximab heavy chain fragment, and amino acids 117-459 contain a heavy chain fragment containing a constant domain of feline IgG1a.

[0195] Canine CTLA-4 and feline IgG1a fusion WIN_DANG (M234A, L235A, G237A; D265A, N297G) SEQ ID NO: 14 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0196] Canine CTLA-4 and feline IgG1a fusion WIN_DANG_PSS (M234A, L235A, G237A; D265A, N297G; P331S) SEQ ID NO: 15 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0197] The canine CTLA-4 and feline IgG1a fusion DANG_PSS (D265A, N297G; P331S) SEQ ID NO: 16 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0198] Canine CTLA-4 and feline IgG1a fusion variant WIN (M234A, L235A, G237A) SEQ ID NO: 17 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0199] Canine CTLA-4 and feline IgG1a fusion D270G (D270G) SEQ ID NO: 18 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0200] Canine CTLA-4 and feline IgG1a fusion D270S (D270S) SEQ ID NO: 19 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0201] Canine CTLA-4 and feline IgG1a fusion variant WIN_PSA (M234A, L235A, G237A; P331A) SEQ ID NO: 20 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0202] Canine CTLA-4 and feline IgG1a fusion Win_GSA (M234A, L235A, G237A; P329G, P331A) SEQ ID NO: 21 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0203] Canine CTLA-4 and feline IgG1a fusion WIN_GSP (M234A, L235A, G237A; P329G) SEQ ID NO: 22 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0204] Canine CTLA-4 and feline IgG1a fusion Win_PSS (M234A, L235A, G237A; P331S) SEQ ID NO: 23 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0205] Canine CTLA-4 and feline IgG1a fusion PSS (P331S) SEQ ID NO: 24 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0206] Canine CTLA-4 and feline IgG1a fusion Win_GSS (M234A, L235A, G237A; P329G, P331S) SEQ ID NO: 25 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0207] Canine CTLA-4 and feline IgG1a fusion variant WIN_GAP (M234A, L235A, G237A; P329G, S330A) SEQ ID NO: 26 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0208] Canine CTLA-4 and feline IgG1a fusion WIN_SAS (M234A, L235A, G237A; P329S, S330A, P331S) SEQ ID NO: 27 Amino acids 21-145 contain the canine CTLA-4 sequence, and amino acids 146-382 contain a fragment of the feline IgG1a heavy chain containing the Fc region.

[0209] Canine IgG The inventors of this application have surprisingly discovered that by substituting one or more amino acid residues in the Fc region of canine IgG2, advantageous properties can be introduced into canine IgG, such as reducing or completely knocking out CDC, ADCC and ADCP activities, or increasing or decreasing binding affinity to canine Fcγ receptors and C1q.

[0210] Therefore, in various embodiments, using EU index numbers such as in Kabat, the Fc region of canine IgG2 may contain one or more mutations or substitutions at positions listed in Table 4, such as those listed in Table 5, and / or substitutions selected from: M234A, L235A, L235Y, G236A, G237A, G237W, S239D, D265A, D270G, D270S, N297G, G298A, K322A, P329G, S330A, S330L, P331A, I332E, E333A, R334A. For example, various substitution combinations in the Fc region of canine IgG2 may include one or more of the following, referred to herein by abbreviated names: 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).

[0211] As another example, mutant canine IgG2 Fc can be contained within a recombinantly modified protein, such as a canine IgG heavy chain containing both the rituximab variable region and the mutation constant region, or a fusion protein containing another effector region (such as a CTLA-4 sequence) fused to a canine IgG heavy chain containing the mutation constant region. Examples of such proteins include the following sequences: Rituximab canine IgG2 GSP (P329G) SEQ ID NO: 28 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0212] Rituximab canine IgG2 GAP (P329G, S330A) SEQ ID NO: 29 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0213] Rituximab canine IgG2 DANG-GAP (D265A, N297G; P329G, S330A) SEQ ID NO: 30 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0214] Rituximab canine IgG2 KAPA (K322A, P331A) SEQ ID NO: 31 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0215] Rituximab canine IgG2 WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) SEQ ID NO: 32 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0216] Rituximab canine IgG2 DANG (D265A, N297G) SEQ ID NO: 33 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0217] Rituximab canine IgG2 AAA (G298A, E333A, R334A) SEQ ID NO: 34 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0218] Rituximab in canines IgG2DLE (S239D, S330L, I332E) SEQ ID NO: 35 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0219] Rituximab canine IgG2DE (S239D, I332E) SEQ ID NO: 36 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0220] Rituximab canine IgG2DAE (G236A, S239D, I332E) SEQ ID NO: 37 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0221] Rituximab in canines IgG2YWA (L235Y, G237W, G298A) SEQ ID NO: 38 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0222] Rituximab canine IgG2EFT (D267E, P268F, N324T) SEQ ID NO: 39 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0223] Rituximab canine IgG2Q345R (Q345R) SEQ ID NO: 40 Amino acids 1-116 contain fragments of the heavy chain variable region of rituximab, and amino acids 117-456 contain mutant canine IgG2 heavy chains containing constant regions.

[0224] Canine CTL4-Canine IgG2 fusion D270G (D270G) SEQ ID NO: 41 Amino acids 1-145 contain fragments of canine CTLA-4 (accession number XP_038303218), and amino acids 146-382 contain fragments of the canine IgG2 heavy chain containing constant regions.

[0225] Canine CTL4-Canine IgG2 fusion D270S (D270S) SEQ ID NO: 42 Amino acids 1-145 contain fragments of canine CTLA-4 (accession number XP_038303218), and amino acids 146-382 contain fragments of the canine IgG2 heavy chain containing constant regions.

[0226] Canine CTL4-canine IgG2 fusion WIN (M234A, L235A, G237A) SEQ ID NO: 43 Amino acids 1-145 contain fragments of canine CTLA-4 (accession number XP_038303218), and amino acids 146-382 contain fragments of the canine IgG2 heavy chain containing constant regions.

[0227] Canine CTL4-canine IgG2 fusion WIN_DANG_GAP (M234A, L235A, G237A; D265A, N297G; P329G, S330A) SEQ ID NO: 44 Amino acids 1-145 contain fragments of canine CTLA-4 (accession number XP_038303218), and amino acids 146-382 contain fragments of the canine IgG2 heavy chain containing constant regions.

[0228] Equine IgG The inventors of this application have surprisingly discovered that by substituting one or more amino acid residues in the Fc region of equine IgG1, advantageous properties can be introduced into equine IgG, such as reducing or completely knocking out CDC, ADCC and ADCP activities, or increasing or decreasing binding affinity to canine Fcγ receptors and C1q.

[0229] Therefore, in various embodiments, the Fc region of equine IgG1 may contain one or more mutations or substitutions, including mutations or substitutions at positions 229, 234, 235, 237, 329, 330, and / or 331. In various embodiments, using EU index numbers such as in Kabat, the Fc region of equine IgG1 may contain one or more mutations and / or substitutions selected from the following: P229S, L234A, L235A, G237A, P329S, Q330A, Q330S, and P331S. For example, various substitution combinations in the Fc region of equine IgG1 may include one or more of the following, which are referred to herein by abbreviated names: SAP (P329S, Q330A), PAP (Q330A), SQP (P229S), WIN (L234A, L235A, G237A), PSS (Q330S, P331S), WIN_SAP (L234A, L235A, G237A; P329S, Q330A) and WIN_PSS (L234A, L235A, G237A; Q330S, P331S).

[0230] As another example, mutant equine IgG1 Fc can be contained within a recombinantly modified protein, such as an equine IgG heavy chain containing both a rituximab variable region and a mutation constant region, or a fusion protein containing an effector region (such as a CTLA-4 sequence) fused to an equine IgG heavy chain containing a mutation constant region. Examples of such proteins include the following sequences: Rituximab in equines IgG1 wt SEQ ID NO: 45 Amino acids 1-121 contain the rituximab variable region, and amino acids 122-458 contain the wild-type sequence of the constant region of the equine IgG1 heavy chain (e.g., accession number CAC44624).

[0231] Rituximab in equines IgG1SAP (P329S, Q330A) SEQ ID NO: 46 Amino acids 1-121 contain the rituximab variable region, and amino acids 122-458 contain the mutant sequence of the constant region of the equine IgG1 heavy chain.

[0232] Rituximab in equines IgG1WIN (L234A, L235A, G237A) SEQ ID NO:47 Amino acids 1-121 contain the rituximab variable region, and amino acids 122-458 contain the mutant sequence of the constant region of the equine IgG1 heavy chain.

[0233] Rituximab in equines IgG1PSS (Q330S, P331S) SEQ ID NO: 48 Amino acids 1-121 contain the rituximab variable region, and amino acids 122-458 contain the mutant sequence of the constant region of the equine IgG1 heavy chain.

[0234] Rituximab in equines IgG1 WIN_SAP (L234A, L235A, G237A; P329S, Q330A) SEQ ID NO:49 Amino acids 1-121 contain the rituximab variable region, and amino acids 122-458 contain the mutant sequence of the constant region of the equine IgG1 heavy chain.

[0235] Rituximab in equines IgG1 WIN_PSS (L234A, L235A, G237A; Q330S, P331S) SEQ ID NO:50 Amino acids 1-121 contain the rituximab variable region, and amino acids 122-458 contain the mutant sequence of the constant region of the equine IgG1 heavy chain.

[0236] Canine CTLA-4 IgG1 fusion PAP (Q330A) SEQ ID NO: 51 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-384 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0237] Canine CTLA-4 IgG1 fusion SQP (P229S) SEQ ID NO: 52 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-384 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0238] The inventors of this application have surprisingly discovered that by substituting one or more amino acid residues in the Fc region of equine IgG4, advantageous properties can be introduced into equine IgG, such as reducing or completely knocking out CDC, ADCC and ADCP activities, or increasing or decreasing binding affinity to canine Fcγ receptors and C1q.

[0239] Therefore, in various embodiments, the Fc region of equine IgG4 may contain one or more mutations or substitutions, including mutations or substitutions at positions 229, 234, 235, 237, 329, 330, and / or 331. In various embodiments, using EU index numbers such as in Kabat, the Fc region of equine IgG4 may contain one or more mutations and / or substitutions selected from the following: L234A, Q235A, G237A, P239S, A330Q, A330S, P331S. For example, various substitution combinations in the Fc region of equine IgG4 may include one or more of the following, which are referred to herein by abbreviated names: PSS (A330S, P331S), SQP (P239S, A330Q), Win (L234A, Q235A, G237A), WinPSS (L234A, Q235A, G237A; A330S, P331S), SAP (P329S), and WinSAP (L234A, Q235A, G237A; P329S).

[0240] As another example, mutant equine IgG4 Fc can be contained within a recombinantly modified protein, such as an equine IgG heavy chain containing both a rituximab variable region and a mutation constant region, or a fusion protein containing an effector region (such as a CTLA-4 sequence) fused to an equine IgG heavy chain containing a mutation constant region. Examples of such proteins include the following sequences: Canine CTLA-4 IgG4 fusion PSS (A330S, P331S) SEQ ID NO: 53 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-374 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0241] Canine CTLA-4 IgG4 fusion SQP (P239S, A330Q) SEQ ID NO: 54 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-374 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0242] Canine CTLA-4 IgG4 fusion variant WIN_PSS (L234A, Q235A, G237A; A330S, P331S) SEQ ID NO: 55 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-374 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0243] Canine CTLA-4 IgG4 fusion variant WIN_SAP (L234A, Q235A, G237A; P329S) SEQ ID NO: 56 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-374 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0244] Canine CTLA-4 IgG4 fusion with equine IgG4, SAP (P329S) SEQ ID NO: 57 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-374 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0245] Canine CTLA-4 IgG4 fusion variant WIN (L234A, Q235A, G237A) SEQ ID NO: 58 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-374 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0246] The inventors of this application have surprisingly discovered that by substituting one or more amino acid residues in the Fc region of equine IgG7, advantageous properties can be introduced into equine IgG, such as reducing or completely knocking out CDC, ADCC and ADCP activities, or increasing or decreasing binding affinity to canine Fcγ receptors and C1q.

[0247] Therefore, in various embodiments, the Fc region of equine IgG7 may contain one or more mutations or substitutions, including mutations or substitutions at positions 229, 234, 235, 237, 329, 330, and / or 331. In various embodiments, using EU index numbers such as in Kabat, the Fc region of equine IgG7 may contain one or more mutations and / or substitutions selected from the following: L234A, S235A, V236G, G237A, P239S, P329S, A330S, A330Q, P331S. For example, various substitution combinations in the Fc region of equine IgG7 may include one or more of the following, referred to herein by abbreviated names: 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).

