Bispecific Binding Agents for Use in Companion Animals

Abstract

Provided are bispecific binding agents (e.g., bispecific antibodies or Fc constructs) for use in companion animals (e.g., dogs and cats), pharmaceutical compositions comprising such bispecific binding agents, and methods of their use.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on January 9, 2024, is named 51682-008WO2_Sequence_Listing_1_9_24.XML and is 328,720 bytes in size.

[0002] The present disclosure relates generally to bispecific binding agents (e.g., bispecific antibodies or Fc constructs) for use in companion animals (e.g., dogs and cats), pharmaceutical compositions comprising such bispecific binding agents, and methods of their use. [Background technology]

[0003] Bispecific binding agents, such as bispecific antibodies, that can bind to two or more antigens have a wide range of therapeutic applications. Bispecific antibodies can contain two binding domains that can target two different antigens or two different epitopes on the same antigen. Various bispecific antibody formats have been developed for human use, such as tetravalent bispecific antibodies formed by fusing an IgG antibody with a single-chain domain, and other bispecific antibody formats in which the antibody core structure (e.g., IgA, IgD, IgE, IgG, or IgM) is no longer retained. For example, diabodies, triabodies, tetrabodies, and minibodies have been developed, as well as several single-chain formats (scFv, Bis-scFv) that can bind to two or more antigens. Such formats may use linkers to either fuse the antibody core (e.g., IgA, IgD, IgE, IgG, or IgM) to the binding protein (e.g., scFv) or, for example, to fuse two Fab fragments or scFvs. Generating bispecific antibodies can be challenging. For example, sophisticated purification procedures may be required to generate bispecific antibodies due to the presence of mismatched by-products.

[0004] While several bispecific antibodies have been approved or are in clinical development for the treatment of humans, no bispecific antibodies are currently approved for companion animals (e.g., dogs and cats). In addition, there is limited guidance in the art regarding how to successfully generate bispecific antibodies based on companion animal antibodies. In particular, increasing the overall yield, homogeneity, and stability of bispecific antibodies remains a challenge.

[0005] Thus, there is a need in the art for bispecific binding agents that can be used to treat companion animals. Summary of the Invention

[0006] Provided herein, inter alia, are bispecific binding agents (e.g., bispecific antibodies or Fc constructs) for use in companion animals (e.g., dogs and cats), nucleic acids encoding such bispecific binding agents, vectors, host cells, methods of making, pharmaceutical compositions comprising such bispecific binding agents, and methods of their use.

[0007] In a first aspect, the present invention provides a bispecific antibody comprising: (a) a first binding domain that binds to a first antigen, the first binding domain being linked to a first companion animal Fc region variant; (b) a second binding domain that binds to a second antigen, the second binding domain being linked to a second companion animal Fc region variant; wherein at least one of the first binding domain and the second binding domain comprises a single domain antibody.

[0008] In some embodiments, the first binding domain and the second binding domain each specifically bind to an antigen independently selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-31R, IL-33, CD3, CD20, CD47, CD52, and complexes of the complement system.

[0009] In some embodiments, the first binding domain and / or the second binding domain comprises an antibody, an antibody fragment, or a ligand-binding portion of a receptor, hi some embodiments, the antibody fragment is selected from the group consisting of Fab, single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, and a diabody.

[0010] In some embodiments, the single domain antibody is linked to the first companion animal Fc region variant or the second companion animal Fc region variant directly or via a peptide linker. In some embodiments, the single domain antibody is a VHH domain. In one embodiment, the VHH domain comprises a C-terminal residue, the first companion animal Fc region variant or the second companion animal Fc region variant comprises an N-terminal residue, and the C-terminal residue of the VHH domain is linked to the N-terminal residue of the first companion animal Fc region variant or the second companion animal Fc region variant directly or via a peptide linker. In another embodiment, the VHH domain comprises an N-terminal residue, the first companion animal Fc region variant or the second companion animal Fc region variant comprises a C-terminal residue, and the N-terminal residue of the VHH domain is linked to the C-terminal residue of the first companion animal Fc region variant or the second companion animal Fc region variant directly or via a peptide linker.

[0011] In some embodiments, the peptide linker is (a) GPGGQ (SEQ ID NO: 38), (b) PKRENGRVPRPPDCPKCP (SEQ ID NO: 363); (c) VPKRENGRVPRPPDCPKCP (SEQ ID NO: 364), (d) FNECRCTDTPPCPVPEP (SEQ ID NO: 22); (e) PKRENGRVPRPPDCPKCPAPEM (SEQ ID NO: 23), (f) AKECECKCNCNNCPCPGCGL (SEQ ID NO: 24), (g) PKESTCKCISPCPVPES (SEQ ID NO: 25), (h) PKESTCKCIPPCPVPES (SEQ ID NO: 26), (i) KTDHPPGPKPCDCPKCP (SEQ ID NO: 27), and (j) KTASTIESKTGEGPKCP (SEQ ID NO: 29) The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0012] In some embodiments, the first companion animal Fc region variant and the second companion animal Fc region variant are canine Fc region variants. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 11. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO:12.

[0013] In some embodiments, the first canid Fc region variant and the second canid Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first canid Fc region variant and the second canid Fc region variant.

[0014] In some embodiments, the first canid Fc region variant and the second canid Fc region variant each comprise a protrusion or a recess; when the first canid Fc region comprises a protrusion, the second canid Fc region comprises a recess, and when the first canid Fc region comprises a recess, the second canid Fc region comprises a protrusion.

[0015] In some embodiments, the first canid Fc region variant and the second canid Fc region variant are (a) S354C and T366W in a first canine Fc region variant and Y349C, T366S, L368A, and Y407V in a second canine Fc region variant; (b) T366W in a first canine Fc region variant and T366S, L368A, and Y407V in a second canine Fc region variant; (c) R392D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (d) S364H and F405A in a first canine Fc region variant and Y349T and T394F in a second canine Fc region variant; (e) F405L in a first canine Fc region variant and K409R in a second canine Fc region variant; (f) T366L, R392L, and T394W in a first canine Fc region variant and L351Y, F405A, and Y407V in a second canine Fc region variant; (g) K360E and K409W in a first canine Fc region variant and S347R, D399V, and F405T in a second canine Fc region variant; (h) Y349C, K360E, and K409W in a first canine Fc region variant and S347R, S354C, D399V, and F405T in a second canine Fc region variant; (i) K370E and K409W in a first canine Fc region variant and E357N, D399V, and F405T in a second canine Fc region variant; (j) K360D, D399M, and Y407A in a first canine Fc region variant and Q345R, S347R, T366V, and K409V in a second canine Fc region variant; (k) Y349S, T366M, K370Y, and K409V in a first canine Fc region variant and E356G, E357D, S364Q, and Y407A in a second canine Fc region variant; (l) L351D and L368E in a first canine Fc region variant and L351K and T366K in a second canine Fc region variant; (m) L368D and K370S in a first canine Fc region variant and E356Q and S364K in a second canine Fc region variant; or (n) T366Y in a first canine Fc region variant and T366S, L368A, and Y407T in a second canine Fc region variant. wherein the amino acid positions are based on EU numbering.

[0016] In some embodiments, the first canid Fc region variant comprises a first charged region and the second canid Fc region variant comprises a second charged region, with which the first charged region forms a charge pair, hi some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0017] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S183D in the first canine Fc region variant and S183K in the second canine Fc region variant. It further comprises a CH1 domain comprising:

[0018] In some embodiments, the first canid Fc region variant and the second canid Fc region variant are (a) K409D in a first canine Fc region variant and D399K in a second canine Fc region variant; (b) K390D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (c) K390D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant; and (d) K370D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0019] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S176K in the first canine Fc region variant and S176D in the second canine Fc region variant. It further comprises a CL domain comprising:

[0020] In some embodiments, the first canid Fc region variant and / or the second canid Fc region variant comprises the following amino acid substitutions: (a) 252Y and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (b) 252M and, optionally, at least one amino acid substitution selected from the group consisting of 251D or 251E; 256D or 256F; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (c)434R, (d) 426Y and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (e) 426H and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (f) 426F and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 426H; and (g) 434R and, optionally, at least one amino acid substitution selected from the group consisting of 286L; 286Y; 312P; and 436H. wherein the amino acid positions are based on EU numbering.

[0021] In other embodiments, the first companion animal Fc region variant and the second companion animal Fc region variant are feline Fc region variants. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 19. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 20. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 21.

[0022] In some embodiments, the first feline Fc region variant and the second feline Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first feline Fc region variant and the second feline Fc region variant.

[0023] In some embodiments, the first feline Fc region variant and the second feline Fc region variant each comprise a protrusion or a recess, and if the first feline Fc region comprises a protrusion, the second feline Fc region comprises a recess, and if the first feline Fc region comprises a recess, the second feline Fc region comprises a protrusion.

[0024] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) T366W in a first feline Fc region variant and T366S, L368A, and Y407V in a second feline Fc region variant; (b) T366W in a first feline Fc region variant and T366S, L368A, and Y398T in a second feline Fc region variant; (c) A354C and T366W in a first feline Fc region variant and Y349C, T366S, L368A, and Y407V in a second feline Fc region variant; (d) R392D and K409D in a first feline Fc region variant and E356K and D399K in a second feline Fc region variant; (e) S364H and F405A in a first feline Fc region variant and Y349T and T394F in a second feline Fc region variant; (f) F405L in a first feline Fc region variant and K409R in a second feline Fc region variant; (g) T366L, R392L, and T394W in a first feline Fc region variant and L351Y, F405A, and Y407V in a second feline Fc region variant; (h) R360E and K409W in a first feline Fc region variant and Q347R, D399V, and F405T in a second feline Fc region variant; (i) Y349C, R360E, and K409W in a first feline Fc region variant and Q347R, A354C, D399V, F405T in a second feline Fc region variant; (j) K370E and K409W in a first feline Fc region variant and E357N, D399V, and F405T in a second feline Fc region variant; (k) R360D, D399M, and Y407A in a first feline Fc region variant and E345R, Q347R, T366V, and K409V in a second feline Fc region variant; (l) Y349S, K370Y, T366M, and K409V in a first feline Fc region variant and E356G, E357D, S364Q, and Y407A in a second feline Fc region variant; (m) L351D and L368E in a first feline Fc region variant and L351K and T366K in a second feline Fc region variant; (n) L368D and K370S in a first feline Fc region variant and E356Q and S364K in a second feline Fc region variant; and (o) T366Y in the first feline Fc region variant and T366S, L368A, and Y407T in the second feline Fc region variant. wherein the amino acid positions are based on EU numbering.