[0248] As another example, mutant equine IgG7 Fc can be contained within a recombinantly modified protein, such as an equine IgG heavy chain containing both a rituximab variable region and a mutation constant region, or a fusion protein containing an effector region (such as a CTLA-4 sequence) fused to an equine IgG heavy chain containing a mutation constant region. Examples of such proteins include the following sequences: Canine CTLA-4 IgG7 fusion PSS (A330S, P331S) SEQ ID NO: 59 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-375 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0249] Canine CTLA-4 IgG7 fusion SAP (P329S) SEQ ID NO: 60 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-375 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0250] Canine CTLA-4 IgG7 fusion SQP (P239S, A330Q) SEQ ID NO: 61 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-375 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0251] Canine CTLA-4 IgG7 fusion WIN (L234A, S235A, V236G, G237A) SEQ ID NO: 62 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-375 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0252] Canine CTLA-4 IgG7 fusion variant WIN_PSS (L234A, S235A, V236G, G237A; A330S, P331S) SEQ ID NO: 63 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-375 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0253] Canine CTLA-4 IgG7 fusion variant WIN_SAP (L234A, S235A, V236G, G237A; P329S) SEQ ID NO: 64 Amino acids 22-145 contain a fragment of canine CTLA-4 (accession number XP_038303218), and amino acids 146-375 contain a fragment of equine IgG1 heavy chain containing a mutation constant region.

[0254] Method for preparing antibody molecules of the present invention Methods for preparing antibody molecules are well known in the art and are 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 herein by reference in their entirety. Any suitable method, process, or technique known to those skilled in the art can be used. Antibody molecules having the variant Fc region of the present invention can be generated according to methods well known in the art. In some embodiments, the variant Fc region may be fused with a selected heterologous polypeptide, such as an antibody variable domain or a receptor or ligand binding domain.

[0255] With the advent of molecular biology methods and recombinant technologies, those skilled in the art can generate antibodies and antibody-like molecules through recombinant means, thereby generating gene sequences of the specific amino acid sequences present in the polypeptide structure encoding the antibody. Such antibodies can be produced by cloning the gene sequence of the polypeptide chain encoding the antibody or by the direct synthesis of the polypeptide chain, wherein the synthesized chain assembles to form an active tetramer (H2L2) structure with affinity for specific epitopes and antigenic determinants. This allows for the readily available generation of antibodies with sequence properties of neutralizing antibodies from different species and sources.

[0256] Regardless of the source of the antibody, how it is recombinantly constructed, or how it is synthesized in vitro or in vivo using transgenic animals, laboratory or commercial-scale large cell cultures, transgenic plants, or through direct chemical synthesis without the use of living organisms at any stage of the process, all antibodies possess a similar overall 3D structure. This structure is typically given as H2L2 and refers to the fact that antibodies generally contain two light (L) amino acid chains and two heavy (H) amino acid chains. Both chains have regions capable of interacting with structurally complementary antigen targets. The regions that interact with the target are called "variable" or "V" regions and are characterized by differences in the amino acid sequences of antibodies from different antigen specificities. The variable regions of the H or L chains contain amino acid sequences capable of specifically binding to the antigen target.

[0257] As used herein, the term "antigen-binding region" refers to the portion of an antibody molecule containing amino acid residues that interact with the antigen and confer the antibody's specificity and affinity for the antigen. The antibody-binding region comprises the "framework" amino acid residues necessary to maintain the correct conformation of the antigen-binding residues. Within the variable region of the H or L chain that provides the antigen-binding region are smaller sequences known as "hypervariates" because they exhibit significant variability between antibodies of different specificities. These hypervariable regions are also called "complementarity-determining regions" or "CDR" regions. These CDR regions illustrate the fundamental specificity of the antibody for a particular antigenic determinant structure.

[0258] CDRs represent discontinuous segments of amino acids within the variable region. However, regardless of species, these key amino acid sequences have been found to occupy similar positions within the variable heavy and light chain regions as within the variable chain's amino acid sequence. All antibodies have three CDR regions each in their variable heavy and light chains, each discontinuous from the others. In all mammalian species, antibody peptides contain constant regions (i.e., highly conserved) and variable regions, and within the latter, there are CDRs and so-called "framework regions" consisting of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

[0259] The present invention further provides a vector comprising at least one of the aforementioned nucleic acids. Due to the degeneracy of the genetic code, more than one codon can be used to encode a specific amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each capable of encoding an amino acid. By considering anomalous base pairings and the frequency of actual use of a specific codon (to encode a specific amino acid) in eukaryotic or prokaryotic cells expressing antibodies or portions, the probability that a specific oligonucleotide will actually constitute the actual coding sequence can be estimated. Such “codon usage rules” are disclosed in: Lathe et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe’s “codon usage rules,” a single nucleotide sequence or set of nucleotide sequences containing the theoretically “most probable” nucleotide sequence capable of encoding a feline, equine, or canine IgG sequence can be identified. Furthermore, it is possible to provide antibody coding regions for use in the present invention by altering existing antibody genes using standard molecular biology techniques that produce 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 antibody or peptide.

[0260] For example, one type of substitution is conserved amino acid substitution. This type of substitution involves replacing a given amino acid in a feline antibody peptide with another amino acid that has similar properties. Substitutions that are generally considered conserved are substitutions between aliphatic amino acids Ala, Val, Leu, and Ile; interchanges of hydroxyl residues Ser and Thr; exchange of acidic residues Asp and Glu; substitutions between amide residues Asn and Gin; exchange of basic residues Lys and Arg; and substitutions between aromatic residues Phe, Tyr, etc. Guidelines on which amino acid changes may be phenotypic silencing can be found in Bowie et al., 247 Science 1306-10 (1990).

[0261] Variant feline antibodies or peptides may be fully functional or lack function in one or more activities. Fully functional variants typically contain only conserved variations or variations in non-critical residues or non-critical regions. Functional variants may also contain substitutions of similar amino acids that result in no or minimal change in function. Alternatively, such substitutions may affect function to some extent, either positively or negatively. Non-functional variants typically contain one or more non-conserved amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or deletions in critical residues or critical regions.

[0262] Essential amino acids can be identified using methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces a single alanine mutation at each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as epitope binding or in vitro ADCC activity. Sites crucial for ligand-receptor binding can also be determined by structural analysis, such as crystallography, NMR, or photoaffinity labeling. Smith et al., 224 Journal of Molecular Biology 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

[0263] Furthermore, polypeptides typically contain amino acids beyond the twenty naturally occurring amino acids. In addition, many amino acids, including terminal amino acids, can be modified through natural processes, such as processing and other post-translational modifications, or through chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP ribosylation, amidation, covalent linkage of flavin, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine ​​residues, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, isopentenylation, racemization, selenization, sulfation, transfer RNA-mediated amino acid addition to proteins such as argininoylation, and ubiquitination. Such modifications are well known to those skilled in the art and have been described in detail in the scientific literature. For example, several particularly common modifications—glycosylation, lipid attachment, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues—are described in most basic texts, such as *Proteins-Structure and Molecular Properties* (2nd ed., TE Creighton, WH Freeman & Co., NY, 1993). There are many detailed reviews on this topic, such as Wold, *Posttranslational Covalent Modification of Proteins*, 1-12 (edited by Johnson, Academic Press, NY, 1983); Seifter et al., 182. Enzymatic Methods 626-46 (1990); and Rattan et al. 663, *Annals of the New York Academy of Sciences* ( Ann. NY Acad. Sci. )》 48-62 (1992).

[0264] In another aspect, the present invention provides antibody derivatives. These "derivatives" of antibodies contain additional chemical portions that are not normally part of a protein. Covalent modifications of proteins are included within the scope of this invention. Such modifications can be introduced into the molecule by reacting the target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side-chain or terminal residues. For example, derivatization with bifunctional agents well known in the art can be used to crosslink antibodies or fragments with a water-insoluble carrier matrix or other macromolecular carrier.

[0265] Derivatives also include labeled, radiolabeled monoclonal antibodies. For example, those using radioactive iodine (251, 1311), carbon (4C), sulfur (35S), indium, and tritium (H) 3 Conjugates of monoclonal antibodies with biotin or avidin, or with enzymes such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucosyl amylase, carboxylic acid dehydratase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase; and conjugates of monoclonal antibodies with bioluminescent agents (e.g., luciferase), chemiluminescent agents (e.g., acridinium ester), or fluorescent agents (e.g., phycobiliproteins).

[0266] Another derivative of the present invention, a bifunctional antibody, is a bispecific antibody produced by combining portions of two separate antibodies that recognize two different groups of antigens. This can be achieved through cross-linking or recombination techniques. Additionally, a portion can be added to the antibody or a portion thereof to increase its in vivo half-life (e.g., by prolonging the time it takes to be cleared from the bloodstream). Such techniques include, for example, adding a PEG portion (also known as polyethylene glycolation), and are well known in the art. See U.S. Patent Application Publication No. 20030031671.

[0267] In some embodiments, the nucleic acid encoding the host antibody is directly introduced into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. After the host nucleic acid is introduced into the cell, the cell is typically incubated, usually at 37°C, sometimes selectively, for a period of approximately 1–24 hours to allow antibody expression. In one embodiment, the antibody is secreted into the supernatant of the cell growth medium. Traditionally, monoclonal antibodies are produced as naturally occurring molecules in murine hybridoma lines. In addition to the techniques described above, this invention provides recombinant DNA expression of antibodies. This allows for the production of antibodies, as well as a spectrum of antibody derivatives and fusion proteins from selected host species.

[0268] The nucleic acid sequence encoding at least one antibody, portion, or polypeptide of the present invention can be recombined with vector DNA using conventional techniques, including flush or staggered ends for ligation, restriction enzyme digestion for providing suitable ends, filling of sticky ends where appropriate, alkaline phosphatase treatment for avoiding unwanted ligation, and ligation with a suitable ligase. Techniques for such operations are disclosed, for example, in Maniatis et al., *Molecular Cloning, Lab Manual* (Cold Spring Harbor Laboratory Press, New York, 1982 and 1989) and Ausubel et al., 1993. Ibid., and can be used to construct nucleic acid sequences encoding antibody molecules or their antigen-binding regions.

[0269] A nucleic acid molecule (such as DNA) is said to be "capable of expressing" a polypeptide if it contains a nucleotide sequence that carries information about the regulation of transcription and translation, and this sequence is "operably linked" to a nucleotide sequence that encodes the polypeptide. An operable link is a link in which the regulatory DNA sequence and the DNA sequence seeking expression are linked in a manner that allows the gene to be expressed in a recoverable amount as a peptide or antibody portion. As is well known in similar fields, the precise nature of the regulatory region required for gene expression can vary from organism to organism. See, for example, Sambrook et al., 2001 ibid.; Ausubel et al., 1993 ibid.

[0270] Therefore, this invention covers the expression of antibodies or peptides in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including in vivo or in situ bacterial, yeast, insect, fungal, avian, and mammalian cells, or host cells derived from mammals, insects, birds, or yeast. Mammalian cells or tissues may be derived from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats. Any other suitable mammalian cells known in the art may also be used.

[0271] In one embodiment, the nucleotide sequence of the present invention is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. For this purpose, any of a variety of vectors may be used. See, for example, Ausubel et al., 1993, ibid. Important factors in choosing a specific plasmid or viral vector include: the ease with which recipient cells containing the vector can be identified and selected from those that do not contain the vector; the desired copy number of the vector in the specific host; and whether it is desired to enable the vector to “shuttle” between host cells of different species.

[0272] Examples of prokaryotic vectors known in the art include plasmids, such as those capable of replicating in *E. coli* (e.g., pBR322, CoIEl, pSCIOl, pACYC 184, .pi.vX). Such plasmids are disclosed, for example, by Maniatis et al., 1989, ibid.; Ausubel et al., 1993, ibid. Bacillus plasmids include pC194, pC221, pT127, etc. These plasmids were disclosed by Gryczan in *The Molecular Biology of Bacillus*, pp. 307-329 (Academic Press, New York, 1982).

[0273] Suitable Streptomyces plasmids include plJIOl (Kendall et al., 169 J. Bacterial. 4177-83 (1987)) and Streptomyces bacteriophages, such as phLC31 (Chater et al., published at the Sixth International Symposium on Actinomycetes BIO. 45-54 (Akademiai Kaido, Budapest, Hungary)). 30 Hungary) 1986). Pseudomonas plasmids are reviewed in the following literature: John et al., 8 Rev. Infect. Dis. 693-704 (1986); Izak:i, 33 Jpn. Journal of Bacteriology 729-42 (1978); and Ausubel et al., 1993 ibid.

[0274] Alternatively, gene expression elements that can be used to express 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 *Molecular Cell Biology* 280 (1983)), the Rous sarcoma virus LTR (Gorman et al., 79 *Proceedings of the National Academy of Sciences*, USA 6777 (1982)), and the Moloney murine leukemia virus LTR (Grosschedl et al., 41 *Cell* 885 (1985)); (b) splice regions and polyadenylation sites, such as those derived from the late SV40 region (Okayarea et al., 1983); and (c) polyadenylation sites such as those in SV40 (Okayama et al., 1983).

[0275] Immunoglobulin cDNA genes can be expressed, as described by Weidle et al., 51 Gene, 21 (1987), using the SV40 early promoter and its enhancer as expression elements, the mouse immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globulin intervention sequence, immunoglobulin and rabbit S-globulin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin genes consisting of part cDNA and part genomic DNA (Whittle et al., Protein Engineering, 499 (1987)), the transcription promoter can be human cytomegalovirus, the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin, and the mRNA splicing and polyadenylation regions can be native chromosomal immunoglobulin sequences.

[0276] In this embodiment, to express the cDNA gene in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancer is either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains introns greater than 31 bp, and the polyadenylation and transcription termination regions are derived from natural chromosomal sequences corresponding to the synthesized immunoglobulin chains. In other embodiments, cDNA sequences encoding other proteins are combined with the above expression elements to achieve protein expression in mammalian cells.

[0277] Each fused gene can be assembled into or inserted into an expression vector. Recipient cells capable of expressing the immunoglobulin chain gene product are then transfected individually with the gene encoding the peptide or H chain or L chain, or co-transfected with both H and L chain genes. Transfected recipient cells are cultured under conditions that allow for the expression of incorporated genes, and the expressed immunoglobulin chain or intact antibody or fragment is recovered from the culture.

[0278] In one embodiment, a gene encoding a peptide or a fusion of H and L chains, or a portion thereof, is assembled in a separate expression vector, which is then used for co-transfection of recipient cells. Alternatively, the gene encoding a fusion of H and L chains can be assembled on the same expression vector. For transfection of the expression vector and antibody production, the recipient cell line can be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin gene and have mechanisms for immunoglobulin glycosylation. Myeloma cells can be grown in cultures or in the peritoneal cavity of mice, where the secreted immunoglobulins are obtained from ascites fluid. Other suitable recipient cells include lymphoid cells, such as B lymphocytes of feline or non-feline origin, hybridoma cells of feline or non-feline origin, or interspecies heterohybridoma cells.

[0279] Expression vectors carrying the antibody constructs or peptides of the present invention can be introduced into suitable host cells by any of a variety of suitable methods, including biochemical methods such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, as well as mechanical methods such as electroporation, direct microinjection, and microbolite bombardment. Johnston et al., 240 Science 1538 (1988).

[0280] Yeast offers a substantial advantage over bacteria in the production of immunoglobulin H and L chains. Yeast undergoes post-translational peptide modifications, including glycosylation. Numerous recombinant DNA strategies exist that utilize strong promoter sequences and high-copy-number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of cloned mammalian gene products and secretes peptides (i.e., propeptides) carrying the leader sequence. (Hitzman et al., 11th International Conference on Yeast, Genetics & Molecular Biology (Montpelier, France, 1982)).

[0281] The levels of peptide, antibody, fragment, and region production, secretion, and stability of yeast gene expression systems can be routinely assessed. Any system from a range of yeast gene expression systems can be utilized, incorporating promoter and terminator elements from genes encoding glycolytic enzymes that are produced in large quantities when yeast is grown in glucose-rich media. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, promoter and terminator signals from the phosphoglycerate kinase (PGK) gene can be utilized. Various methods can be employed to evaluate the optimal expression plasmid for expressing cloned immunoglobulin cDNAs in yeast. See Volume II, DNA Cloning, 45-66, (ed. Glover), IRL Press, Oxford, UK, 1985.

[0282] Bacterial strains can also be used as hosts for producing the antibody molecules or peptides described in this invention. Plasmid vectors containing replicons and control sequences derived from a species compatible with the host cell are used in conjunction with these bacterial hosts. The vectors carry replication sites and specific genes capable of providing phenotypic selection in transformed cells. Many methods can be used to evaluate the production of antibodies, fragments, and regions or antibody chains encoded by cDNA of cloned immunoglobulins in bacteria from expression plasmids (see Glover, 1985 ibid.; Ausubel, 1993 ibid.; Sambrook, 2001 ibid.; Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994–2001); Colligan et al., eds., Current Protocols in Protein Science (1997–2001)).

[0283] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications for immunoglobulin protein molecules, including leader peptide removal, folding and assembly of H and L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. In addition to the lymphoid-derived cells mentioned above, mammalian cells that can be used as hosts for antibody protein production include fibroblast-derived cells, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vector systems can be used to express cloned peptide H and L chain genes in mammalian cells (see Glover, 1985, ibid.). Different methods can be used to obtain complete H2L2 antibodies. H and L chains can be co-expressed in the same cells to achieve intracellular association and ligation of H and L chains into complete tetrameric H2L2 antibodies and / or peptides. Co-expression can be achieved by using the same or different plasmids in the same host. The genes for H and L chains and / or peptides can be placed in the same plasmid, which is then transfected into cells, thereby directly selecting cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one strand, such as the L chain, and then the resulting cell line can be transfected with an H chain plasmid containing a second optional marker. Cell lines that produce peptides and / or H2L2 molecules via either pathway can be transfected with plasmids encoding peptides, H, L, or H plus L chains, combined with additional optional markers, to produce cell lines with enhanced properties, such as higher yields of assembled H2L2 antibody molecules or enhanced stability of the transfected cell lines.

[0284] To achieve long-term, high-yield production of recombinant antibodies, stable expression can be used. For example, cell lines that stably express antibody molecules can be engineered. Host cells can be transformed with immunoglobulin expression cassettes and selective markers, instead of using expression vectors containing viral replication origins. After introducing foreign DNA, engineered cells can be grown in enriched media for 1–2 days, then switched to selective media. The optional markers in the recombinant plasmid confer resistance to the selected medium and allow cells to stably integrate the plasmid into the chromosome and grow to form lesions, which can then be cloned and expanded into cell lines. Such engineered cell lines can be particularly useful for screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0285] Once the antibodies of the present invention are generated, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and fractionation column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In many embodiments, the antibodies are secreted from cells into a culture medium and harvested from the culture medium.

[0286] Antibodies having one or more substitutions as described herein can be any suitable antibody known to those skilled in the art. In one example, the antibody is an anti-IL31 antibody. In another example, the antibody is an anti-NGF antibody.

[0287] Anti-IL31 antibodies without the substitutions described herein are well known in the art and are fully described in the following documents: for example, U.S. Patents 10,526,405, 10,421,807, 9,206,253, and 8,790,651. Furthermore, anti-NGF antibodies without the substitutions described herein are also well known in the art and are fully described in the following documents: for example, U.S. Patents 10,125,192, 10,093,725, 9,951,128, 9,617,334, and 9,505,829.

[0288] In one embodiment, the anti-IL31 antibody of the present invention (i.e., an antibody with substitution) reduces, inhibits, or neutralizes IL-31-mediated itching or allergic symptoms. In another embodiment, the anti-IL31 antibody of the present invention reduces, inhibits, or neutralizes IL-31 activity. The VL, VH, and CDR sequences of the anti-IL31 antibody are well known in the art and are fully described in the following documents: for example, U.S. Patents 11,530,262, 10,526,405, 10,421,807, 9,206,253, and 8,790,651. In one embodiment, the anti-IL31 antibody of the present invention may include a variable light chain comprising the amino acid sequence shown therein. In another embodiment, the anti-IL31 antibody of the present invention may include a variable heavy chain comprising the amino acid sequence shown therein.

[0289] In one embodiment, the mutant anti-NGF antibody of the present invention (i.e., an antibody with substitution) reduces, inhibits, or neutralizes NGF activity in animals, and / or enhances the ability to inhibit NGF binding to Trk A and p75, in order to treat NGF-mediated pain or symptoms. The VL, VH, and CDR sequences of the anti-NGF antibody are also well known in the art and are fully described in the following documents: 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 present invention may include at least one of the complementarity-determining region (CDR) sequences shown therein.

[0290] Drug and veterinary applications The present invention also provides a pharmaceutical composition comprising the molecules of the present invention and one or more pharmaceutically acceptable carriers. More specifically, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and an antibody or peptide according to the present invention as an active ingredient.

[0291] "Pharmaceutically acceptable carriers" include any excipient that is non-toxic to cells or animals exposed thereto at the doses and concentrations used. The pharmaceutical composition may include one or more therapeutic agents.

[0292] "Pharmaceutical acceptable" means compounds, materials, compositions, and / or dosage forms that, within a reasonable medical judgment, are suitable for contact with animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problematic complications, in proportion to a reasonable benefit / risk ratio.

[0293] Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, insecticides, chelating agents, antioxidants, isotonic agents, and absorption retardants.

[0294] Pharmaceutically acceptable carriers include: water; saline; phosphate-buffered saline; dextran; glycerol; alcohols such as ethanol and isopropanol; phosphoric acid, citric acid, 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 dextrin; EDTA; counterion-forming salts such as sodium; and / or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS; isotonic agents such as sugars; polyols such as mannitol and sorbitol; and sodium chloride; and combinations thereof.

[0295] The pharmaceutical compositions of the present invention can 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 infusionable solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills, or powders. In some embodiments, the composition is in the form of an injectable or infusionable solution. The composition can be in a form suitable for intravenous, intra-arterial, intramuscular, subcutaneous, parenteral, mucosal, oral, topical, or transdermal administration. The composition can be formulated as an immediate-release, controlled-release, sustained-release, or delayed-release composition.

[0296] The compositions of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents. They can be administered alone, but are typically administered with a drug carrier chosen based on the selected route of administration and standard pharmaceutical practice. The administration of the antibodies disclosed herein can be performed by any suitable manner, including parenteral injection (e.g., intraperitoneal, subcutaneous, or intramuscular), oral administration, or by topical application of the antibody (typically carried in a pharmaceutical formulation) to the airway surface. Topical application to the airway surface can be performed via intranasal administration (e.g., using a dropper, swab, or inhaler). Topical application of antibodies to the airway surface can also be performed by inhalation, such as by generating inhalable particles (including both solid clones and liquid particles) of the pharmaceutical formulation containing the antibody in the form of an aerosol suspension, and then having the subject inhale the inhalable particles. Methods and devices for administering inhalable particles of the pharmaceutical formulation are well known and can employ any conventional technique.

[0297] In some preferred embodiments, the antibody is administered via parenteral injection. For parenteral administration, the antibody or molecule can be formulated as a solution, suspension, emulsion, or lyophilized powder associated with a pharmaceutically acceptable parenteral carrier. For example, the carrier can be a solution of the antibody or a mixture thereof dissolved in an acceptable carrier, such as an aqueous carrier, like water, saline, Ringer's solution, dextran solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, etc. Liposomes and non-aqueous carriers, such as fixed oils, can also be used. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized using conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable excipients close to those required for physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibodies in these formulations can vary considerably, for example from less than about 0.5% by weight, typically or at least about 1% to as high as 15% or 20% by weight, and will be selected primarily based on fluid volume, viscosity, etc., depending on the specific administration method chosen. The mediator or lyophilized powder may contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and additives to enhance chemical stability (e.g., buffers and preservatives). The formulations are sterilized using common techniques. Practical methods for preparing compositions suitable for parenteral administration will be known or obvious to those skilled in the art and are described in more detail, for example, in *Remington's Pharmaceutical Sciences* (15th edition, Mack Pub. Co., Easton, Pa., 1980).

[0298] The antibodies or molecules of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective against conventional immunoglobulins. Any suitable lyophilization and reconstitution technique can be employed. Those skilled in the art will understand that lyophilization and reconstitution may result in varying degrees of loss of antibody activity, and the level of use may need to be adjusted to compensate for this. Compositions containing the antibodies of the present invention or mixtures thereof can be administered for the prevention and / or therapeutic treatment of relapse of existing diseases. Suitable drug carriers are described in the latest edition of Remington Pharmaceutical Sciences, which is the standard reference text in this art. In therapeutic applications, the composition is administered to a subject with the disease in an amount sufficient to cure or at least partially suppress or alleviate the disease and its complications.

[0299] The effective dose of the compositions of the present invention for treating the symptoms or diseases described herein varies depending on many different factors, including, but not limited to, the pharmacokinetic properties of the particular agent and its mode and route of administration; the target site; the physiological state of the animal; other drugs administered; whether the treatment is preventive or therapeutic; the age, health and weight of the recipient; the nature and severity of the symptoms; the types of concurrent treatments; the frequency of treatment and the desired effect.

[0300] The composition can be administered once or multiple times by the treating veterinarian selecting the dosage level and pattern. In any case, the pharmaceutical formulation should provide an amount sufficient to effectively treat the subject with the antibody of the present invention. In some embodiments, the composition is administered once every two months, once every three months, once every four months, once every five months, once every six months, or once every seven months.

[0301] Therapeutic doses can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy. The pharmaceutical compositions of the present invention may include a “therapeutic effective amount.” A “therapeutic effective amount” refers to an effective amount, expressed in doses, that is sustained for the required duration to achieve the desired therapeutic outcome. Therapeutic effective amounts of a molecule can vary depending on factors such as an individual’s disease state, age, sex, and weight, as well as the molecule’s ability to elicit the desired response in an individual. A therapeutic effective amount is also the amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the molecule.