[0025] In some embodiments, the first feline Fc region variant comprises a first charged region and the second feline Fc region variant comprises a second charged region, and the first charged region forms a charge pair with the second charged region. In some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0026] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S183D in the first feline Fc region variant and S183K in the second feline Fc region variant. It further comprises a CH1 domain comprising:

[0027] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) K409D in a first feline Fc region variant and D399K in a second feline Fc region variant; and (b) K370D and K409D in a first feline Fc region variant and E357K and D399K in a second feline Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0028] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S176K in the first feline Fc region variant and S176D in the second feline Fc region variant. It further comprises a CL domain comprising:

[0029] In some embodiments, the first feline Fc region variant and / or the second feline Fc region variant comprises the following amino acid substitutions: (a) at least one amino acid substitution selected from the group consisting of 286E, 311V, and 428Y; (b) two or more amino acid substitutions selected from the group consisting of 286E, 311V, and 428Y, and (c) 286E, 311V, and 428Y wherein the amino acid positions are based on EU numbering.

[0030] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) a first polypeptide comprising a first companion animal Fc region variant; (b) a second polypeptide comprising a second companion animal Fc region variant; and wherein a first companion animal Fc region variant and a second companion animal Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first companion animal Fc region variant and the second companion animal Fc region variant, and wherein the first polypeptide or the second polypeptide does not comprise an antibody.

[0031] In some embodiments, the Fc construct further comprises a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1, VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, and a thrombopoietin binding peptide.

[0032] In some embodiments, the first companion animal Fc region variant and the second companion animal Fc region variant are canine Fc region variants, and in some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12.

[0033] In some embodiments, the first canid Fc region variant and the second canid Fc region variant each comprise a protrusion or a recess; when the first canid Fc region comprises a protrusion, the second canid Fc region comprises a recess, and when the first canid Fc region comprises a recess, the second canid Fc region comprises a protrusion.

[0034] In some embodiments, the first canid Fc region variant and the second canid Fc region variant each comprise: (a) S354C and T366W in a first canine Fc region variant and T366S, L368A, Y407V, and Y349C in a second canine Fc region variant; (b) T366W in a first canine Fc region variant and T366S, L368A, and Y407V in a second canine Fc region variant; (c) R392D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (d) S364H and F405A in a first canine Fc region variant and Y349T and T394F in a second canine Fc region variant; (e) F405L in a first canine Fc region variant and K409R in a second canine Fc region variant; (f) T366L, R392L, and T394W in a first canine Fc region variant and L351Y, F405A, and Y407V in a second canine Fc region variant; (g) K360E and K409W in a first canine Fc region variant and S347R, D399V, and F405T in a second canine Fc region variant; (h) Y349C, K360E, and K409W in a first canine Fc region variant and S347R, S354C, D399V, and F405T in a second canine Fc region variant; (i) K370E and K409W in a first canine Fc region variant and E357N, D399V, and F405T in a second canine Fc region variant; (j) K360D, D399M, and Y407A in a first canine Fc region variant and Q345R, S347R, T366V, and K409V in a second canine Fc region variant; (k) Y349S, T366M, K370Y, and K409V in a first canine Fc region variant and E356G, E357D, S364Q, and Y407A in a second canine Fc region variant; (l) L351D and L368E in a first canine Fc region variant and L351K and T366K in a second canine Fc region variant; (m) L368D and K370S in a first canine Fc region variant and E356Q and S364K in a second canine Fc region variant; or (n) T366Y in a first canine Fc region variant and T366S, L368A, and Y407T in a second canine Fc region variant. wherein the amino acid positions are based on EU numbering.

[0035] In some embodiments, the first canid Fc region variant comprises a first charged region and the second canid Fc region variant comprises a second charged region, with which the first charged region forms a charge pair, hi some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0036] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S183D in the first canine Fc region variant and S183K in the second canine Fc region variant. It further comprises a CH1 domain comprising:

[0037] In some embodiments, the first canid Fc region variant and the second canid Fc region variant are (a) K409D in a first canine Fc region variant and D399K in a second canine Fc region variant; (b) K390D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (c) K390D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant; and (d) K370D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0038] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S176K in the first canine Fc region variant and S176D in the second canine Fc region variant. It further comprises a CL domain comprising:

[0039] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: (a) 252Y and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (b) 252M and, optionally, at least one amino acid substitution selected from the group consisting of 251D or 251E; 256D or 256F; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (c)434R, (d) 426Y and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (e) 426H and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (f) 426F and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 426H; and (g) 434R and, optionally, at least one amino acid substitution selected from the group consisting of 286L; 286Y; 312P; and 436H. wherein the amino acid positions are based on EU numbering.

[0040] In other embodiments, the first companion animal Fc region variant and the second companion animal Fc region variant are feline Fc region variants. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21.

[0041] In some embodiments, the first feline Fc region variant and the second feline Fc region variant each comprise a protrusion or a recess, and if the first feline Fc region comprises a protrusion, the second feline Fc region comprises a recess, and if the first feline Fc region comprises a recess, the second feline Fc region comprises a protrusion.

[0042] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) T366W in a first feline Fc region variant and T366S, L368A, and Y407V in a second feline Fc region variant; (b) T366W in a first feline Fc region variant and T366S, L368A, and Y398T in a second feline Fc region variant; (c) A354C and T366W in a first feline Fc region variant and Y349C, T366S, L368A, and Y407V in a second feline Fc region variant; (d) R392D and K409D in a first feline Fc region variant and E356K and D399K in a second feline Fc region variant; (e) S364H and F405A in a first feline Fc region variant and Y349T and T394F in a second feline Fc region variant; (f) F405L in a first feline Fc region variant and K409R in a second feline Fc region variant; (g) T366L, R392L, and T394W in a first feline Fc region variant and L351Y, F405A, and Y407V in a second feline Fc region variant; (h) R360E and K409W in a first feline Fc region variant and Q347R, D399V, and F405T in a second feline Fc region variant; (i) Y349C, R360E, and K409W in a first feline Fc region variant and Q347R, A354C, D399V, F405T in a second feline Fc region variant; (j) K370E and K409W in a first feline Fc region variant and E357N, D399V, and F405T in a second feline Fc region variant; (k) R360D, D399M, and Y407A in a first feline Fc region variant and E345R, Q347R, T366V, and K409V in a second feline Fc region variant; (l) Y349S, K370Y, T366M, and K409V in a first feline Fc region variant and E356G, E357D, S364Q, and Y407A in a second feline Fc region variant; (m) L351D and L368E in a first feline Fc region variant and L351K and T366K in a second feline Fc region variant; (n) L368D and K370S in a first feline Fc region variant and E356Q and S364K in a second feline Fc region variant; and (o) T366Y in the first feline Fc region variant and T366S, L368A, and Y407T in the second feline Fc region variant. wherein the amino acid positions are based on EU numbering.

[0043] In some embodiments, the first feline Fc region variant comprises a first charged region and the second feline Fc region variant comprises a second charged region, and the first charged region forms a charge pair with the second charged region. In some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0044] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S183D in the first feline Fc region variant and S183K in the second feline Fc region variant. It further comprises a CH1 domain comprising:

[0045] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) K409D in a first feline Fc region variant and D399K in a second feline Fc region variant; and (b) K370D and K409D in a first feline Fc region variant and E357K and D399K in a second feline Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0046] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S176K in the first feline Fc region variant and S176D in the second feline Fc region variant. It further comprises a CL domain comprising:

[0047] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: (a) at least one amino acid substitution selected from the group consisting of 286E, 311V, and 428Y; (d) two or more amino acid substitutions selected from the group consisting of 286E, 311V, and 428Y; and (c) 286E, 311V, and 428Y wherein the amino acid positions are based on EU numbering.

[0048] In another aspect, the invention features a pharmaceutical composition including (i) any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein, and (ii) a pharmaceutically acceptable carrier.

[0049] In another aspect, the invention features one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein.

[0050] In another aspect, the invention features one or more expression vectors including one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein.

[0051] In another aspect, the invention features a host cell containing one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein, or one or more expression vectors containing one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein.

[0052] In another aspect, the present invention provides a method of making a bispecific antibody or Fc construct, comprising the steps of: (a) providing one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein; (b) expressing any one of the one or more nucleic acids disclosed herein in a host cell culture, thereby producing a bispecific antibody or an Fc construct; and optionally (c) recovering the bispecific antibody or Fc construct produced in (ii) from the host cell culture; The present invention is characterized by a method comprising:

[0053] In some embodiments, the host cell culture comprises (i) one host cell population expressing both a first companion animal Fc region variant and a second companion animal Fc region variant, or (ii) two host cell populations comprising a first population expressing a first companion animal Fc region variant and a second population expressing a second companion animal Fc region variant.

[0054] In another aspect, the invention features a method of treating or preventing a companion animal disease or disorder in a companion animal in need thereof, comprising administering an effective amount of a composition comprising any one of the bispecific antibodies disclosed herein, any one of the Fc constructs comprising a canine Fc region variant disclosed herein, or a pharmaceutical composition comprising same.

[0055] In another aspect, the invention features a method of treating or preventing a canine disease or disorder in a dog in need thereof, comprising administering an effective amount of a composition comprising any one of the bispecific antibodies comprising a canine Fc region variant disclosed herein, any one of the Fc constructs comprising a canine Fc region variant disclosed herein, or a pharmaceutical composition comprising same.

[0056] In some embodiments, the canine disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer. In some embodiments, the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0057] In another aspect, the invention features any one of the bispecific antibodies comprising a canine Fc region variant disclosed herein, any one of the Fc constructs comprising a canine Fc region variant disclosed herein, or a pharmaceutical composition comprising same, for use in treating or preventing a canine disease or disorder in a dog in need thereof.

[0058] In some embodiments, the canine disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer. In some embodiments, the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0059] In another aspect, the invention features a method of treating or preventing a feline disease or disorder in a cat in need thereof, comprising administering an effective amount of a composition comprising any one of the bispecific antibodies comprising a feline Fc region variant disclosed herein, any one of the Fc constructs comprising a feline Fc region variant disclosed herein, or a pharmaceutical composition comprising same.

[0060] In some embodiments, the feline disease or disorder is an allergic disease, chronic pain, acute pain, an inflammatory disease, an autoimmune disease, an endocrine disease, a gastrointestinal disease, a cardiovascular disease, a renal disease, a reproductive-related disorder, an infectious disease, or cancer. In some embodiments, the feline disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0061] In another aspect, the invention features any one of the bispecific antibodies comprising a feline Fc region variant disclosed herein, any one of the Fc constructs comprising a feline Fc region variant disclosed herein, or a pharmaceutical composition comprising same, for use in treating or preventing a feline disease or disorder in a cat in need thereof.

[0062] In some embodiments, the feline disease or disorder is an allergic disease, chronic pain, acute pain, an inflammatory disease, an autoimmune disease, an endocrine disease, a gastrointestinal disease, a cardiovascular disease, a renal disease, a reproductive-related disorder, an infectious disease, or cancer. In some embodiments, the feline disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity. [Brief explanation of the drawings]

[0063] [Figure 1] Analytical size-exclusion chromatography (aSEC) chromatograms of a canine knob-in-hole bispecific antibody containing two Fab-Fc chains (006 and 225) with 006 knob + 225 hole or 006 hole + 225 knob mutations in the Fc domain. K = knob, H = hole, AU = absorbance units. [Figure 2] Deconvoluted subunit mass spectra of a canine knob-in-hole bispecific antibody containing two Fab-Fc chains (006 and 225) with 006 knob + 225 hole or 006 hole + 225 knob mutations in the Fc domain, analyzed by liquid chromatography-mass spectrometry (LC-MS) under reducing or non-reducing conditions, treated with peptide-N-glycosidase F (PNGase F). HC = heavy chain, LC = light chain, GalNAc = N-acetylgalactosamine, Gal = galactose, amu = atomic mass unit. [Figure 3] Schematic representation of canine bispecific antibodies determined by non-reduced intact mass spectrometry. Both species identified contain knob-in-hole mutations. [Figure 4]Dual-reference-subtracted BIACORE™ sensorgrams are shown for a canine knob-in-hole bispecific antibody containing two Fab-Fc chains (006 and 225) with 006 knob + 225 hole or 006 hole + 225 knob mutations in the Fc domain, binding to each of the respective antigens. [Figure 5] aSEC chromatograms of a canine VHH-Fc (02F09R3, with or without a linker) and a VHH-Fc knob-in-hole bispecific antibody containing two VHH-Fc chains (02F09R3 and 01E03R3) with 02F09R3-knob + 01E03R3-hole mutations in the Fc domain. [Figure 6] Deconvoluted subunit mass spectra of LC-MS analysis under reducing or non-reducing conditions for a canine VHH-Fc (02F09R3, with or without a linker) and a VHH-Fc knob-in-hole bispecific antibody containing two VHH-Fc chains (02F09R3 and 01E03R3) harboring the 02F09R3-knob + 01E03R3-hole mutations in the Fc domain, treated with PNGase F. PyroQ = pyroglutamate. [Figure 7] Schematic representation of canine VHH-Fc and VHH-Fc knob-in-hole bispecific antibodies as determined by non-reducing intact mass spectrometry. [Figure 8] aSEC chromatograms of canine monoclonal antibody (mAb) / VHH-Fc knob-in-hole bispecific antibodies containing a Fab-Fc chain (006) and a VHH-Fc chain (02F09R3 or 01E03R3) with 006-knob + 01E03R3-hole or 02F09R3-knob + 006-hole mutations in the Fc domain. [Figure 9]Deconvoluted subunit mass spectra of LC-MS analysis of a canine mAb / VHH-Fc knob-in-hole bispecific antibody containing a Fab-Fc chain (006) and a VHH-Fc chain (02F09R3 or 01E03R3) with 006-knob + 01E03R3-hole or 02F09R3-knob + 006-hole mutations in the Fc domain, treated with PNGase F, under reducing or non-reducing conditions. [Figure 10] Schematic of canine mAb / VHH-Fc knobs-in-hole bispecific antibodies as determined by non-reduced intact mass spectrometry. Both species identified contained knobs-in-hole mutations. [Figure 11] Dual-reference-subtracted BIACORE™ sensorgrams are shown for a canine mAb / VHH-Fc knob-in-hole bispecific antibody comprising a Fab-Fc chain (006) and a VHH-Fc chain (02F09R3) with 02F09R3-knob + 006-hole mutations in the Fc domain, binding to each of their respective antigens. [Figure 12] Shown is the aSEC chromatogram of a fully monoclonal canine IgGB antibody (006_GGS_02F09R3) with a C-terminal linker followed by a VHH. [Figure 13] Deconvoluted subunit mass spectra of LC-MS analysis under reducing or non-reducing conditions for a fully monoclonal canine IgGB antibody (006_GGS_02F09R3) with a C-terminal linker followed by a VHH, treated with PNGase F. [Figure 14] FIG. 1 is a schematic diagram of a fully monoclonal canine IgGB antibody with a C-terminal linker followed by a VHH as determined by non-reduced intact mass spectrometry. [Figure 15] 1 shows dual reference subtracted BIACORE™ sensorgrams of a fully monoclonal canine IgGB antibody (006_GGS_02F09R3) with a C-terminal linker followed by a VHH binding to each of the respective antigens. [Figure 16]aSEC chromatograms of feline knob-in-hole bispecific antibodies containing two Fab-Fc chains (076 and 023) with the following mutations in the Fc domain: 076 knob + 023 hole, 076 hole + 023 knob, 076 knob A354C + 023 hole Y349C, or 076 hole Y349C + 023 knob A354C. C = cysteine ​​mutation (A354C or Y349C). [Figure 17] Deconvoluted subunit mass spectra from LC-MS analysis under non-reducing conditions of a feline knob-in-hole bispecific antibody containing two Fab-Fc chains (076 and 023) with the 076 knob + 023 hole, 076 hole + 023 knob, 076 knob A354C + 023 hole Y349C, or 076 hole Y349C + 023 knob A354C mutations in the Fc domain, treated with PNGase F. DETAILED DESCRIPTION OF THE INVENTION

[0064] The invention described herein relates to bispecific binding agents (e.g., bispecific antibodies or Fc constructs) for use in companion animals (e.g., dogs and cats), pharmaceutical compositions comprising such bispecific binding agents, and methods of their use. These bispecific binding agents can be used for a variety of therapeutic and diagnostic purposes. In some examples, the disclosure features bispecific binding agents with complementary dimerization selectivity modules that promote dimer formation between polypeptide chains, thereby increasing yield and stability.

[0065] definition When values ​​are described as ranges, the description should be understood to include disclosure of all possible subranges within such ranges and specific numerical values ​​falling within such ranges, whether or not a specific numerical value or specific subrange is expressly set forth. D, temperatures, times, concentrations, and molecular weights are approximations that are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variance of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about" and that numerical designations may include values ​​that are rounded to the nearest significant figure. It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0066] Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context or clearly indicated otherwise, singular terms shall include the plural and plural terms shall include the singular. In the event of any discrepancy in definitions among various sources or publications, the definitions provided herein shall prevail.

[0067] It is understood that embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" multiple aspects and embodiments. As used herein, the singular forms "a," "an," and "the" include plural referents (e.g., at least one, one or more) unless otherwise indicated. The use of the word "or" herein means "and / or" and is not intended to imply that alternatives are mutually exclusive unless otherwise specified.

[0068] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is intended to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0069] As used herein, "percent (%) amino acid sequence identity," "% identical," and "homology" with respect to a nucleic acid or polypeptide sequence are defined as the percentage of nucleotides or amino acid residues in a reference sequence that are identical to the nucleotides or amino acid residues in a particular nucleic acid or polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved by various means within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any parameters needed to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, a variant has at least 50% sequence identity with a reference nucleic acid molecule or polypeptide, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, not counting any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least 50% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the sequence of the reference nucleic acid or polypeptide.

[0070] The term "companion animal" refers to an animal suitable for human companionship. In some embodiments, a companion animal is a small mammal, such as a canine, feline, horse, rabbit, ferret, guinea pig, rodent, etc. In some embodiments, a companion animal is a canine (e.g., a dog) or a feline (e.g., a cat). In some embodiments, a companion animal is a livestock animal, such as a horse, cow, pig, etc.

[0071] The term "dimerization selectivity module" refers to a sequence of an Fc domain monomer that facilitates preferred pairing between two Fc domain monomers. A "complementary" dimerization selectivity module is a dimerization selectivity module that promotes or supports the selective interaction of two Fc domain monomers with each other. Complementary dimerization selectivity modules can have the same or different sequences. Exemplary complementary dimerization selectivity modules are described herein.

[0072] As used herein, the term "hole" or "recess" refers to at least one of the original amino acid residues in the CH3 domain of an Fc domain monomer being replaced with a different amino acid residue having a smaller side chain volume than the original amino acid residue, thus creating a three-dimensional hole or recess in the CH3 domain. The term "original amino acid residue" refers to the naturally occurring amino acid residue encoded by the genetic code of the wild-type CH3 domain.

[0073] As used herein, the term "knob" or "protrusion" refers to at least one of the original amino acid residues in the CH3 domain of an Fc domain monomer being replaced with a different amino acid residue having a larger side chain volume than the original amino acid residue, thus creating a three-dimensional knob or protrusion in the CH3 domain. The term "original amino acid residue" refers to the naturally occurring amino acid residue encoded by the genetic code of the wild-type CH3 domain.

[0074] As used herein, the term "knob-in-hole (KiH)" refers to an Fc domain or Fc region variant comprising two Fc domain monomers, where the first Fc domain monomer comprises a "hole" or "recess" in its CH3 domain and the second Fc domain monomer comprises a "knob" or "protrusion" in its CH3 domain. In a KiH pair, the knob in the CH3 domain of the first Fc domain monomer is positioned to interact with the hole in the CH3 domain of the second Fc domain monomer without significantly interfering with the normal association of the dimers at the interface between the CH3 domains, thereby promoting heterodimerization of the two Fc domain monomers.

[0075] As used herein, the term "charged region" refers to amino acid substitutions in an Fc domain monomer within the ring of charged residues at the interface between CH3 domains that promote dimerization of the Fc domain monomers by forming charge pairs. The term "charge pair" refers to electrostatic pairing of amino acid residues with opposite charges, e.g., pairing between a basic amino acid residue and an acidic amino acid residue.

[0076] The term "amino acid substitution" refers to the replacement of an amino acid in a polypeptide with another amino acid. In some embodiments, the amino acid substitution is a conservative substitution. The amino acid substitution can be introduced into a polypeptide that has been screened for a desired activity, such as maintaining or improving FcRn binding, maintaining or improving antigen binding, reducing immunogenicity, improving ADCC or CDC, or improving pharmacokinetics.