[0302] On the other hand, the compositions of the present invention can be used, for example, to treat various diseases and conditions. As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, including preventive or preventive measures, wherein the aim is to prevent or mitigate (reduce) undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of the severity of the disease or condition, stabilization of the disease or condition (i.e., no worsening of the disease or condition), delay or slowing of the progression of the disease or condition, improvement or mitigation of the disease or condition, and remission of the disease or condition (whether partial or complete), whether detectable or undetectable. Those requiring treatment include those who already have the disease or condition, those who are susceptible to the disease or condition, or those who wish to prevent the disease or condition.

[0303] Compositions containing the mutant molecules of the present invention can be used to treat any suitable disease or condition. For example, the mutant anti-IL31 antibody of the present invention can be used to treat IL-31-mediated pruritus or allergic symptoms. Examples of IL-31-mediated pruritus symptoms include, but are not limited to, atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. Examples of IL-31-mediated allergic symptoms include, but are not limited to, allergic dermatitis, summer eczema, urticaria, vomiting, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and inflammatory processes caused by autoimmunity.

[0304] The mutant anti-NGF antibody of the present invention can be used to treat NGF-mediated pain or symptoms. Examples of pain include, but are not limited to, chronic pain, inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, osteoporotic fracture pain, postherpetic neuralgia, cancer pain, burn pain, wound-related pain, trauma-related pain, neuropathic pain, pain associated with musculoskeletal disorders, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism, periarticular disorders, or peripheral neuropathy. In a particular embodiment, the pain is osteoarthritis pain.

[0305] All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0306] The following examples are provided to illustrate, rather than limit, the claimed embodiments. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and those skilled in the art will recognize that various parameters may be changed without departing from the spirit of this disclosure or the scope of the appended claims.

[0307] Example Example 1—Detection of IgG Fc effector function in felines based on CDC cells A 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 within the subclasses. The assay can be used to identify key residues in the Fc region that determine the CDC activity of feline IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 and bind to CTLA4 on the Fc fusion protein. These target cells have been used in previous canine ADCC assays and CDC assays due to their reliability (Bergeron, McCandless et al. 2014).

[0308] Incubating target cells bound to the fusion protein with serum containing preserved feline complement induces the binding of Fc to the fusion protein and the complement component C1q, which initiates the complement cascade, ultimately forming a membrane attack complex. This pore-forming complex mediates cell lysis of the target cells, which is measured by loss of cell viability. If Fc does not bind to C1q, no eventual cell lysis / death occurs.

[0309] Methods. CD80-expressing CHO cells (target cells) were plated at 40,000 cells / well in CD CHO medium in 96-well round-bottom plates. The titrated fusion protein in the CD CHO medium was added to the target cells and allowed to bind at 37°C for 60 min. Serum containing feline complement (25% in CD CHO medium) was added to the plates at 37°C for 60 min. Cell viability was then measured using CellTiter-Glo, and the data are expressed as “cell viability, % relative to control” calculated using a serum control without fusion protein + containing complement.

[0310] Result. For example Figure 1 As shown in Table 1, the feline IgG1a and IgG1b Fc subclasses exhibited robust and potent CDC activity, with EC50 values ​​of 66.0 ng / mL and 68.9 ng / mL, respectively. The feline IgG2 Fc subclass showed no CDC effector functional activity. The feline IgG1a and IgG1b Fc subclasses exhibited CDC activity. The feline IgG2 Fc subclass showed no CDC activity.

[0311] Table 1. CDC effect induced by wild-type subtype of feline IgG Fc.

[0312] like Figure 2A and 2B As shown, the IgG1a Fc subclass in wild-type felines exhibits robust and effective CDC activity, but several Fc mutations studied all showed varying degrees of reduced CDC activity. Figure 2A In this study, both the feline IgG1a WT FcCTLA4 fusion protein and the IgG1a WT FcCTLA4 fusion protein exhibited robust concentration-dependent CDC activity. The Win mutation showed a slight rightward shift in the concentration-response curve. Figure 2B In the study, all seven listed Win plus mutations showed significant (60-75%) knockdown of CDC effector function.

[0313] like Figure 3As shown, wild-type feline IgG1a Fc subclass exhibits robust and potent CDC activity, while several of the tested Fc mutations all show attenuated CDC activity to varying degrees. Similarly, the Win mutation shows a slight right shift of the concentration-response curve. CDC effector function is sequentially knocked down as the mutations progress from DANG < DANG PSS < WinDANG < WinDANGPSS.

[0314] As Figure 4 shown, the wild-type IgG1a Fc subclass exhibits robust and potent CDC activity. However, several of the tested Fc mutations all show attenuated CDC activity to varying degrees. Similarly, the Win mutation shows a slight right shift of the concentration-response curve. Mutations GSP, GAP, DANG_GAP and WinDANG_GAP all knocked down CDC effector function by 60-70%.

[0315] As Figure 5 shown, the wild-type IgG1a Fc subclass exhibits robust and potent CDC activity. However, the tested Fc mutations show different effects on CDC effector function, ranging from no effect to complete knockout of CDC effector function. Both mutations KAPA and WinKAPA show significant knockdown of CDC effector function, with WinKAPA showing complete knockout of effector function. However, mutations D270G and D270S have little effect on CDC effector function.

[0316] Example 2 - ADCP Assay for Feline IgG Effector Function The antibody-dependent cell phagocytosis (ADCP) assay uses engineered CHO target cells that express canid CD80 and bind to canid CTLA4 on an Fc fusion protein. The Fc region of the fusion protein can then bridge the complex to Fcγ receptors on activated macrophage effector cells, which have the ability to phagocytose target cells. ADCP is measured by the fluorescence intensity and / or fluorescence area within the effector macrophage population in the co-culture of target cells stained with a pH-sensitive fluorescent dye, wherein fluorescent cells represent effector cells that have successfully internalized the target cells into acidic lysosomes.

[0317] Methods. Canine CD80-expressing CHO cells (CD80 target cells) or CD80-non-expressing wild-type CHO cells (parental target cells) were stained with pHrodo red dye for 30 min at 37 °C. The stained cells were then incubated with the feline CTLA4-Fc fusion protein for 60 min to mediate CTLA4:CD80 binding. 30,000 target cells were then added to 7,000 pre-coated Fcwf-4 effector cells (feline macrophage line). Co-cultures were maintained at 37 °C for 6–8 h while monitoring and analyzing the co-cultures using the SX5 Incucyte live-cell imaging system. Fluorescence area and / or intensity were quantified and expressed using predefined treatments defined in the Incucyte software.

[0318] Result. Figure 5 As shown, wild-type IgG1a exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed little or no effect on ADCP activity or a slight knockdown. GSP mutations in Fc showed a significant knockdown of ADCP activity.

[0319] like Figure 6 As shown, wild-type IgG1a exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed different effects on ADCP activity. The WinDANG_PSS mutation in Fc showed knockdown of ADCP activity. However, the WinPSS mutation in Fc showed enhanced ADCP activity.

[0320] like Figure 7 As shown, wild-type IgG1a exhibits concentration-dependent ADCP activity. Several Fc mutations studied showed little or no effect on ADCP activity compared to wild-type. The Win and WinDANG mutations had little or no effect on ADCP activity compared to wild-type IgG1a, but the WinDANG_GAP mutation in Fc showed ADCP activity knockdown.

[0321] like Figure 8 As shown, wild-type IgG1a exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed little or no effect on ADCP activity or significant knockdown. The KAPA mutation in Fc showed significant ADCP activity knockout. The D270G and D270S mutations showed no effect on ADCP activity.

[0322] like Figure 9As shown, wild-type IgG1a exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed moderate knockdown of ADCP activity. The DANG_GAP and WinKAPA mutations in Fc showed significant knockdown of ADCP activity.

[0323] Example 3—ADCC determination of IgG effector function in felines We developed and employed an ADCC-based assay to characterize the effectiveness of feline IgG1a subclass Fc rituximab chimeric IgG in mediating ADCC and to investigate Fc region mutations in subclass IgG1a. This will help define key residues in the Fc region that determine ADCC activity in feline IgG subclasses. The assay utilizes specially engineered HiBiT Ramos target cells (Promega) that express human CD20 that binds to our rituximab chimeric IgG. These target cells have been used in previous human ADCC assays (Promega).

[0324] Incubating rituximab-chimeric target cells with cultured activated PBMCs induces Fc binding to FcγRIII on IgG, mediating the release of granzyme and perforin from NK cells within the PBMC population. The action of these proteins leads to cytotoxicity and lysis of IgG-bound target cells. Upon killing target cells, the HiBiT fusion protein is released and binds extracellular LgBiT to produce a functional NanoBiT® luciferase (Promega). Luminescence is measured using the luciferase substrate and the GloMax® Discover system (Promega). If Fc does not bind to FcγRIII, final target cell death is not measured.

[0325] Methods. HiBiT Ramos cells (target cells) were seeded at 1,000 cells / well in EMEM medium in 384-well plates. The titrated fusion protein in EMEM medium was added to the target cells and allowed to bind at 37°C for 60 min. Single-donor canine PBMCs (effective cells) cultured overnight in RPMI 1640 medium + IL-2 and IL-15 were added to the plates at a 25:1 effector cell:target cell ratio (E:F ratio) for 5 h at 37°C. Cell death was then quantified using the Promega Nano-Glo HiBiT extracellular detection system. Data are expressed as “fold induction” relative to control normalization (minus effector PBMCs). Figure 13 This paper summarizes the mutations studied using ADCC assays.

[0326] Result. Figure 10As shown, wild-type feline IgG1a exhibits concentration-dependent ADCC activity. Compared to wild-type, the studied Fc mutations showed slightly knocked-down or enhanced ADCC activity. The Win mutation in Fc showed knocked-down ADCC activity. Both KAPA and DE mutations showed enhanced ADCC activity, especially the DE mutation.

[0327] like Figure 11 As shown, wild-type IgG1a exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed varying knockdown to knockout effects on ADCC activity. The DANG and DANG_GAP mutations in Fc showed ADCC activity knockdown. The WinDANG_GAP mutation in Fc showed ADCC activity knockout.

[0328] like Figure 12 As shown, wild-type IgG1a exhibits concentration-dependent ADCC activity. Compared to wild-type, the studied Fc mutations showed knockdown of ADCC activity. The WinKAPA, WinKA, and WinPA mutations in Fc showed knockdown of ADCC activity.

[0329] Example 4—Computer simulation modeling of the IgG Fc region in felines First, the Fc regions of the corresponding CH2 and CH3 regions were designed for the feline skeletal subclasses IgG1a and IgG1b. Protein modeling features of Alphafold 2.2, developed by DeepMind, were implemented to model the 3D structure of each feline skeletal subclass and each of the wild-type (WT) and mutant constructs of allogeneic variants.

[0330] Methods. The Molecular Operation Environment (MOE), developed by the Chemical Computation Group (MOE2019.0102), provides a flexible and automated graphical user interface for protein modeling. To analyze structural differences, the sequence-spectrum alignment algorithm uses a scoring algorithm to rank sequence templates, and a score above 85% ensures the selection of protein templates with physically realistic structures. The same pipeline is then used to optimize the model, and the structural stability of the model is validated using Ramachandran plots, which examine the stereochemical quality of the protein structures.

[0331] This method was applied to wild-type (WT) constructs and mutations at the following positions: M234, L235, G237, S239, D265, D270, N297, K322, P329, S330, P331, and I332. A stacking model was used, and the mutated residues were displayed in ball-and-stick form. Figure 14A). Generate RMSD plots to calculate the root mean square deviation of the two WT structures, feline-IgG1a_WT and feline-IgG1b_WT, relative to each other ( ). Figure 14B An RMSD value of 2.0 Å or lower was considered a standard for considering two structurally similar structures. The results showed that the feline IgG1a construct was fold identical to the feline IgG1b subclass, with the average RMSD value of that construct being 1.05 Å. Table 2A also notes the RMSD values ​​at twelve sites where mutation scans were performed, ranging from 0.2 to 0.9 Å.

[0332] Table 2A. Comparison of root mean square deviation (RMSD) of mutation locations in protein models of IgG1a and IgG1b in WT felines.

[0333] The above method was applied to wild-type (WT) constructs and mutations at the following positions: G236, G267, P268, S298, N324, E333, R334, and E345. A stacking model was used, and the mutated residues were displayed in ball-and-stick form. Figure 14C The root mean square deviations (RMSDs) of the feline-IgG1a_WT and feline-IgG1b_WT constructs relative to each other were calculated. Table 2B shows the RMSDs at twelve sites for mutation scanning, ranging from 0.1 to 0.7 Å. RMSD values ​​of 2.0 Å or lower were considered a standard for considering the similarity between the two structures. The results indicate that the feline IgG1a construct is fold identical to the feline IgG1b subclass, with the mean RMSD of the constructs being 1.05 Å.