[0077] The term "conservative substitution," as used herein, refers to the substitution of one amino acid residue for another having similar properties, e.g., charge, hydrophobicity, and / or size. For example, amino acids can be grouped according to common side chain properties: Hydrophobic: norleucine (Nle), Met, Ala, Val, Leu, Ile, Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, Acidic: Asp, Glu, Basic: His, Lys, Arg, Rigidity: Gly, Pro, Aromatic: Trp, Tyr, Phe.

[0078] Conservative substitution involves exchanging a member of one of these classes for another member of the same class. Non-conservative substitution involves exchanging a member of one of these classes for another class. In some embodiments, a conservative amino acid substitution refers to a substitution that results in similar properties or functions as another amino acid substitution. For example, a conservative amino acid substitution for A426Y can be A426F, A426T, or A426W. Further non-limiting examples of conservative amino acid substitutions are shown in Table 1. [Table 1]

[0079] The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or receptor) and its binding partner (e.g., an antigen or ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen, receptor and ligand). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ) Affinity can be measured by common protein-protein interaction tools known in the art, such as immunoblot, enzyme-linked immunosorbent assay (ELISA), equilibrium exclusion assay (KinExA), biolayer interferometry (BLI), or surface plasmon resonance (SPR) devices. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0080] "Surface plasmon resonance (SPR)" refers to an optical phenomenon that allows for the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore™ system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al., 1993, Ann. Biol. Clin. 51:19-26.

[0081] The term "amino acid sequence" refers to the sequence of amino acid residues in a peptide or protein. The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by definition. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to a protein containing modifications (generally conservative in nature) to the native sequence, such as deletions, additions, and substitutions, so long as the protein maintains a desired activity. These modifications may be intentional, via site-directed mutagenesis, or accidental, for example, through mutations of hosts producing the protein or errors resulting from PCR amplification.

[0082] The term "antibody" as used herein is used in the broadest sense and refers to a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab), so long as they exhibit the desired antigen-binding activity.

[0083] The term "bispecific antibody" refers to an antibody derivative having variable regions that recognize two different epitopes within the same antibody molecule. Bispecific antibodies may be antibodies that recognize two different antigens or two different epitopes on the same antigen.

[0084] The term "antibody fragment" refers to a molecule other than a full-length antibody that contains a portion of a full-length antibody that binds to the antigen to which the full-length antibody binds. In some embodiments, antibody fragments include, but are not limited to, Fab, single-chain variable fragments (e.g., scFv), Fv, Fab', Fab'-SH, F(ab')2, nanobodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0085] The terms "full length antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain with a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0086] The terms "nanobody," "VHH," "VHH domain," "VHH antibody fragment," and "single-domain antibody," used interchangeably herein, refer to the variable domain of a single heavy chain of a class of antibodies found in the family Camelidae, typically found in a naturally occurring form lacking light chains. Suitable nanobodies are well known to those skilled in the art, and illustrative examples include nanobodies from camel, dromedary, llama, and alpaca. However, single-domain antibodies can also be derived from sources outside the family Camelidae.

[0087] The term "binding domain" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab, scFv, Fv, Fab', Fab'-SH, F(ab')2, nanobodies, and diabodies), receptors or fragments thereof (e.g., the extracellular domains of canine or feline receptor proteins), ligands, aptamers, and other molecules with identified binding partners.

[0088] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0089] The terms "Fc region," "Fc domain," and "Fc polypeptide" refer to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term "Fc region variant" refers to a variant of a companion animal Fc region having one or more substitutions relative to a wild-type companion animal Fc region. The term "wild-type canine IgG Fc domain" refers to a native Fc region of a canine antibody. The term "canine Fc region variant" refers to a variant of a canine antibody Fc region having one or more substitutions relative to a wild-type canine Fc region. In some embodiments, the canine Fc region sequence is derived from a canine (e.g., dog) IgG (e.g., IgGA, IgGB, IgGC, or IgGD). The term "wild-type feline IgG Fc domain" refers to a native Fc region of a feline antibody. The term "feline Fc region variant" refers to a variant of a feline antibody Fc region having one or more substitutions relative to a wild-type feline Fc region. In some embodiments, the feline Fc region sequence is derived from a feline (e.g., cat) IgG (e.g., IgG1a, IgG1b, or IgG2). In some embodiments, the IgG Fc polypeptide comprises a hinge, CH2, and CH3, but not a CH1 or a CL. In some embodiments, the IgG Fc polypeptide comprises a CH2 and CH3, but not a CH1, hinge, or CL. In some embodiments, the IgG Fc polypeptide comprises a CH1, a hinge, CH2, and CH3, with or without a CL. In some embodiments, the IgG Fc polypeptide comprises a CH1, a hinge, CH2, CH3, and a CL. For example, the CL may be linked to CH1 via a disulfide bridge. In some embodiments, an Fc polypeptide, such as an IgG Fc polypeptide, lacks one or more C-terminal amino acids, e.g., 1-20, 1-15, 1-10, 1-5, or 1-2 amino acids, yet retains biological activity. In some embodiments, the biological activity of an Fc polypeptide is the ability to bind to FcRn.Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0090] The term "Fc domain monomer" refers to a polypeptide chain comprising at least a hinge domain and second and third antibody constant domains (CH2 and CH3) or functional fragments thereof (e.g., a fragment that is capable of (i) dimerizing with another Fc domain monomer to form an Fc domain, and (ii) binding to an Fc receptor). The Fc domain monomer may be of any immunoglobulin antibody isotype, including, for example, IgG. In addition, the Fc domain monomer may be an IgG subtype, e.g., IgGA, IgGB, IgGC, or IgGD in dogs, or IgG1a, IgG1b, or IgG2 in cats. In some embodiments, the Fc domain monomer does not comprise any portion of an immunoglobulin capable of antigen binding, e.g., a variable domain or a complementarity-determining region (CDR). In some embodiments, the Fc domain monomer comprises a portion of an immunoglobulin capable of acting as an antigen recognition region, e.g., a variable domain or a CDR. In some embodiments, the Fc domain monomer comprises a single domain antibody, e.g., a VHH domain.

[0091] As used herein, the term "Fc construct" refers to an assembled polypeptide chain comprising an Fc domain monomer or Fc region variant described herein (e.g., an Fc construct comprising an Fc domain monomer or an Fc region variant). The Fc constructs described herein can comprise Fc domain monomers having the same or different sequences. In some embodiments, an Fc construct does not comprise any portion of an immunoglobulin capable of antigen binding, e.g., a variable domain or a complementarity-determining region (CDR). In some embodiments, an Fc construct comprises a portion of an immunoglobulin capable of acting as an antigen recognition region, e.g., a variable domain or a CDR. In some embodiments, an Fc construct comprises a single domain antibody, e.g., a VHH domain.

[0092] The term "wild-type" refers to a non-mutated form of a polypeptide occurring in nature, or a fragment thereof. A wild-type polypeptide may be recombinantly produced. In some embodiments, a wild-type canine (e.g., dog) IgG Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 9-12. In other embodiments, a wild-type feline (e.g., cat) IgG Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 19-21.

[0093] The term "disease" or "disorder" refers to any condition that may benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder.

[0094] The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, myeloma, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma (e.g., angiosarcoma, osteosarcoma, soft tissue sarcoma, and histiocytic sarcoma), leukemia, head and neck squamous cell carcinoma, salivary gland carcinoma, breast cancer, mast cell tumor, melanoma, lung cancer (e.g., small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal carcinoma, hepatocellular carcinoma, squamous cell carcinoma, meningioma, glioma, gastric cancer, intestinal cancer, colon cancer, colorectal cancer, pancreatic adenocarcinoma, glioblastoma, cervical cancer, endometrial or uterine cancer, ovarian cancer, bladder cancer, prostate cancer, kidney or renal cancer, vulvar cancer, thyroid cancer, and transitional cell carcinoma.

[0095] The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0096] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0097] An "effective amount" of a composition, e.g., a polypeptide (e.g., a bispecific binding agent such as a bispecific antibody or Fc construct) of the present disclosure or a composition thereof (e.g., a pharmaceutical composition), refers to at least the minimum amount needed to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder (e.g., any disorder affecting a canine or feline, e.g., a cell proliferative disorder, e.g., cancer). An effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the animal, and the ability of the polypeptide (e.g., a bispecific binding agent such as a bispecific antibody or Fc construct) to elicit a desired response in the animal. An effective amount is also one in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of prophylactic use, beneficial or desired results include those that eliminate or reduce the risk of, reduce the severity of, or delay the onset of, the disease, including its biochemical, histological, and / or behavioral symptoms, its complications, and intermediate pathological phenotypes manifested during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as a reduction in one or more symptoms caused by a disease, an increase in the quality of life of a person suffering from a disease, a reduction in the dose of another medication required to treat the disease, an improvement in the effectiveness of another medication through targeting or the like, a delay in disease progression, and / or an increase in survival time. An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if a desired result can be or is achieved in combination with one or more other agents.

[0098] The terms "host cell" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include bacterial cells (e.g., E. coli cells) and eukaryotic cells. In some embodiments, host cells include yeast cells (e.g., Pichia (see, e.g., Powers et al., 2001, J Immunol Methods. 251:123-135), Hanseula, or Saccharomyces). In some embodiments, host cells also include "transformants" and "transformed cells," including the primary transformed cell line (e.g., CHO, 293E, COS, 293T, and HeLa) and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0099] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies within the population are identical and / or bind the same epitope, except for possible variant antibodies (such variants are generally present in minor amounts), e.g., containing naturally occurring mutations or arising during production of the monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0100] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional components that have unacceptable toxicity to the subject to which the formulation may be administered.

[0101] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0102] As used herein, the term "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention in an attempt to alter the natural course of the treated individual and can be carried out for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis. In some embodiments, the bispecific binding agents of the invention are used to delay the onset of disease or slow the progression of disease.

[0103] As used herein, the term "delaying progression" of a disorder or disease means to postpone, prevent, slow, inhibit, stabilize, and / or postpone the onset of the disease or disorder (e.g., a cell proliferative disorder, e.g., cancer). This delay can be of varying lengths of time depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0104] The term "epitope" refers to one or more specific sites on an antigen molecule to which an antibody or other binding agent binds. For example, an epitope can be a linear epitope or a conformational epitope.

[0105] As used herein, the terms "reduce" and "inhibit" refer to being able to cause an overall reduction of, for example, 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more, for example, when compared to a reference or control.

[0106] The terms "increase" and "improve" refer to being able to cause an overall increase of, for example, 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more, for example, when compared to a reference or control.

[0107] The terms "variable region" and "variable domain" refer to the domains of an antibody heavy or light chain that are involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., 2007, Kuby Immunology, 6th ed. W.H. Freeman and Co., page 91. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain derived from an antibody that binds to the antigen. See, e.g., Portolano et al., 1993, J. Immunol. 150:880-887, and Clarkson et al., 1991, Nature 352:624-628.