[0334] Table 2B. Comparison of root mean square deviation (RMSD) of mutation locations in WT feline IgG1a and IgG1b protein models.

[0335] Results. Molecular modeling and validation of all mutations in the two IgG1 allotypes IgG1a and IgG1b of the feline skeleton were performed using MOE2019.0102. Fc folds and residue conformations between subclasses were shown as RMSDs of less than 2 Å, indicating very high structural identity of the proteins, and therefore mutations at positions in IgG1a would function similarly when extrapolated to IgG1b.

[0336] Example 5—Detection of IgG Fc effector function in canines based on CDC cells A 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 within the subclasses. The assay can be used to identify key residues in the Fc region that determine the CDC activity of the feline IgG subclasses. The assay utilizes CHO target cells engineered to express canine CD80 and bind to CTLA4 on the Fc fusion protein. These target cells have been used in previous canine ADCC assays and CDC assays due to their reliability (Bergeron, McCandless et al. 2014).

[0337] Methods. Incubating target cells bound to the fusion protein with serum containing preserved feline complement induced the binding of Fc to the fusion protein and the complement component C1q, which initiates the complement cascade, ultimately forming a membrane attack complex. The pore-forming complex mediates cell lysis of the target cells, which is measured by loss of cell viability. If Fc does not bind to C1q, no eventual cell lysis / death occurs.

[0338] CD80-expressing CHO cells (target cells) were seeded at 40,000 cells / well in CD CHO medium in 96-well round-bottom plates. The titrated fusion protein from the CD CHO medium was added to the target cells and allowed to bind at 37°C for 60 min. Serum containing complement (at a final concentration of 25% in CD CHO medium) was then added to the plates at 37°C for 60 min. Cell viability was then measured using CellTiter-Glo, and the data are expressed as “cell viability, % relative to control” calculated using a serum control without fusion protein + containing complement.

[0339] Result. Figure 15 As shown in Table 3, the canine IgG2 Fc subclass exhibited robust and effective CDC activity, with an EC50 value of 298.7 ng / mL. The canine IgG1 Fc subclass showed no CDC effector functional activity.

[0340] Table 3. CDC effects induced by wild-type subtypes of IgG1 and IgG2 Fc in canines.

[0341] like Figure 16As shown, the wild-type canine IgG2 Fc subclass exhibits robust and effective CDC activity. The studied Fc mutations showed no effect, reduced CDC activity, or complete knockout. The D270S mutation in Fc showed no effect on CDC activity. The Win mutation showed a slight rightward shift in the concentration-response curve. The D70G mutation showed knockdown of CDC activity. Six mutations—GSP, GAP, DANG_GAP, WIN_DANG_GAP, KAPA, and WIN_KAPA—showed complete knockout of CDC effector function.

[0342] Example 6—ADCP assay for IgG effector function in canines Antibody-dependent phagocytosis (ADCP) assays utilize CHO target cells engineered to express canine CD80 and bind to canine CTLA4 on an Fc fusion protein. The Fc region of the fusion protein then bridges the complex to an Fcγ receptor on activated macrophage effector cells capable of phagocytizing the target cells. ADCP is measured by the fluorescence intensity and / or fluorescent area of ​​target cells within a population of effector macrophages in a co-culture, stained with a pH-sensitive fluorescent dye, where fluorescent cells represent effector cells that have successfully internalized the target cells into acidic lysosomes.

[0343] Methods. CHO cells expressing canine CD80 (CD80 target cells) or wild-type CHO cells not expressing CD80 (parental target cells) were stained with pHrodo red dye for 30 min at 37 °C. The stained cells were then incubated with the canine CTLA4-Fc fusion protein for 60 min to mediate CTLA4:CD80 binding. 30,000 target cells were then added to 7,000 pre-coated DH-82 cells (canine macrophage line). The co-cultures were maintained at 37 °C for 6–8 h and monitored and analyzed using the SX5 Incucyte live-cell imaging system. Fluorescence area and / or intensity were quantified using predefined treatments defined in the Incucyte software and expressed accordingly.

[0344] Result. Figure 17 As shown, wild-type IgG2 exhibits concentration-dependent ADCP activity. Compared to wild-type, all studied Fc mutations showed ADCP activity knockout. The Win and GAP mutations in Fc showed ADCP activity knockout.

[0345] like Figure 18 As shown, wild-type IgG2 exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed a knockdown effect on ADCP activity. The DANG_GAP and WIN_DANG_GAP mutations in Fc showed ADCP activity knockdown.

[0346] like Figure 19 As shown, wild-type IgG2 exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed ADCP activity knockdown or elimination. The KAPA mutation in Fc showed ADCP activity knockdown. The GSP mutation showed ADCP activity elimination.

[0347] like Figure 20 As shown, wild-type IgG2 exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed moderate knockdown of ADCP activity. The DANG_GAP and WinKAPA mutations in Fc showed moderate to almost complete knockdown of ADCP activity, respectively.

[0348] like Figure 21 As shown, wild-type IgG2 exhibits concentration-dependent ADCP activity. Compared to wild-type, the studied Fc mutations showed either knockdown of ADCP activity or minimal effect on activity. The D270G mutation in Fc showed knockdown of ADCP activity. The D270S mutation showed no effect on ADCP activity.

[0349] Example 7—ADCC determination of IgG effector function in canines We developed and employed an ADCC-based assay to characterize the effectiveness of canine subclass IgG2a Fc rituximab chimeric IgG in mediating ADCC and to investigate Fc region mutations in this subclass. This will help define key residues in the Fc region that determine ADCC activity in feline IgG subclasses. The assay utilizes specially engineered HiBiT Ramos target cells (Promega) expressing human CD20 that binds to our rituximab chimeric IgG. These target cells have been used in previous human ADCC assays (Promega).

[0350] Incubating rituximab-chimeric target cells with cultured activated PBMCs induces Fc binding to FcγRIII on IgG, mediating the release of granzyme and perforin from NK cells within the PBMC population. The action of these proteins leads to cytotoxicity and lysis of IgG-bound target cells. Upon killing target cells, the HiBiT fusion protein is released and binds extracellular LgBiT to produce a functional NanoBiT® luciferase. Luminescence is measured using the luciferase substrate and the GloMax® Discover system. If Fc does not bind to FcγRIII, final target cell death will not be measured.

[0351] Methods. HiBiT Ramos cells (target cells) were seeded at 1,000 cells / well in EMEM medium in 384-well plates. The titrated fusion protein in EMEM medium was added to the target cells and allowed to bind at 37°C for 60 min. Single-donor canine PBMCs (effective cells) cultured overnight in RPMI 1640 medium + IL-2 and IL-15 were added to the plates at a 25:1 effector cell:target cell ratio (E:F ratio) for 5 h at 37°C. Cell death was then quantified using the Promega Nano-Glo HiBiT extracellular detection system. Data are expressed as “fold induction” relative to control normalization (minus effector PBMCs).

[0352] Result. Figure 22 As shown, wild-type canine IgG2 exhibits concentration-dependent ADCC activity. Compared to wild-type, the studied Fc mutation showed slightly reduced or knocked-out ADCC activity. The GAP mutation showed reduced ADCC activity. The Win, DANG, DANG_GAP, and WinDANG_GAP mutations showed knocked-out ADCC activity.

[0353] like Figure 23 As shown, wild-type IgG2 exhibits concentration-dependent ADCC activity. Compared to wild-type, the studied Fc mutations showed varying knockdown to knockout effects on ADCC activity. The Win mutation in Fc showed ADCC activity knockout. The KAPA, DE, DAE, and DLE mutations showed slightly enhanced ADCC activity.

[0354] like Figure 24 As shown, wild-type IgG2 exhibits concentration-dependent ADCC activity. All studied Fc mutations showed knockdown of ADCC activity compared to wild-type. The WinKAPA, WinKA, and WinPA mutations in Fc showed knockdown of ADCC activity, with WinKAPA showing the most effective knockout.

[0355] Example 8—Determination of Fcγ receptor and C1q binding in canines.

[0356] The binding of canine WT IgG2 (IgG 65) and mutant Fc was measured by surface plasmon resonance. IgG was used as a ligand and captured into a sensor chip for analysis of canine Fcγ receptor and C1q binding. 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20 at pH 7.4 were used as titration and method run buffers. Canine Fcγ receptor and human C1q were used as analytes and flowed onto the captured Fc mutant IgG. Mutations were performed at several locations listed in Table 4.

[0357] Table 4 lists the mutation sites in canine IgG2 that have been determined to affect the binding of canine Fcγ receptor and C1q, using EU index numbers such as those in Kabat.

[0358] Table 5 lists the mutations in canine IgG2 that have been determined to affect the binding of canine Fcγ receptor and C1q, using EU index numbers such as those in Kabat.

[0359] Kinetics and affinity were determined using Cytiva 8K evaluation software. Blank runs containing only buffer were subtracted from all runs. Flow cytometry cells were generated again using 10 mM glycine at pH 1.5. Runs were performed at room temperature. Mutations generated at the corresponding canine mutant Fc IgG sites (Table 5) significantly affected the kinetics and / or affinity of IgG for canine Fcγ receptors and / or human C1q when compared to the corresponding WT Fc kinetic and / or affinity values ​​(Table 6).

[0360] Table 6—Binding kinetics and affinities of canine Fcγ receptors and / or human C1q to WT IgG2 with specified substitutions in Fc and mutant canine IgG2. (LS = low signal. NBO = no binding observed.) Example 9—Detection of IgG Fc effector function in equines based on CDC cells A CDC-based assay was developed and employed to characterize the effectiveness of nine CTLA4-mediated CDC in equine IgG subclasses of Fc fusion proteins and to investigate Fc region mutations within these subclasses. This will help define key residues in the Fc region that determine the CDC activity of equine IgG subclasses. The assay utilized CHO target cells engineered to express canine CD80 and bind to CTLA4 on the Fc fusion proteins. These target cells have been used in previous canine ADCC assays and CDC assays due to their reliability.

[0361] Incubating target cells bound to the fusion protein with serum containing preserved complement induces the binding of Fc to the fusion protein and the complement component C1q, which initiates the complement cascade, ultimately forming a membrane attack complex. This pore-forming complex mediates cell lysis of the target cells, which is measured by loss of cell viability. If Fc does not bind to C1q, no eventual cell lysis / death occurs.

[0362] Methods. CD80-expressing CHO cells (target cells) were plated at 40,000 cells / well in CD CHO medium in 96-well round-bottom plates. The titrated fusion protein in CD CHO medium was added to the target cells and allowed to bind at 37°C for 60 min. Serum containing equine complement (at a final concentration of 20% in CD CHO medium) was added to the plates at 37°C for 45 min. Cell viability was then measured using CellTiter-Glo, and data are expressed as “cell viability, % relative to control” calculated using a serum control without fusion protein + containing complement.

[0363] Result. Figure 26 As shown in Table 7, four of the six wild-type Fc subclasses of equines exhibited robust and potent CDC activity. IgG2 and IgG5 were exceptions, both showing no CDC activity. EC50 values ​​ranged from 0.19 µg / mL to 0.29 µg / mL.

[0364] Table 7. CDC effect induced by wild-type IgG Fc subtype fusion protein in equines.

[0365] like Figure 27 As shown, the wild-type IgG1 Fc subclass exhibited robust and potent CDC activity. The investigated Fc mutations showed varying effects on CDC activity. Compared to WT, the PAP mutation in Fc showed no effect on CDC activity. The PSS mutation showed a slight enhancement of CDC activity. The Win and WinPSS mutations showed knockdown of CDC activity. The SQP, SAP, and WinSAP mutations most effectively knocked out CDC activity.

[0366] like Figure 28 As shown, the wild-type IgG4 Fc subclass exhibits robust and potent CDC activity. The investigated Fc mutations showed varying effects on CDC activity. Compared to WT, the Win, PSS, and WinPSS mutations in Fc showed slightly lower CDC activity. The SQP, SAP, and WinSAP mutations knocked out CDC activity.

[0367] like Figure 29 As shown, the wild-type IgG7 Fc subclass exhibited robust and potent CDC activity. The investigated Fc mutations showed varying effects on CDC activity. Compared to WT, the Win mutation in Fc showed no effect on CDC activity. The PSS mutation showed moderate knockdown of CDC activity. The SQP, SAP, WinSAP, and WinPSS mutations knocked out CDC activity.

[0368] Example 10—ADCP Determination of IgG Effector Function in Equine Antibody-dependent phagocytosis (ADCP) assays utilize CHO target cells engineered to express canine CD80 and bind to canine CTLA4 on an Fc fusion protein. The Fc region of the fusion protein then bridges the complex to an Fcγ receptor on activated macrophage effector cells capable of phagocytizing the target cells. ADCP is measured by the fluorescence intensity and / or fluorescent area of ​​a pH-sensitive fluorescent dye within a population of effector macrophages in a co-culture, where fluorescent cells represent effectors that have successfully internalized fluorescently labeled target cells into acidic lysosomes.