[0108] A "variant" is a polypeptide that differs from a reference polypeptide by single or multiple non-natural amino acid substitutions, deletions, and / or additions. In some embodiments, a variant retains at least one biological activity of the reference polypeptide. In some embodiments, a variant has a biological activity that the reference polypeptide substantially lacks. A "canid Fc region variant" comprises an amino acid sequence that differs from that of a wild-type canid Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, a canid Fc region variant has at least one amino acid substitution compared to the wild-type canid Fc region, e.g., 1 to 10 amino acid substitutions, preferably 1 to 5 amino acid substitutions in the wild-type canid Fc region. A canid Fc region variant herein preferably has at least 80% homology to the wild-type canid Fc region, most preferably at least 90% homology, and more preferably at least 95% homology. In some embodiments, the canine Fc region is a canine IgGA Fc region variant, a canine IgGB Fc region variant, a canine IgGC Fc region variant, or a canine IgGD Fc region variant. A "feline Fc region variant" comprises an amino acid sequence that differs from the amino acid sequence of a wild-type feline Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the feline Fc region variant has at least one amino acid substitution compared to the wild-type feline Fc region, e.g., 1 to 10 amino acid substitutions, preferably 1 to 5 amino acid substitutions in the wild-type feline Fc region. A feline Fc region variant herein preferably has at least 80% homology with the wild-type feline Fc region, most preferably at least 90% homology, and more preferably at least 95% homology. In some embodiments, the feline IgG Fc region is a feline IgG1a Fc region variant, a feline IgG1b Fc region variant, or a feline IgG2 Fc region variant.In some embodiments, the Fc region variant (e.g., a canine or feline Fc region variant) comprises a hinge, CH2, and CH3, but does not comprise a CH1 or a CL. In some embodiments, the Fc region variant (e.g., a canine or feline Fc region variant) comprises a CH2 and CH3, but does not comprise a CH1, a hinge, or a CL. In some embodiments, the Fc region variant (e.g., a canine or feline Fc region variant) comprises a CH1, a hinge, CH2, and CH3, with or without a CL. In some embodiments, the Fc region variant (e.g., a canine or feline Fc region variant) comprises a CH1, a hinge, CH2, CH3, and a CL. For example, the CL may be linked to the CH1 via a disulfide bridge.

[0109] The term "vector," as used herein, refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0110] As used herein, "administering" refers to a method of providing a subject with a dosage of a compound (e.g., a bispecific binding agent of the present disclosure, e.g., a bispecific antibody or Fc construct) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a bispecific binding agent of the present disclosure). Compositions utilized in the methods described herein can be administered, for example, parenterally, intramuscularly, intravenously, intradermally, transdermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrathoracically, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion directly bathing target cells, by catheter, by lavage, in a cream, or in a lipid composition. Administration can be local or systemic. The method of administration can vary depending on various factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated.

[0111] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) administration and sequential or consecutive administration in any order. The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent occurs within a short period of time relative to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered approximately a specified number of minutes or less apart. The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents, where the administration of one or more agents continues after the administration of one or more other agents is discontinued, or the administration of one or more agents begins before the administration of one or more other agents. For example, the administration of two or more therapeutic agents is administered approximately a specified number of minutes or more apart. As used herein, "in combination with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to administering one therapeutic modality to an animal before, during, or after administering another therapeutic modality.

[0112] Bispecific Binding Agents Bispecific antibodies: The present disclosure provides a bispecific antibody, comprising: (a) a first binding domain that binds to a first antigen, the first binding domain being linked to a first companion animal Fc region variant; (b) a second binding domain that binds to a second antigen, the second binding domain being linked to a second companion animal Fc region variant. In some embodiments, at least one of the first binding domain and the second binding domain comprises a single domain antibody.

[0113] For example, the present disclosure provides a bispecific antibody comprising: (a) a first binding domain that binds to a first antigen, the first binding domain being linked to a first companion animal Fc region variant; (b) a second binding domain that binds to a second antigen, the second binding domain being linked to a second companion animal Fc region variant; and wherein at least one of the first binding domain and the second binding domain comprises a single domain antibody. In some examples, the first binding domain comprises a single domain antibody. In some examples, the second binding domain comprises a single domain antibody. In some examples, the first binding domain and the second binding domain each comprise a single domain antibody.

[0114] The first binding domain or the second binding domain may bind to any suitable antigen(s). In some embodiments, the first binding domain and the second binding domain each specifically bind to an antigen independently selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-31R, IL-33, CD3, CD20, CD47, CD52, and complexes of the complement system.

[0115] In some embodiments, the first binding domain and / or the second binding domain comprise a ligand-binding portion of an antibody, an antibody fragment, or a receptor. In some examples, the first binding domain or the second binding domain comprise a ligand-binding portion of an antibody, an antibody fragment, or a receptor. In some examples, the first binding domain and the second binding domain each comprise a ligand-binding portion of an antibody, an antibody fragment, or a receptor. In some embodiments, the antibody fragment is selected from the group consisting of Fab, single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, and a diabody.

[0116] In some embodiments, the single domain antibody is linked to the first companion animal Fc region variant or the second companion animal Fc region variant directly or via a peptide linker. In some examples, the single domain antibody is linked to the first companion animal Fc region variant directly. In some examples, the single domain antibody is linked to the first companion animal Fc region variant via a peptide linker. In some examples, the single domain antibody is linked to the second companion animal Fc region variant directly. In some examples, the single domain antibody is linked to the second companion animal Fc region variant via a peptide linker.

[0117] In some embodiments, the single-domain antibody is a VHH domain. In one embodiment, the VHH domain comprises a C-terminal residue, the first companion animal Fc region variant or the second companion animal Fc region variant comprises an N-terminal residue, and the C-terminal residue of the VHH domain is linked to the N-terminal residue of the first companion animal Fc region variant or the second companion animal Fc region variant, either directly or via a peptide linker. In another embodiment, the VHH domain comprises an N-terminal residue, the first companion animal Fc region variant or the second companion animal Fc region variant comprises a C-terminal residue, and the N-terminal residue of the VHH domain is linked to the C-terminal residue of the first companion animal Fc region variant or the second companion animal Fc region variant, either directly or via a peptide linker.

[0118] In some embodiments, the peptide linker is (a) GPGGQ (SEQ ID NO: 38), (b) PKRENGRVPRPPDCPKCP (SEQ ID NO: 363); (c) VPKRENGRVPRPPDCPKCP (SEQ ID NO: 364), (d) FNECRCTDTPPCPVPEP (SEQ ID NO: 22); (e) PKRENGRVPRPPDCPKCPAPEM (SEQ ID NO: 23), (f) AKECECKCNCNNCPCPGCGL (SEQ ID NO: 24), (g) PKESTCKCISPCPVPES (SEQ ID NO: 25), (h) PKESTCKCIPPCPVPES (SEQ ID NO: 26), (i) KTDHPPGPKPCDCPKCP (SEQ ID NO: 27), and (j) KTASTIESKTGEGPKCP (SEQ ID NO: 29) The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0119] In one embodiment, the first companion animal Fc region variant and the second companion animal Fc region variant are canine Fc region variants. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 11. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO:12.

[0120] In some embodiments, the first canid Fc region variant and the second canid Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first canid Fc region variant and the second canid Fc region variant. Any suitable complementary dimerization selectivity module may be used.

[0121] In one embodiment, the first canid Fc region variant and the second canid Fc region variant each comprise a protrusion or a recess; when the first canid Fc region comprises a protrusion, the second canid Fc region comprises a recess, and when the first canid Fc region comprises a recess, the second canid Fc region comprises a protrusion.

[0122] In some embodiments, the first canid Fc region variant and the second canid Fc region variant are (a) S354C and T366W in a first canine Fc region variant and Y349C, T366S, L368A, and Y407V in a second canine Fc region variant; (b) T366W in a first canine Fc region variant and T366S, L368A, and Y407V in a second canine Fc region variant; (c) R392D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (d) S364H and F405A in a first canine Fc region variant and Y349T and T394F in a second canine Fc region variant; (e) F405L in a first canine Fc region variant and K409R in a second canine Fc region variant; (f) T366L, R392L, and T394W in a first canine Fc region variant and L351Y, F405A, and Y407V in a second canine Fc region variant; (g) K360E and K409W in a first canine Fc region variant and S347R, D399V, and F405T in a second canine Fc region variant; (h) Y349C, K360E, and K409W in a first canine Fc region variant and S347R, S354C, D399V, and F405T in a second canine Fc region variant; (i) K370E and K409W in a first canine Fc region variant and E357N, D399V, and F405T in a second canine Fc region variant; (j) K360D, D399M, and Y407A in a first canine Fc region variant and Q345R, S347R, T366V, and K409V in a second canine Fc region variant; (k) Y349S, T366M, K370Y, and K409V in a first canine Fc region variant and E356G, E357D, S364Q, and Y407A in a second canine Fc region variant; (l) L351D and L368E in a first canine Fc region variant and L351K and T366K in a second canine Fc region variant; (m) L368D and K370S in a first canine Fc region variant and E356Q and S364K in a second canine Fc region variant; or (n) T366Y in a first canine Fc region variant and T366S, L368A, and Y407T in a second canine Fc region variant. wherein the amino acid positions are based on EU numbering.

[0123] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions S354C and T366W, and the second canine Fc region variant comprises the amino acid substitutions T366S, L368A, Y407V, and Y349C.

[0124] In some embodiments, the first canine Fc region variant comprises the amino acid substitution T366W and the second canine Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407V.

[0125] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions R392D and K409D, and the second canine Fc region variant comprises the amino acid substitutions E356K and D399K.

[0126] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions S364H and F405A, and the second canine Fc region variant comprises the amino acid substitutions Y349T and T394F.

[0127] In some embodiments, the first canine Fc region variant comprises the amino acid substitution F405L and the second canine Fc region variant comprises the amino acid substitution K409R.

[0128] In some embodiments, the first canine Fc region variant comprises amino acid substitutions T366L, R392L, and T394W, and the second canine Fc region variant comprises amino acid substitutions L351Y, F405A, and Y407V.

[0129] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K360E and K409W, and the second canine Fc region variant comprises the amino acid substitutions S347R, D399V, and F405T.

[0130] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions Y349C, K360E, and K409W, and the second canine Fc region variant comprises the amino acid substitution S354C.

[0131] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K370E and K409W, and the second canine Fc region variant comprises the amino acid substitutions E357N, D399V, and F405T.

[0132] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K360D, D399M, and Y407A, and the second canine Fc region variant comprises the amino acid substitutions Q345R, S347R, T366V, and K409V.

[0133] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions Y349S, T366M, K370Y, and K409V, and the second canine Fc region variant comprises the amino acid substitutions E356G, E357D, S364Q, and Y407A.

[0134] In some embodiments, the first canine Fc region variant comprises amino acid substitutions L351D and L368E, and the second canine Fc region variant comprises amino acid substitutions L351K and T366K.

[0135] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions L368D and K370S, and the second canine Fc region variant comprises the amino acid substitutions E356Q and S364K.

[0136] In some embodiments, the first canine Fc region variant comprises the amino acid substitution T366Y and the second canine Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407T.

[0137] In another embodiment, the first canid Fc region variant comprises a first charged region and the second canid Fc region variant comprises a second charged region, wherein the first charged region is charge-paired with the second charged region. In some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0138] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S183D in the first canine Fc region variant and S183K in the second canine Fc region variant. It further comprises a CH1 domain comprising:

[0139] In some embodiments, the first canid Fc region variant and the second canid Fc region variant are (a) K409D in a first canine Fc region variant and D399K in a second canine Fc region variant; (b) K390D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (c) K390D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant; and (d) K370D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0140] In some embodiments, the first canine Fc region variant comprises the amino acid substitution K409D and the second canine Fc region variant comprises the amino acid substitution D399K.

[0141] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K390D and K409D, and the second canine Fc region variant comprises the amino acid substitutions E356K and D399K.

[0142] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K390D and K409D, and the second canine Fc region variant comprises the amino acid substitutions E357K and D399K.

[0143] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K370D and K409D, and the second canine Fc region variant comprises the amino acid substitutions E357K and D399K.

[0144] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S176K in the first canine Fc region variant and S176D in the second canine Fc region variant. It further comprises a CL domain comprising:

[0145] In some embodiments, the first canid Fc region variant and / or the second canid Fc region variant further comprise at least one amino acid substitution that increases half-life, including any amino acid substitution disclosed in U.S. Patent Application Publication Nos. 2020 / 0216536 and 2020 / 0362035, U.S. Patent Application No. 17 / 875,934, and U.S. Patent No. 11,434,276, each of which is incorporated by reference herein in its entirety.

[0146] In some embodiments, the first canid Fc region variant and / or the second canid Fc region variant comprises the following amino acid substitutions: (a) 252Y and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (b) 252M and, optionally, at least one amino acid substitution selected from the group consisting of 251D or 251E; 256D or 256F; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (c)434R, (d) 426Y and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (e) 426H and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (f) 426F and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 426H; and (g) 434R and, optionally, at least one amino acid substitution selected from the group consisting of 286L; 286Y; 312P; and 436H. wherein the amino acid positions are based on EU numbering.

[0147] In another embodiment, the first companion animal Fc region variant and the second companion animal Fc region variant are feline Fc region variants. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 19. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 20. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 21.

[0148] In some embodiments, the first feline Fc region variant and the second feline Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first feline Fc region variant and the second feline Fc region variant.

[0149] In one embodiment, the first feline Fc region variant and the second feline Fc region variant each comprise a protrusion or a recess, and if the first feline Fc region comprises a protrusion, the second feline Fc region comprises a recess, and if the first feline Fc region comprises a recess, the second feline Fc region comprises a protrusion.

[0150] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) T366W in a first feline Fc region variant and T366S, L368A, and Y407V in a second feline Fc region variant; (b) T366W in a first feline Fc region variant and T366S, L368A, and Y398T in a second feline Fc region variant; (c) A354C and T366W in a first feline Fc region variant and Y349C, T366S, L368A, and Y407V in a second feline Fc region variant; (d) R392D and K409D in a first feline Fc region variant and E356K and D399K in a second feline Fc region variant; (e) S364H and F405A in a first feline Fc region variant and Y349T and T394F in a second feline Fc region variant; (f) F405L in a first feline Fc region variant and K409R in a second feline Fc region variant; (g) T366L, R392L, and T394W in a first feline Fc region variant and L351Y, F405A, and Y407V in a second feline Fc region variant; (h) R360E and K409W in a first feline Fc region variant and Q347R, D399V, and F405T in a second feline Fc region variant; (i) Y349C, R360E, and K409W in a first feline Fc region variant and Q347R, A354C, D399V, F405T in a second feline Fc region variant; (j) K370E and K409W in a first feline Fc region variant and E357N, D399V, and F405T in a second feline Fc region variant; (k) R360D, D399M, and Y407A in a first feline Fc region variant and E345R, Q347R, T366V, and K409V in a second feline Fc region variant; (l) Y349S, K370Y, T366M, and K409V in a first feline Fc region variant and E356G, E357D, S364Q, and Y407A in a second feline Fc region variant; (m) L351D and L368E in a first feline Fc region variant and L351K and T366K in a second feline Fc region variant; (n) L368D and K370S in a first feline Fc region variant and E356Q and S364K in a second feline Fc region variant; and (o) T366Y in the first feline Fc region variant and T366S, L368A, and Y407T in the second feline Fc region variant. wherein the amino acid positions are based on EU numbering.

[0151] In some embodiments, the first feline Fc region variant comprises the amino acid substitution T366W and the second feline Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407V.

[0152] In some embodiments, the first feline Fc region variant comprises the amino acid substitution T366W and the second feline Fc region variant comprises the amino acid substitutions T366S, L368A, and Y398T.

[0153] In some embodiments, the first feline Fc region variant comprises amino acid substitutions A354C and T366W, and the second feline Fc region variant comprises amino acid substitutions Y349C, T366S, L368A, and Y407V.

[0154] In some embodiments, the first feline Fc region variant comprises the amino acid substitutions R392D and K409D, and the second feline Fc region variant comprises the amino acid substitutions E356K and D399K.

[0155] In some embodiments, the first feline Fc region variant comprises amino acid substitutions S364H and F405A, and the second feline Fc region variant comprises amino acid substitutions Y349T and T394F.

[0156] In some embodiments, the first feline Fc region variant comprises the amino acid substitution F405L and the second feline Fc region variant comprises the amino acid substitution K409R.

[0157] In some embodiments, the first feline Fc region variant comprises amino acid substitutions T366L, R392L, and T394W, and the second feline Fc region variant comprises amino acid substitutions L351Y, F405A, and Y407V.

[0158] In some embodiments, the first feline Fc region variant comprises amino acid substitutions R360E and K409W, and the second feline Fc region variant comprises amino acid substitutions Q347R, D399V, and F405T.

[0159] In some embodiments, the first feline Fc region variant comprises amino acid substitutions Y349C, R360E, and K409W, and the second feline Fc region variant comprises amino acid substitutions Q347R, A354C, D399V, F405T.

[0160] In some embodiments, the first feline Fc region variant comprises the amino acid substitutions K370E and K409W, and the second feline Fc region variant comprises the amino acid substitutions E357N, D399V, and F405T.

[0161] In some embodiments, the first feline Fc region variant comprises amino acid substitutions R360D, D399M, and Y407A, and the second feline Fc region variant comprises amino acid substitutions E345R, Q347R, T366V, and K409V.

[0162] In some embodiments, the first feline Fc region variant comprises amino acid substitutions Y349S, K370Y, T366M, and K409V, and the second feline Fc region variant comprises amino acid substitutions E356G, E357D, S364Q, and Y407A.

[0163] In some embodiments, the first feline Fc region variant comprises amino acid substitutions L351D and L368E, and the second feline Fc region variant comprises amino acid substitutions L351K and T366K.

[0164] In some embodiments, the first feline Fc region variant comprises amino acid substitutions L368D and K370S, and the second feline Fc region variant comprises amino acid substitutions E356Q and S364K.

[0165] In some embodiments, the first feline Fc region variant comprises the amino acid substitution T366Y and the second feline Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407T.

[0166] In another embodiment, the first feline Fc region variant comprises a first charged region and the second feline Fc region variant comprises a second charged region, and the first charged region forms a charge pair with the second charged region. In some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0167] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S183D in the first feline Fc region variant and S183K in the second feline Fc region variant. It further comprises a CH1 domain comprising:

[0168] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) K409D in a first feline Fc region variant and D399K in a second feline Fc region variant; and (b) K370D and K409D in a first feline Fc region variant and E357K and D399K in a second feline Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0169] In some embodiments, the first feline Fc region variant comprises the amino acid substitution K409D and the second feline Fc region variant comprises the amino acid substitution D399K.

[0170] In some embodiments, the first feline Fc region variant comprises the amino acid substitutions K370D and K409D, and the second feline Fc region variant comprises the amino acid substitutions E357K and D399K.

[0171] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S176K in the first feline Fc region variant and S176D in the second feline Fc region variant. It further comprises a CL domain comprising:

[0172] In some embodiments, the first feline Fc region variant and / or the second feline Fc region variant further comprise at least one amino acid substitution that increases half-life, including any amino acid substitution disclosed in U.S. Patent Application Publication No. 2022 / 0259282, U.S. Patent Application No. 18 / 046,082, and U.S. Patent No. 11,498,953, each of which is incorporated by reference herein in its entirety.

[0173] In some embodiments, the first feline Fc region variant and / or the second feline Fc region variant comprises the following amino acid substitutions: (a) at least one amino acid substitution selected from the group consisting of 286E, 311V, and 428Y; (b) two or more amino acid substitutions selected from the group consisting of 286E, 311V, and 428Y, and (c) 286E, 311V, and 428Y wherein the amino acid positions are based on EU numbering.

[0174] Fc construct: The present disclosure also provides (a) a first polypeptide comprising a first companion animal Fc region variant; (b) a second polypeptide comprising a second companion animal Fc region variant; and wherein the first and second companion animal Fc region variants comprise complementary dimerization selectivity modules that promote dimerization between the first and second companion animal Fc region variants. In some embodiments, the first polypeptide or the second polypeptide does not comprise an antibody.

[0175] For example, the present disclosure also provides: (a) a first polypeptide comprising a first companion animal Fc region variant; (b) a second polypeptide comprising a second companion animal Fc region variant; and The present invention provides an Fc construct comprising: a first companion animal Fc region variant and a second companion animal Fc region variant, each of which comprises complementary dimerization selectivity modules that promote dimerization between the first companion animal Fc region variant and the second companion animal Fc region variant; and the first polypeptide or the second polypeptide does not comprise an antibody.

[0176] In some embodiments, the Fc construct further comprises a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1, VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, and a thrombopoietin binding peptide.