[0369] Methods. Canine CHO cells expressing CD80 (CD80 target cells) or wild-type CHO cells not expressing CD80 (parental target cells) were stained with pHrodo red dye for 30 min at 37 °C. The stained cells were then incubated with the equine CTLA4-Fc fusion protein for 60 min to mediate CTLA4:CD80 binding. 40,000 target cells were then added to 8,000 pre-coated equine activated macrophages isolated and differentiated from PMBCs. The co-cultures were maintained at 37 °C for 6–8 h while monitoring and analyzing the co-cultures using the SX5 Incucyte live-cell imaging system. Fluorescence area and / or intensity were quantified and expressed using predefined treatments defined in the Incucyte software.

[0370] Result. For example Figure 30 As shown, the wild-type equine IgG1 Fc subclass exhibits robust and potent ADCP activity. The studied Fc mutations showed varying degrees of attenuation of ADCP activity. The Win and SAP mutations in Fc showed knockdown of ADCP activity.

[0371] like Figure 31 As shown, the wild-type IgG4 Fc subclass exhibits robust and potent ADCP activity. The studied Fc mutations showed varying degrees of attenuation of ADCP activity. The Win and SAP mutations in Fc showed knockdown of ADCP activity.

[0372] like Figure 32 As shown, the wild-type IgG4 Fc subclass exhibits robust and potent ADCP activity. The studied Fc mutations did not affect or knock out ADCP activity. The WinSAP mutation in Fc showed ADCP activity knockout.

[0373] like Figure 33 As shown, the wild-type IgG7 Fc subclass exhibits robust and potent ADCP activity. The studied Fc mutations showed varying degrees of attenuation of ADCP activity. The Win and SAP mutations in Fc showed knockdown of ADCP activity.

[0374] like Figure 34 As shown, the wild-type IgG7 Fc subclass exhibits robust and potent ADCP activity. The studied Fc mutations showed reduced ADCP activity. WinSAP and SQP mutations in Fc showed knockdown of ADCP activity.

[0375] Example 11—ADCC determination of IgG effector function in equines.

[0376] An ADCC-based assay was developed and employed to characterize the effectiveness of nine CTLA4-mediated equine IgG subclass Fc fusion proteins in mediating ADCC, and to investigate Fc region mutations within these subclasses. This will help define key residues in the Fc region that determine ADCC activity in equine IgG subclasses. The assay utilized CHO target cells engineered to express canine CD80 and bind to CTLA4 on the Fc fusion proteins. These target cells have been used in previous canine ADCC assays and in CDC assays due to their reliability.

[0377] Methods. CD80-expressing CHO cells (target cells) were plated at 20,000 cells / well in CD CHO medium in 96-well round-bottom plates. The titrated fusion protein in the CD CHO medium was added to the target cells and allowed to bind at 37°C for 60 min. Single-donor equine PBMCs (effective cells) cultured overnight in RPMI 1640 medium + IL-2 and IL-15 were added to the plates at a 40–50:1 effector cell:target cell ratio (E:F ratio) for 18–20 h at 37°C. Cells were then fixed and stained, and analyzed by flow cytometry to quantify the live / dead staining of target cells. Data are expressed as “% of dead target cells” relative to the fusion protein alone (minus the effector PBMCs).

[0378] Result. For example Figure 35 As shown in Table 8, four of the six wild-type Fc subclasses of equines exhibited robust and potent ADCC activity. IgG2 and IgG5 were exceptions, showing either no ADCC activity or significantly reduced ADCC activity, respectively. EC50 values ​​ranged from 16.1 ng / mL to 108.6 ng / mL. The equine IgG1, 3, 4, and 7 Fc CTLA4 fusion proteins all showed varying degrees of ADCC activity. IgG2 and 5 showed no ADCC activity.

[0379] Table 8. ADCC effect induced by CTLA4 fusion protein of wild-type IgG Fc subtype in equines.

[0380] like Figure 36 and 37 As shown, the wild-type IgG1 subclass exhibits robust and efficient ADCC activity. The studied Fc mutations showed different effects on ADCC activity. The Win, PSS, WinPSS, and SQP mutations in Fc knocked down ADCC effector activity. Figure 36 SAP and WinSAP mutation knockout ADCC effector functional activity ( Figure 37Compared to wild-type IgG1 Fc, the PAP mutation appears to have a small effect on the functional activity of this ADDC effector. Figure 37 ).

[0381] like Figure 38 As shown, the wild-type IgG4 subclass exhibits robust and efficient ADCC activity. The investigated Fc mutations showed varying effects on ADCC activity. Compared to wild-type IgG4 Fc, the Win, PSS, WinPSS, and SQP mutations in Fc showed no effect on ADCC effector activity. The SAP mutation knocked down ADCC effector activity, and the WinSAP mutation knocked out ADCC effector activity.

[0382] like Figure 39 As shown, the wild-type IgG7 subclass exhibits robust and efficient ADCC activity. The investigated Fc mutations showed varying effects on ADCC activity. Compared to wild-type IgG7 Fc, the Win, SAP, and PSS mutations in Fc knocked down ADCC effector activity. WinSAP and Win PSS showed significantly reduced ADCC activity. The SQP mutation most effectively knocked out ADCC effector activity.

[0383] Example 12—Computer simulation modeling of the IgG Fc region in equines First, the Fc regions of four equine allotypes IgG1, IgG4a, IgG4b, IgG7a, and IgG7b were designed using the corresponding CH2 and CH3 regions. Protein modeling features of Alphafold 2.2, developed by DeepMind, were implemented to model the 3D structure of the equine FcRn and each of the wild-type (WT) and mutant constructs of each equine allotype.

[0384] The Molecular Operation Environment (MOE), developed by the Chemical Computation Group (MOE2019.0102), provides a flexible and automated graphical user interface for protein modeling. To analyze structural differences, the sequence-spectrum alignment algorithm uses a scoring algorithm to rank sequence templates, and a score above 85% ensures the selection of protein templates with physically realistic structures. The model is then optimized using the same pipeline, and its structural stability is verified using Laplace plots, which examine the stereochemical quality of the protein structures.

[0385] The above method was used on wild-type (WT) equine constructs, and the following residues were mutated in IgG1: L234, L235, G237, P329, Q330, and P331. The location of the mutant library is... Figure 40The results are presented in ball-and-stick format. 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, A330, and P331. The locations of the mutant libraries are... Figure 41 and Figure 42 It is presented in the form of a baseball bat.

[0386] RMSD plots were generated to calculate the root mean square deviation of WT structure, equine-IgG4a, equine-IgG4b, equine-IgG7a, and equine-IgG7b relative to each other. Figure 43 An RMSD value of 2.0 Å or lower was considered a standard for considering the similarity between the two structures. The results showed that the equine IgG4a construct was allotypically identical to the equine IgG4b construct, with a mean RMSD value of 0.44 Å for the structures described (RMSD at a single location in Table 9). The equine IgG7a construct was allotypically identical to the equine IgG7b construct, with a mean RMSD value of 0.9 Å (RMSD at a single location in Table 10).

[0387] Table 9. Comparison of root mean square deviation (RMSD) of residues in protein models of IgG4a and IgG4b in WT equines.

[0388] Table 10. Comparison of root mean square deviation (RMSD) of residues in protein models of IgG7a and IgG7b in WT equines.

[0389] Example 13—CDC cell-based assay of IgG1a, IgG1b, IgG2 and IgG3 subclasses in felines A CDC-based assay was developed and employed to characterize the effectiveness of feline IgG subclasses IgG1a, IgG1b, IgG2, and IgG3 Fc mAbs in mediating CDC, and to investigate Fc region mutations within these subclasses. This will contribute to the identification of residues in the Fc region that determine the CDC activity of feline IgG subclasses. The assay utilizes CLBL-1 target cells expressing canine CD20 that binds to the anti-canine CD20 test mAb. These target cells have been used in previous canine CDC assays and have proven reliable.

[0390] Incubating mAb-bound target cells with serum containing preserved complement induces the binding of Fc to the mAb and C1q, a complement component that initiates the complement cascade, ultimately forming a membrane attack complex. This pore-forming complex mediates cell lysis of the target cells, which is measured by loss of cell viability. If Fc does not bind to C1q, no eventual cell lysis / death occurs.

[0391] In summary, CLBL-1 cells expressing canine CD20 (target cells) were plated at 40,000 cells / well in complete RPMI 1640 medium in 96-well round-bottom plates. The titrated fusion protein in complete RPMI 1640 medium was added to the target cells and allowed to bind at 37°C for 60 min. Serum containing feline complement (25% in complete RPMI 1640 medium) was added to the plates at 37°C for 60 min. Cell viability was then measured using CellTiter-Glo, and the data are expressed as “cell viability, % relative to control” calculated using a serum control without fusion protein + containing complement.

[0392] Result. Figure 44 As shown, the IgG1a Fc subclass in wild-type felines exhibits robust and potent CDC activity. The Fc mutations studied showed well-complete knockout of CDC activity. All Fc mutations tested in this figure showed CDC knockout, with the WinKAPA and WinPA mutations showing maximal knockout.

[0393] like Figure 45 As shown, the wild-type IgG1b Fc subclass exhibited robust and potent CDC activity. All Fc mutations studied showed little effect on CDC activity. Four mutations showed slight knockdown of CDC activity, with M234A, L235A, and G237A mutations showing the largest knockdown. S239D and I332E mutations showed slightly enhanced CDC activity. Feline IgG1b WT exhibited robust concentration-dependent CDC activity. All tested Fc mutations showed very little effect on CDC activity. Four mutations showed slight knockdown of CDC activity, with M234A, L235A, and G237A mutations showing the largest knockdown. S239D and I332E mutations showed slightly enhanced CDC activity.

[0394] like Figure 46As shown, the wild-type feline IgG1b Fc subclass exhibited robust and potent CDC activity. All Fc mutations studied showed varying degrees of CDC activity knockout. Feline IgG1b WT demonstrated robust concentration-dependent CDC activity. Eight tested Fc mutations showed CDC activity knockout. All five mutations tested showed CDC activity knockdown, with the M234A, L235A, G237A, K332A, and P331S mutations showing the most significant CDC activity knockdown.

[0395] like Figure 47 As shown, the wild-type (WT) subclass of IgG2 Fc in wild-type felines and the Fc mutations in all eight studies showed no CDC activity.

[0396] like Figure 48 As shown, the IgG3 Fc subclass in wild-type felines exhibits robust and effective CDC activity. All five Fc mutations studied showed little effect on CDC activity.

[0397] like Figure 49 As shown, the four other feline IgG3 Fc subclass mutations studied had little effect on CDC activity, and one showed moderate knockdown of CDC activity. The D265A_N297G mutation showed moderate knockdown of CDC activity.

[0398] Conclusion. Among the Fc mutations identified above, the Fc mutations with the strongest knockout effect on feline IgG1a subclass-mediated feline CDC are WinKAPA and WinKA, which produce a larger knockout compared to WinPA. The optimal Fc mutation for knockout of feline IgG1b subclass-mediated feline CDC is M234A_L235A_G237A_K332A_P331S. The optimal Fc mutation for knockout of IgG3 subclass-mediated feline CDC is D265A_N297G.

[0399] Example 14—ADCP assay for IgG effector function in felines As described in Example 2, antibody-dependent phagocytosis (ADCP) was measured in feline IgG1a variants.

[0400] Result. Figure 50 As shown, wild-type feline IgG1a exhibits concentration-dependent ADCP activity. Compared to wild-type, the Fc mutants WinKAPA, WinKA, and WinPA showed moderate to strong knockdown of ADCP activity, with the WinPA mutation showing the greatest knockdown.

[0401] Example 15—Detection of IgG Fc effector function in canines based on CDC cells A CDC-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 these subclasses. This will help define key residues in the Fc region that determine the CDC activity of canine IgG subclasses. The assay utilizes CLBL-1 target cells expressing canine CD20 that binds to the anti-canine CD20 test mAb. These target cells have been used in previous canine CDC assays and have proven reliable.

[0402] Incubating mAb-bound target cells with serum containing preserved canine complement induces the binding of Fc to the mAb and C1q, a complement component that initiates the complement cascade, ultimately forming a membrane attack complex. This pore-forming complex mediates cell lysis of the target cells, which is measured by loss of cell viability. If Fc does not bind to C1q, no eventual cell lysis / death occurs.

[0403] Methods. Briefly, CLBL-1 cells expressing canine CD20 (target cells) were seeded at 40,000 cells / well in complete RPMI 1640 medium in 96-well round-bottom plates. Titration mAbs in complete RPMI 1640 medium were added to the target cells and allowed to bind at 37°C for 60 min. Serum containing canine complement (25% in complete RPMI 1640 medium) was added to the plates at 37°C for 60 min. Cell viability was then measured using CellTiter-Glo, and data are expressed as “cell viability, % relative to control” calculated using a serum control without fusion protein + containing complement.

[0404] Result. Figure 51 As shown, the wild-type canine IgG2 Fc subclass exhibited robust and potent CDC activity. The Win, KAPA, WinKAPA, WinKA, and WinPA mutations in Fc showed CDC activity knockout. This differs slightly from previous canine CDC assays using CD-80-expressing CHO target cells, CTLA4 fusion protein assay products, and serum containing preserved feline complement. In those assays, the Win mutation only showed a slight rightward shift in the response curve and only partially knocked down CDC activity, whereas here, the Win mutation showed almost complete CDC activity knockout.

[0405] like Figure 52 As shown, the D270S mutation in canine IgG2 does not knock out CDC activity, but the D270G mutation does. GSP, GAP, DANG, Win DANG GAP, and DANG GAP mutations all show CDC activity knockout. Figure 53As shown, of the mutations tested in this group, two showed enhanced CDC activity, four showed little effect on CDC activity, and one (DLE) showed CDC activity knockout. The Q345R mutation showed a maximum enhancement of approximately half a logarithmic increase in CDC potency. Figure 54 As shown, the mutations tested in this group showed varying degrees of CDC activity knockdown, with the triple mutant D276K_T299E_N324P showing the largest knockdown in the group.

[0406] like Figure 55 As shown, mutations in IgG2 tested in this group showed no effect to slight knockdown of CDC activity, including triple mutants. This can be compared with... Figure 56 In contrast, all mutations tested in this group showed CDC activity knockout, whether they were single mutation sites around residues 263, 264, 265, 266, or 267, or combinations of mutation sites added to V263W. Figure 57 As shown, all mutations tested in this group exhibited CDC knockout activity, whether they were single mutation sites around aa 299 or 328, or combinations of mutation sites added to T299E. Figure 58 As shown, all mutations tested in this group exhibited CDC knockout activity, whether at a single mutation site or a combination of mutation sites. Figure 59 As shown, at residue N297, all mutations tested in this group showed CDC activity knockdown, with N297A, N297D, and N297K mutations showing the greatest knockdown. Figure 60 As shown, among the mutations tested in this group, the Q295G mutation showed little effect on CDC activity. All other mutations tested showed enhanced CDC activity, with the P268W mutation showing the largest enhancement of approximately one log unit in potency so far.

[0407] like Figure 61 As shown, among the IgG2 mutations tested in this group, most of the tested combined mutations involving V262, V264, and P268 sites showed enhanced CDC activity. The combined mutations V262L_Q295N_N324P_Q345R and V264F_F269A_N324P_Q345R showed a slightly lower CDC activity. So far, the P268W_Q345R double mutation has shown the largest enhancement of approximately one log unit.

[0408] like Figure 62 As shown, mutations in IgG3 tested in this group exhibited no effect on CDC activity or significantly enhanced activity. Figure 63As shown, most of the mutations tested in this group had no effect on CDC activity. The P268W mutation showed a significant enhancement of CDC activity, approaching a one-log-unit increase in potency.

[0409] These assays show that the optimal Fc mutations in canine CDC mediated by the IgG2 subclass are: Win, WinKAPA, WinKA, Win PA, D270G, GSP, GAP, DLE, and mutations around positions 238, 263, 265, 266, 267, 294, 299, and 328.

[0410] The optimal knockout Fc mutations in 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 optimal enhancement Fc mutations in canine CDC are: P268W and P268W_Q345R of the IgG2 subclass, and P268W of the IgG3 subclass.

[0411] Example 16—ADCP assay for IgG effector function in canines Antibody-dependent phagocytosis (ADCP) assays utilize CHO target cells engineered to express canine CD80 or CLBL-1 target cells expressing canine CD20. These cells are stained with a pH-sensitive fluorescent dye that can image the cells as they are phagocytosed. Our test products are a CD80-binding canine CTLA4 IgG2 Fc fusion protein or an anti-canine CD20 chimeric monoclonal IgG2 antibody (mAb). The Fc region of the fusion protein or mAb then bridges the target cell / test product complex to Fcγ receptors on DH82 effector cells (a canine macrophage line) capable of phagocytizing stained target cells. ADCP is measured by the fluorescence intensity and / or fluorescent area of ​​the pH-sensitive fluorescent dye within a population of effector DH82 cells in a co-culture, where fluorescent cells represent effector cells that have successfully internalized the fluorescently labeled target cells into acidic lysosomes.

[0412] Methods. Briefly, target cells were stained with pHrodo red dye at 37°C for 30 min and then incubated with our test product for 60 min to mediate test product:target cell binding. The complex was then added at 30,000 cells / well to DH-82 cells pre-coated in 7,000 cells / well plates. The plates were maintained at 37°C for 24 h and monitored and analyzed using the SX5 Incucyte live-cell imaging system. Fluorescence area and / or intensity were quantified using predefined treatment definitions in the Incucyte software and expressed accordingly.

[0413] like Figure 64 As shown, wild-type canine IgG2 exhibits concentration-dependent ADCP activity. Compared to IgG2 WT, the Fc mutations studied in this group showed either knockdown of ADCP activity or minimal effect on activity. The Win, WinKAPA, WinKA, and WinPA mutations in Fc showed well-inhibited ADCP activity. The KAPA mutation showed no effect on ADCP activity. Figure 65 As shown, among the mutations tested in this group, the N297P, N297A, and N297D mutations in Fc exhibited ADCP knockdown. Figure 66 As shown, among the mutations tested in this group, the N297F and N297K mutations in Fc showed ADCP activity knockout. The N297G mutation showed ADCP activity knockdown. Figure 67 As shown, among the mutations tested in this group, the T299I, T299E, and T299Q mutations in Fc showed ADCP activity knockdown, with T299I showing the lowest knockdown.

[0414] like Figure 68 As shown, among the canine IgG2 mutations tested in this group, the T299R mutation in Fc showed complete ADCP activity knockout. The N324P mutation robustly enhanced ADCP activity. The L328K mutation showed no effect on ADCP activity. Figure 69 As shown, among the mutations tested in this group, the P238L and V264F mutations in Fc showed knockdown of ADCP activity. The L328P mutation showed no effect on ADCP activity. Figure 70 As shown, among the mutations tested in this group, the L266P, E269P, and E294H mutations in Fc showed ADCP knockdown. Figure 71 As shown, among the mutations tested in this group, the Q295G and Q295N mutations in Fc showed knockdown of ADCP activity. The E294N mutation showed no effect on ADCP activity.

[0415] like Figure 72As shown, among the canine IgG2 mutations tested in this group, the F296A and F296S mutations in the Fc region exhibited ADCP activity knockout. The Q295W mutation showed no effect on ADCP activity. Figure 73 As shown, among the mutations tested in this group, the G298M mutation in Fc showed a slight knockdown of ADCP activity. The M234D, G236E, and S239L mutations showed ADCP activity knockout. The L266G, G298L, and Y300Q mutations showed no effect on ADCP activity. Figure 74 As shown, among the mutations tested in this group, the M234E and V262L mutations in Fc showed ADCP activity knockdown. The M234E and L325D mutations showed ADCP activity knockout. Figure 75 As shown, among the mutations tested in this group, the D267K, T299E mutation in Fc showed almost complete knockout of ADCP activity. The D267K, N324P mutation and the T299E, N324P mutation showed complete knockout of ADCP activity.

[0416] like Figure 76 As shown, among the canine IgG2 mutations tested in this group, the V240S, V262L, and E294M mutations in the Fc region showed ADCP activity knockdown. The P268W mutation showed no effect on ADCP activity. The V240Q, P268W, and G298I mutations showed ADCP activity knockout. Figure 77 As shown, among the mutations tested in this group, the D2267K, T299E, N324P, and V263W, T299Q mutations in Fc showed ADCP activity knockdown, with the latter showing a better knockdown effect. The V263W, N297A mutation showed no effect on ADCP activity. Figure 78 As shown, among the mutations tested in this group, the V263H mutation in Fc showed a slight knockdown of ADCP activity. Other mutations showed no effect on ADCP activity. Figure 79 As shown, among the mutations tested in this group, the D265F mutation in Fc showed knockdown of ADCP activity. Compared to WT, the V263W and V264R mutations showed almost no effect on ADCP activity.

[0417] like Figure 80 As shown, among the canine IgG2 mutations tested in this group, the D265V, L266K, and D267K mutations in the Fc region exhibited moderate ADCP knockdown. Figure 81As shown, among the mutations tested in this group, the Win mutation and the M234D, I332E mutation in Fc showed well-inhibited ADCP activity. The M234D, L328F mutation in Fc showed moderately-inhibited ADCP activity. Figure 82 As shown, among the mutations tested in this group, the S239L,I332E mutation, L328F,I332E mutation, and M234D,S239L,I332E mutation in Fc showed moderate to well-inhibited ADCP activity. Figure 83 As shown, among the mutations tested in this group, the M234D, L328F, I332E and M234D, S239L, L328F, I332E mutations in Fc showed ADCP activity knockdown. The S239L, L328F, I332E mutation showed no effect on ADCP activity. Figure 84 As shown, among the mutations tested in this group, the L325D, I332E mutation showed moderate knockdown of ADCP activity. The L325D, S239L and L235D, S239L, I332E mutations in Fc showed very low knockdown to near-knockout of ADCP activity. Figure 85 As shown, among the mutations tested in this group, the L235D, L328F, I332E, L235D, S239L, L328F, I332E, and G236W, S239L mutations in Fc showed slightly to moderately low ADCP activity.

[0418] like Figure 86 As shown, among the canine IgG2 mutations tested in this group, the G236W,I332E, G236W,S239L,I332E, and G236W,L328F,I332E mutations in Fc showed moderate ADCP knockdown. Figure 87 As shown, among the mutations tested in this group, the G236W, S239L, L328F, I332E mutations, M234D, S239L mutations, and G236E, S239L mutations in Fc showed slightly to moderate knockdown of ADCP activity. Figure 88 As shown, among the mutations tested in this group, the L235D, G236W and G236W, L328F mutations in Fc showed moderate ADCP activity knockdown. L235D, G236W, and L328F showed slight ADCP activity knockdown. Figure 89 As shown, among the mutations tested in this group, the L266G, G298L and L266G, S330K mutations in Fc showed little effect on ADCP activity. The G298L, Y300Q mutation showed a moderate knockdown of ADCP activity. Figure 90As shown, among the mutations tested in this group, the G298L,S330K and L266G,G298L,S330K mutations in Fc showed ADCP activity knockdown. The Y300Q,S330K mutation showed no effect on ADCP activity.

[0419] like Figure 91 As shown, among the canine IgG2 mutations tested in this group, the L266G, Y300Q, S330K and L266G, G298L, Y300Q, S330K mutations in the Fc region showed ADCP activity knockdown. The G298L, Y300Q, S330K mutation showed almost no effect on ADCP activity. Figure 92 As shown, among the mutations tested in this group, the L266V,G298K, G298K,S330L, L266V,S330L and L266V,G298K,S330L mutations in Fc showed ADCP activity knockdown, with the L266V,G298K,S330L mutation showing the largest knockdown.

[0420] like Figure 91 As shown, wild-type IgG3 exhibited concentration-dependent ADCP activity. Among the canine IgG3 mutations tested in this group, the N324P mutation in Fc showed no effect on ADCP activity. The L234A, L235A, G237A, L234A, L235A, G237A, K322A, P331A, L234A, L235A, G237A, K322A, P331A, L234A, L235A, G237A, P331A, and T299R mutations in Fc all showed ADCP knockout.

[0421] These assays show that the optimal Fc mutations for knocking out canine ADCP mediated by the IgG2 subclass are: Win, WinKAPA, WinKA, Win PA, N297F, N297K, T299E, T299Q, T299R, FF296A, F296S, M234D_G236E_S239L, M234E_L325D, D267K_N324P, T299E_N324P, and V240Q_P268W_G298I. The optimal Fc mutations for knocking out canine ADCP mediated by the IgG3 subclass are: L234A_L235A_G237A, L234A_L235A_G237A_K322A_P331A, L234A_L235A_G237A_K322A_P331A, L234A_L235A_G237A_P331A, and T299R. For the IgG2 subclass, the optimal Fc mutation for enhancing canine ADCP is N324P.

[0422] Example 17—ADCC Determination of IgG Effector Function in Canines An assay based on canine FcγRIII binding (ADCC) cells was developed and employed to characterize the effectiveness of canine subclass IgG2 Fc anti-canine CD20 chimeric IgG in mediating ADCC, and to investigate Fc region mutations in said subclass. This will help define key residues in the Fc region that determine ADCC activity in canine IgG2 subclasses. Figure 94 The protocol for the ADCC assay is presented. The assay utilizes specially engineered Jurkat "effective cells" (Promega), in which canine FcRγIII is expressed extracellularly and an NFAT response element linked to a luciferase reporter gene is expressed intracellularly. The "target cells" are CLBL-1 cells expressing canine CD20. Our test product is a chimeric construct of canine IgG2 wild-type (WT) Fc and a mutant canine IgG2 WT Fc, designed to combat canine CD20.