[0177] In one embodiment, the first companion animal Fc region variant and the second companion animal Fc region variant are canine Fc region variants. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 11. In some embodiments, the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO:12.

[0178] In one embodiment, the first canid Fc region variant and the second canid Fc region variant each comprise a protrusion or a recess; when the first canid Fc region comprises a protrusion, the second canid Fc region comprises a recess, and when the first canid Fc region comprises a recess, the second canid Fc region comprises a protrusion.

[0179] In some embodiments, the first canid Fc region variant and the second canid Fc region variant each comprise: (a) S354C and T366W in a first canine Fc region variant and T366S, L368A, Y407V, and Y349C in a second canine Fc region variant; (b) T366W in a first canine Fc region variant and T366S, L368A, and Y407V in a second canine Fc region variant; (c) R392D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (d) S364H and F405A in a first canine Fc region variant and Y349T and T394F in a second canine Fc region variant; (e) F405L in a first canine Fc region variant and K409R in a second canine Fc region variant; (f) T366L, R392L, and T394W in a first canine Fc region variant and L351Y, F405A, and Y407V in a second canine Fc region variant; (g) K360E and K409W in a first canine Fc region variant and S347R, D399V, and F405T in a second canine Fc region variant; (h) Y349C, K360E, and K409W in a first canine Fc region variant and S347R, S354C, D399V, and F405T in a second canine Fc region variant; (i) K370E and K409W in a first canine Fc region variant and E357N, D399V, and F405T in a second canine Fc region variant; (j) K360D, D399M, and Y407A in a first canine Fc region variant and Q345R, S347R, T366V, and K409V in a second canine Fc region variant; (k) Y349S, T366M, K370Y, and K409V in a first canine Fc region variant and E356G, E357D, S364Q, and Y407A in a second canine Fc region variant; (l) L351D and L368E in a first canine Fc region variant and L351K and T366K in a second canine Fc region variant; (m) L368D and K370S in a first canine Fc region variant and E356Q and S364K in a second canine Fc region variant; or (n) T366Y in a first canine Fc region variant and T366S, L368A, and Y407T in a second canine Fc region variant. wherein the amino acid positions are based on EU numbering.

[0180] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions T366W and S354C, and the second canine Fc region variant comprises the amino acid substitutions T366S, L368A, Y407V, and Y349C.

[0181] In some embodiments, the first canine Fc region variant comprises the amino acid substitution T366W and the second canine Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407V.

[0182] In some embodiments, the first canine Fc region variant comprises amino acid substitutions K409D and R392D, and the second canine Fc region variant comprises amino acid substitutions D399K and E356K.

[0183] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions S364H and F405A, and the second canine Fc region variant comprises the amino acid substitutions Y349T and T394F.

[0184] In some embodiments, the first canine Fc region variant comprises the amino acid substitution F405L and the second canine Fc region variant comprises the amino acid substitution K409R.

[0185] In some embodiments, the first canine Fc region variant comprises amino acid substitutions T366L, R392L, and T394W, and the second canine Fc region variant comprises amino acid substitutions L351Y, F405A, and Y407V.

[0186] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K360E and K409W, and the second canine Fc region variant comprises the amino acid substitutions S347R, D399V, and F405T.

[0187] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K360E, K409W, and Y349C, and the second canine Fc region variant comprises the amino acid substitution S354C.

[0188] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K370E and K409W, and the second canine Fc region variant comprises the amino acid substitutions E357N, D399V, and F405T.

[0189] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K360D, D399M, and Y407A, and the second canine Fc region variant comprises the amino acid substitutions Q345R, S347R, T366V, and K409V.

[0190] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions Y349S, K370Y, T366M, and K409V, and the second canine Fc region variant comprises the amino acid substitutions E356G, E357D, S364Q, and Y407A.

[0191] In some embodiments, the first canine Fc region variant comprises amino acid substitutions L351D and L368E, and the second canine Fc region variant comprises amino acid substitutions L351K and T366K.

[0192] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions L368D and K370S, and the second canine Fc region variant comprises the amino acid substitutions E356Q and S364K.

[0193] In some embodiments, the first canine Fc region variant comprises the amino acid substitution T366Y and the second canine Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407T.

[0194] In another embodiment, the first canid Fc region variant comprises a first charged region and the second canid Fc region variant comprises a second charged region, wherein the first charged region is charge-paired with the second charged region. In some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0195] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S183D in the first canine Fc region variant and S183K in the second canine Fc region variant. It further comprises a CH1 domain comprising:

[0196] In some embodiments, the first canid Fc region variant and the second canid Fc region variant are (a) K409D in a first canine Fc region variant and D399K in a second canine Fc region variant; (b) K390D and K409D in a first canine Fc region variant and E356K and D399K in a second canine Fc region variant; (c) K390D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant; and (d) K370D and K409D in a first canine Fc region variant and E357K and D399K in a second canine Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0197] In some embodiments, the first canine Fc region variant comprises the amino acid substitution K409D and the second canine Fc region variant comprises the amino acid substitution D399K.

[0198] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K390D and K409D, and the second canine Fc region variant comprises the amino acid substitutions E356K and D399K.

[0199] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K390D and K409D, and the second canine Fc region variant comprises the amino acid substitutions E357K and D399K.

[0200] In some embodiments, the first canine Fc region variant comprises the amino acid substitutions K370D and K409D, and the second canine Fc region variant comprises the amino acid substitutions E357K and D399K.

[0201] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: S176K in the first canine Fc region variant and S176D in the second canine Fc region variant. It further comprises a CL domain comprising:

[0202] In some embodiments, the first canid Fc region variant and / or the second canid Fc region variant further comprise at least one amino acid substitution that increases half-life, including any amino acid substitution disclosed in U.S. Patent Application Publication Nos. 2020 / 0216536 and 2020 / 0362035, U.S. Patent Application No. 17 / 875,934, and U.S. Patent No. 11,434,276, each of which is incorporated by reference herein in its entirety.

[0203] In some embodiments, the first canid Fc region variant and the second canid Fc region variant have the following amino acid substitutions: (a) 252Y and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (b) 252M and, optionally, at least one amino acid substitution selected from the group consisting of 251D or 251E; 256D or 256F; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (c)434R, (d) 426Y and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (e) 426H and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (f) 426F and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 426H; and (g) 434R and, optionally, at least one amino acid substitution selected from the group consisting of 286L; 286Y; 312P; and 436H. wherein the amino acid positions are based on EU numbering.

[0204] In another embodiment, the first companion animal Fc region variant and the second companion animal Fc region variant are feline Fc region variants. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 19. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 20. In some embodiments, the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 21.

[0205] In one embodiment, the first feline Fc region variant and the second feline Fc region variant each comprise a protrusion or a recess, and if the first feline Fc region comprises a protrusion, the second feline Fc region comprises a recess, and if the first feline Fc region comprises a recess, the second feline Fc region comprises a protrusion.

[0206] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) T366W in a first feline Fc region variant and T366S, L368A, and Y407V in a second feline Fc region variant; (b) T366W in a first feline Fc region variant and T366S, L368A, and Y398T in a second feline Fc region variant; (c) A354C and T366W in a first feline Fc region variant and Y349C, T366S, L368A, and Y407V in a second feline Fc region variant; (d) R392D and K409D in a first feline Fc region variant and E356K and D399K in a second feline Fc region variant; (e) S364H and F405A in a first feline Fc region variant and Y349T and T394F in a second feline Fc region variant; (f) F405L in a first feline Fc region variant and K409R in a second feline Fc region variant; (g) T366L, R392L, and T394W in a first feline Fc region variant and L351Y, F405A, and Y407V in a second feline Fc region variant; (h) R360E and K409W in a first feline Fc region variant and Q347R, D399V, and F405T in a second feline Fc region variant; (i) Y349C, R360E, and K409W in a first feline Fc region variant and Q347R, A354C, D399V, F405T in a second feline Fc region variant; (j) K370E and K409W in a first feline Fc region variant and E357N, D399V, and F405T in a second feline Fc region variant; (k) R360D, D399M, and Y407A in a first feline Fc region variant and E345R, Q347R, T366V, and K409V in a second feline Fc region variant; (l) Y349S, K370Y, T366M, and K409V in a first feline Fc region variant and E356G, E357D, S364Q, and Y407A in a second feline Fc region variant; (m) L351D and L368E in a first feline Fc region variant and L351K and T366K in a second feline Fc region variant; (n) L368D and K370S in a first feline Fc region variant and E356Q and S364K in a second feline Fc region variant; and (o) T366Y in the first feline Fc region variant and T366S, L368A, and Y407T in the second feline Fc region variant. wherein the amino acid positions are based on EU numbering.

[0207] In some embodiments, the first feline Fc region variant comprises the amino acid substitution T366W and the second feline Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407V.

[0208] In some embodiments, the first feline Fc region variant comprises the amino acid substitution T366W and the second feline Fc region variant comprises the amino acid substitutions T366S, L368A, and Y398T.

[0209] In some embodiments, the first feline Fc region variant comprises amino acid substitutions A354C and T366W, and the second feline Fc region variant comprises amino acid substitutions Y349C, T366S, L368A, and Y407V.

[0210] In some embodiments, the first feline Fc region variant comprises the amino acid substitutions R392D and K409D, and the second feline Fc region variant comprises the amino acid substitutions E356K and D399K.

[0211] In some embodiments, the first feline Fc region variant comprises amino acid substitutions S364H and F405A, and the second feline Fc region variant comprises amino acid substitutions Y349T and T394F.

[0212] In some embodiments, the first feline Fc region variant comprises the amino acid substitution F405L and the second feline Fc region variant comprises the amino acid substitution K409R.

[0213] In some embodiments, the first feline Fc region variant comprises amino acid substitutions T366L, R392L, and T394W, and the second feline Fc region variant comprises amino acid substitutions L351Y, F405A, and Y407V.

[0214] In some embodiments, the first feline Fc region variant comprises amino acid substitutions R360E and K409W, and the second feline Fc region variant comprises amino acid substitutions Q347R, D399V, and F405T.

[0215] In some embodiments, the first feline Fc region variant comprises amino acid substitutions Y349C, R360E, and K409W, and the second feline Fc region variant comprises amino acid substitutions Q347R, A354C, D399V, F405T.

[0216] In some embodiments, the first feline Fc region variant comprises the amino acid substitutions K370E and K409W, and the second feline Fc region variant comprises the amino acid substitutions E357N, D399V, and F405T.

[0217] In some embodiments, the first feline Fc region variant comprises amino acid substitutions R360D, D399M, and Y407A, and the second feline Fc region variant comprises amino acid substitutions E345R, Q347R, T366V, and K409V.