[0423] Upon binding to the Fc region of the anti-canine CD20 canine IgG2 assay product bound to CLBL-1 "target cells," ADCC bioassay effector cells expressing canine FcγRIII transduce intracellular signals, resulting in NFAT-mediated luciferase activity, which can be easily quantified using the BioGlo™ luciferase assay kit (Promega) and a luminescence meter reader. The more the Fc of our assay product binds to FcγRIII on the "effector cells," the higher the luminescence. If the Fc does not bind to FcγRIII, no final luminescence will be measured.

[0424] Methods. Briefly, CLBL-1 cells (target cells) were seeded at approximately 6,000 cells / well in RPMI 1640 medium in 96-well plates. Canine mAb titration assays in RPMI 1640 medium were added to the target cells and allowed to bind at 37°C for 60 min. Engineered “effect cells” were counted and added to the plates at a 10:1 effector cell:target cell ratio (E:F ratio) for 6 h. FcγRIII binding was then quantified using luminescent detection with Promega’s Bio-Glo luciferase assay reagent. Data are expressed as a “fold increase” relative to control normalization (minus effector cells).

[0425] Result. Figure 95As shown, wild-type canine IgG2 exhibits concentration-dependent and robust conversion to canine FcγRIII binding for ADCC activity. All studied Fc mutations showed complete knockout of canine FcγRIII binding, indicating conversion to ADCC activity. Canine IgG2 Fc exhibits ADCC activity through strong canine FcRγIII binding. All mutations tested in this figure showed complete knockout of ADCC activity.

[0426] like Figure 96 As shown, the Fc mutations studied in this group exhibited complete knockout, knockdown, or enhancement of FcγRIII binding, indicating conversion to ADCC activity in canines. The WinKAPA, WinKA, and WinPA mutations all showed complete knockout of FcγRIII binding. The KAPA and DAE mutations showed knockdown of FcγRIII binding. The DLE and DE mutations showed enhanced FcγRIII binding.

[0427] like Figure 97 As shown, the Fc mutations studied in this group exhibited complete knockout, knockdown, no effect, or slight enhancement of FcγRIII binding, indicating conversion to ADCC activity in canines. The D270G, D270S, and EFT mutations in Fc showed knockdown of FcγRIII binding. The YWA mutation showed complete knockout of FcγRIII binding. Compared to WT, the Q345R mutation showed no effect on FcγRIII binding, and the AAA mutation showed enhanced FcγRIII binding.

[0428] like Figure 98 As shown, the Fc mutations studied in this group exhibited either complete knockout or enhanced FcγRIII binding, indicating conversion to ADCC activity in canines. The Win and L266K mutations in Fc completely knocked out FcγRIII binding, while the V240S_V262L_E294M triplet mutation showed enhanced FcγRIII binding.

[0429] Conclusions. These assays revealed that the optimal Fc mutations for knocking out FcγRIII binding in canine IgG2 subclass-mediated ADCC are: Win, WinKAPA, WinKA, Win PA, GSP, GAP, YWA, and L266K. According to the report here, the optimal Fc mutations for enhancing canine FcγRIII binding in canine IgG2 subclass-mediated ADCC are: DLE, DE, AAA, and V240S_V262L_E294M.

Claims

1. An Fc region of feline IgG1a, using EU index numbering as in Kabat, said Fc region 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.

2. The Fc region of feline IgG1a according to claim 1, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitutions selected from the following: 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.

3. The Fc region of feline IgG1a according to claim 1, using EU indexing as in Kabat, wherein the Fc region comprises one or more of the following substitution combinations: (M234A, L235A, G237A), (P329G), (P331S), (P329G, S330A), (D265A, N297G), (K322A, P331A), (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, L235 A, G237A, P329S, S330A, P331S), (S298A, E333A, R334A), (S239D, S330L, I332E), DE (S239D, I332E), (G236A, S239D, I332E), (L235Y, G237W, S298A), (G267E, P268F, N324T), (D270G), (D270S) and (E345R).

4. An Fc region of feline IgG1b, said Fc region comprising one or more substitutions or combinations of substitutions at positions corresponding to those in feline IgG1a according to any one of claims 1 to 3.

5. An Fc region of feline IgG3, said Fc region comprising one or more substitutions or combinations of substitutions at positions corresponding to those in feline IgG1a according to any one of claims 1 to 3.

6. An Fc region of feline IgG3, said Fc region comprising substitutions at positions D265A and N297G.

7. An Fc region of canine IgG2, using EU index numbering as in Kabat, said Fc region containing one or more mutations selected from the following locations: 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.

8. The Fc region of canine IgG2 according to claim 7, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitutions selected from: A231C, 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, P 232Y, E233A, E233C, E233D, E233F, E233G, E233H, E233I, E233K, E233L, E23 3M, E233N, E233P, E233Q, E233R, E233S, E233T, E233V, E233W, E233Y, M234A, M234C, M234D, M234E, M234F, M234G, M234H, M234I, M234K, M234L, M234N, M2 34P, M234Q, M234R, M234S, M234T, M234W, M234Y, L235A, L235C, L235D, L235E , L235F, L235G, L235H, L235I, L235K, L235M, L235N, L235P, L235Q, L235R, L 235S, L235T, L235V, L235W, L235Y, G236A, G236C, G236D, G236E, G236F, G23 6H, G236I, G236K, G236L, G236M, G236N, G236P, G236Q, G236R, G236S, G236T , G236V, G236W, G236Y, G237A, G237C, G237D, G237E, G237F, G237H, G237I, G2 37K, G237L, G237M, G237N, G237P, G237Q, G237R, G237S, G237T, G237V, G237 W, G237Y, P238A, P238C, P238D, P238E, P238F, P238G, P238H, P238I, P238K, P 238L, P238M, P238N, P238Q, P238R, P238S, P238T, P238V, P238W, P238Y, S23 9A, S239C, S239D, S239E, S239F, S239G, S239H, S239I, S239K, S239L, S239M,S239N、S239P、S239Q、S239R、S239T、S239V、S239W、S239Y、V240A、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、V263A、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、D265A、D265C、D265E、D265F、D265G、D265H、D265I、D265K、D265L、D265M、D265N、D265P、D265Q、D265R、D265S、D265T、D265V、D265W、D265Y、L266A、L266C、L266D、L266E、L266F、L266G、L266H、L266I、L266K、L266M、L266N、L266P、L266Q、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、E269A、E269C、E269D、E269F、E269G、E269H、E269I、E269K、E269L、E269M、E269N、E269P、E269Q、E269R、E269S、E269T、E269V、E269W、E269Y、D270A、D270C、D2 70F、D270G、D270H、D270I、D270K、D270L、D270M、D270N、D270P、D270Q、D270 R、D270S、D270V、D270W、D270Y、P271C、P271F、P271G、P271H、P271I、P271K、 P271L、P271M、P271N、P271Q、P271R、P271S、P271T、P271Y、E294A、E294C、E29 4D、E294F、E294G、E294H、E294I、E294K、E294L、E294M、E294N、E294P、E294Q 、E294R、E294S、E294T、E294V、E294W、E294Y、Q295A、Q295D、Q295E、Q295F、Q 295G、Q295K、Q295L、Q295N、Q295P、Q295R、Q295S、Q295T、Q295V、Q295W、Q29 5Y、F296A、F296C、F296D、F296E、F296G、F296H、F296I、F296K、F296L、F296M、 F296N, F296P, F296Q, F296R, F296S, F296T, F296V, N297A, N297C, N297D, N297E, N297F, N297G, N297H, N297I, N297K, N297L, N297M, N297P, N297Q, N297 R、N297S、N297T、N297V、N297W、N297Y、G298A、G298C、G298D、G298E、G298F、 G298H、G298I、G298K、G298L、G298M、G298N、G298P、G298Q、G298R、G298S、G29 8T, G298V, G298W, G298Y, T299A, T299C, T299D, T299E, T299F, T299G, T299H, T299I, T299K, T299L, T299M, T299N, T299P, T299Q, T299R, T299S, T299V, T 299W、T299Y、Y300A、Y300C、Y300D、Y300E、Y300H、Y300I、Y300K、Y300L、Y30 0M、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, V323 L, V323M, V323N, V323P, V323Q, V323R, V323S, V323T, V323W, V323Y, N324A, N324C, N324D, N324E, N324F, N324G, N324H, N324I, N324K, N324L, N324M, N32 4P, N324Q, N324R, N324S, N324T, N324V, N324W, N324Y, N325A, N325C, N325D, N325E, N325F, N325G, N325H, N325I, N325K, N325L, N325M, N325P, N325Q, N 325R, N325S, N325T, N325V, N325W, N325Y, K326A, K326D, K326E, K326F, K326I, K326L, K326N, K326R, K326S, K326T, K326V, K326W, A327C, A327D, A327E A327F, A327G, A327H, A327I, A327K, A327L, A327M, A327N, A327P, A327Q, A327R, A327S, A327T, A327V, A327W, A327Y, L328A, L328C, L328D, L328E, L328 F、L328G、L328H、L328I、L328K、L328M、L328N、L328P、L328Q、L328R、L328S、 L328T、L328V、L328W、L328Y、K322A、K322C、K322D、K322E、K322F、K322G、K32 2H, K322I, K322L, K322M, K322N, K322P, K322Q, K322R, K322S, K322T, K322V, K322W, K322Y, P329A, P329C, P329D, P329E, P329F, P329G, P329H, P329I, P 329K, P329L, P329M, P329N, P329Q, P329R, P329S, P329T, P329V, P329W, P329Y, S330A, S330C, S330D, S330E, S330F, S330G, S330H, S330I, S330K, S330LS330M, 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, A339V, A339W, A339Y, R340G, T422K, T422R, F423P and L441P.

9. The Fc region of canine IgG2 according to claim 7, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitutions or combinations thereof selected from: (P329G), (P329G, S330A), (D265A, N297G), (D265A, N297G; P329G, S330A), (M234A, L235A, G237A), (M234A, L235A, G237A), (M234A, L235A, G237A). 7A; 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).

10. An Fc region of canine IgG3, using EU index numbering as in Kabat, said Fc region comprising one or more substitutions or combinations of substitutions at positions corresponding to those in feline IgG2 according to any one of claims 7 to 9.

11. An Fc region of canine IgG3, using EU index numbering as in Kabat, said Fc region comprising one or more substitutions or combinations of substitutions selected from the following positions: 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.

12. An Fc region of equine IgG1, using EU index numbering as in Kabat, said Fc region containing mutations or substitutions at one or more of the following locations: 229, 234, 235, 237, 329, 330, and 331.

13. The Fc region of equine IgG1 according to claim 12, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitutions selected from the following: P229S, L234A, L235A, G237A, P329S, Q330A, Q330S and P331S.

14. The Fc region of equine IgG1 according to claim 12, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitution combinations selected from the following: (P329S, Q330A), (Q330A), (P229S), (L234A, L235A, G237A), (Q330S, P331S), (L234A, L235A, G237A; P329S, Q330A) and (L234A, L235A, G237A, Q330S, P331S).

15. An Fc region of equine IgG4, using EU index numbering as in Kabat, said Fc region containing one or more mutations and / or substitutions selected from the following locations: 234, 235, 237, 239, 330, and 331.

16. The Fc region of equine IgG4 according to claim 15, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitutions selected from the following: L234A, Q235A, G237A, P239S, A330Q, A330S, P331S.

17. The Fc region of equine IgG4 according to claim 15, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitutions or combinations thereof selected from the Fc region of equine IgG4: (A330S, P331S), (P239S, A330Q), (L234A, Q235A, G237A), (L234A, Q235A, G237A; A330S, P331S), (P329S), and (L234A, Q235A, G237A; P329S).

18. An Fc region of equine IgG7, using EU index numbering as in Kabat, said Fc region containing one or more mutations and / or substitutions selected from the following locations: 234, 235, 236, 237, 239, 330, and 331.

19. The Fc region of equine IgG7 according to claim 18, using EU index numbering as in Kabat, wherein the Fc region comprises one or more substitutions selected from the following: L234A, S235A, V236G, G237A, P239S, P329S, A330S, A330Q, P331S.

20. The Fc region of equine IgG7 according to claim 18, wherein the Fc region comprises a substitution or combination of substitutions selected from the following in the Fc region of equine IgG7: (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 IgG Fc region according to any one of claims 1 to 20.

22. A recombinant antibody or molecule comprising a feline, canine, or equine IgG Fc region according to any one of claims 1 to 20.

23. A method for producing or manufacturing an antibody or molecule, the method comprising: providing a vector or host cell having a nucleic acid sequence encoding an antibody or molecule according to claim 22.

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