[0218] In some embodiments, the first feline Fc region variant comprises amino acid substitutions Y349S, K370Y, T366M, and K409V, and the second feline Fc region variant comprises amino acid substitutions E356G, E357D, S364Q, and Y407A.

[0219] In some embodiments, the first feline Fc region variant comprises amino acid substitutions L351D and L368E, and the second feline Fc region variant comprises amino acid substitutions L351K and T366K.

[0220] In some embodiments, the first feline Fc region variant comprises amino acid substitutions L368D and K370S, and the second feline Fc region variant comprises amino acid substitutions E356Q and S364K.

[0221] In some embodiments, the first feline Fc region variant comprises the amino acid substitution T366Y and the second feline Fc region variant comprises the amino acid substitutions T366S, L368A, and Y407T.

[0222] In another embodiment, the first feline Fc region variant comprises a first charged region and the second feline Fc region variant comprises a second charged region, and the first charged region forms a charge pair with the second charged region. In some embodiments, the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

[0223] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S183D in the first feline Fc region variant and S183K in the second feline Fc region variant. It further comprises a CH1 domain comprising:

[0224] In some embodiments, the first feline Fc region variant and the second feline Fc region variant are (a) K409D in a first feline Fc region variant and D399K in a second feline Fc region variant; and (b) K370D and K409D in a first feline Fc region variant and E357K and D399K in a second feline Fc region variant. The CH3 domain comprises an amino acid substitution selected from the group consisting of:

[0225] In some embodiments, the first feline Fc region variant comprises the amino acid substitution K409D and the second feline Fc region variant comprises the amino acid substitution D399K.

[0226] In some embodiments, the first feline Fc region variant comprises the amino acid substitutions K370D and K409D, and the second feline Fc region variant comprises the amino acid substitutions E357K and D399K.

[0227] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: S176K in the first feline Fc region variant and S176D in the second feline Fc region variant. It further comprises a CL domain comprising:

[0228] In some embodiments, the first feline Fc region variant and / or the second feline Fc region variant further comprise at least one amino acid substitution that increases half-life, including any amino acid substitution disclosed in U.S. Patent Application Publication No. 2022 / 0259282, U.S. Patent Application No. 18 / 046,082, and U.S. Patent No. 11,498,953, each of which is incorporated by reference herein in its entirety.

[0229] In some embodiments, the first feline Fc region variant and the second feline Fc region variant have the following amino acid substitutions: (a) at least one amino acid substitution selected from the group consisting of 286E, 311V, and 428Y; (d) two or more amino acid substitutions selected from the group consisting of 286E, 311V, and 428Y; and (c) 286E, 311V, and 428Y wherein the amino acid positions are based on EU numbering.

[0230] Binding domain: In some embodiments, the bispecific binding agents (e.g., bispecific antibodies and Fc constructs) described herein comprise an antibody hinge region. The hinge region may be located between the antigen or ligand binding domain and the Fc region variant. In some embodiments, the hinge region binds to the C-terminus of the cytokine, growth factor, enzyme, or peptide, and the hinge region binds to the N-terminus of the Fc region variant. Exemplary hinge region sequences for canine antibodies are shown below: IgGA: FNECRCTDTPPCPVPEP (SEQ ID NO: 22), IgGB:PKRENGRVPRPPDCPKCPAPEM (SEQ ID NO: 23), IgGC:AKECECKCNCNNCPCPGCGL (SEQ ID NO: 24), IgGD:PKESTCKCISPCPVPES (SEQ ID NO: 25), and IgGDmut:PKESTCKCIPPCPVPES (SEQ ID NO: 26). Exemplary hinge region sequences for feline antibodies are shown below: IgG1a: KTDHPPGPKPCDCPKCP (SEQ ID NO: 27), IgG1b: KTDHPPGPKPCDCPKCP (SEQ ID NO: 28), and IgG2: KTASTIESKTGEGPKCP (SEQ ID NO: 29).

[0231] When used, the hinge region in a bispecific binding agent of the present disclosure can contain 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, or 6) amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 22-29. In some embodiments, the hinge region used in a recombinant protein of the present disclosure is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 22-29.

[0232] In some embodiments, a linker sequence may be used in place of an antibody hinge sequence to connect one or more polypeptides (e.g., antibodies, single domain antibodies, ligand-binding domains of receptors, enzymes, ligands, peptides) to a companion animal (e.g., canine or feline) Fc region variant disclosed herein. In certain embodiments, the linker is composed of 1 to 20 amino acids linked by peptide bonds, where the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as will be appreciated by those skilled in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is composed predominantly of amino acids that do not sterically hinder, such as glycine and alanine. Examples of peptide linkers include Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO: 30); Ser Gly Gly Gly (SEQ ID NO: 31); Gly Gly Gly Gly Ser (SEQ ID NO: 32); Ser Gly Gly Gly Gly (SEQ ID NO: 33); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 34); Ser Gly Gly Gly Gly Gly (SEQ ID NO: 35); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 36); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 37); Gly Pro Gly Gly Gln (SEQ ID NO: 38); (Gly Gly Gly Gly Ser) n (SEQ ID NO: 32) (wherein n is an integer of 1 or more (e.g., 1, 2, 3, 4, 5)); and (Ser Gly Gly Gly Gly) n (SEQ ID NO: 33) (wherein n is an integer of 1 or more (e.g., 1, 2, 3, 4, 5)).

[0233] Non-peptide linkers may also be used to link one or more polypeptides of interest to the Fc region variants disclosed herein. For example, -NH(CH2)n Alkyl linkers such as C(O)- (where n=2 to 20) can be used. These alkyl linkers may be further substituted with any group that does not cause steric hindrance, such as lower alkyl (e.g., C1 to C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0234] Bispecific binding agents (e.g., bispecific antibodies or Fc constructs) of the present disclosure can comprise a binding domain. The binding domain can specifically bind to a selected target protein, subunit, domain, motif, and / or epitope described herein. In some embodiments, the binding domain comprises a single-domain antibody. In some embodiments, the single-domain antibody is a VHH domain. In some embodiments, the binding domain comprises an antibody, antibody fragment, or ligand-binding portion of a receptor. In some embodiments, the antibody or antibody fragment comprises six complementarity-determining regions (CDRs) of an immunoglobulin molecule. In other embodiments, the antibody fragment is selected from the group consisting of Fab, single-chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab')2, nanobody, and diabody. In other embodiments, the ligand-binding portion of a receptor comprises a ligand-binding domain of a receptor protein or an extracellular domain of a receptor protein. In some embodiments, the one or more polypeptides (e.g., fusion polypeptides) can comprise a protein, wherein the protein is a therapeutic protein described herein. In some embodiments, the target (e.g., as a target of a binding domain) or therapeutic protein (e.g., as a fusion polypeptide) is 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIBALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, IgE, angiotensin type 1 (AT1) receptor , angiotensin type 2 (AT2) receptor, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CC1, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4 , CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, C D5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30 , CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD 74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 , CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-related Antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, G CSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, GLP1, GLP2, glucagon, Glut4, glycoprotein IIb / IIIa (GP IIb / IIIa), GM-CSF, gp130, gp72, GRO, GnRH, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, cardiac myosin, cytomegalovirus (CMV), growth hormone (GH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE , IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL -9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL-1R, IL- 2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-3 1R, IL-33R), interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin Alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis Y antigen, Lewis Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, Mer Taroproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NAV1.7, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, oncostatin M receptor (OSMR), OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PA RP, PBR, PBSF, PCAD, P-cadherin, PCNA, PD1, PDL1, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RA NKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, T fR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-betaR1 (ALK-5), TGF-betaR11, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1Apo-2, DR4), TNFRSF10B(TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3DcR1, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF R1CD120a, p55-60), TNFRSF1B(TNFRII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DCTRAIL R2 TNFRH2), TNFRST23(DCTRAIL R1TNFRH1), TNFRSF25(DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, UPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antiviral The protein is selected from the group consisting of integrins, VLA, VLA-1, VLA-4, VNR, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and hormone and growth factor receptors.

[0235] In some embodiments, the antibody or antibody fragment comprises one or more complementarity determining regions (CDRs) having an amino acid sequence selected from Table 2 below. For example, the antibody or antibody fragment may comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2. For example, the antibody or antibody fragment may comprise all six CDRs of an antibody listed in Table 2 as binding to a particular target. In some embodiments, the antibody or antibody fragment is selected from the group consisting of antibodies and fragments of U.S. Patent Application Publication Nos. US2020 / 0062840, US2022 / 0251209, US2021 / 0040223, US2022 / 0204615, US2022 / 0251230, US2021 / 0163618, US2021 / 0253722, US2022 / 0119513, US2022 / 0106391, or The antibody or antibody fragment may be any antibody or antibody fragment disclosed in U.S. Patent No. US2022 / 0177594, U.S. Patent No. US11,091,556, U.S. Patent No. US11,447,561, U.S. Patent No. US10,040,849, or U.S. Patent No. US9,951,128, and International Patent Application Publication No. WO2020 / 056393, WO2022 / 079138, WO2021 / 123092, or WO2022 / 029447A1. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9]

[0236] In some embodiments, the antibody or antibody fragment comprises one or more complementarity determining regions (CDRs) having an amino acid sequence selected from Table 3 below. For example, the antibody or antibody fragment may comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 3. For example, the antibody or antibody fragment may comprise all six CDRs of an antibody listed in Table 3 as binding to a particular target. In some embodiments, the antibody or antibody fragment may be any antibody or antibody fragment disclosed in U.S. Patent Application Publication Nos. US2020 / 0062840, US2022 / 0119513, US2022 / 0106391, US2022 / 0177594, or US2022 / 0127351, U.S. Patent No. US9,328,164, and International Patent Application Publication No. WO2020 / 056393. [Table 3-1] [Table 3-2] [Table 3-3]

[0237] In some embodiments, the binding domain binds to one or more therapeutic targets or antigens in companion animals (e.g., canines or felines), such as, but not limited to, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (myostatin), GLP1, GLP2, GnRH, growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL -15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL -1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1Specifically binds to bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OSMR, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-betaR1 (ALK-5), TGF-betaR11, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (flt-1), VEGF, VEGFR, and VEGFR-3 (flt-4).

[0238] In some embodiments, the bispecific binding agent can comprise a protein that is a therapeutic protein, such as EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonist, or thrombopoietin-binding peptide. In some embodiments, the therapeutic protein is ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, follicle-stimulating hormone, GDF-8 (myostatin), GLP1, GLP2, GnRH, growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL -15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL -1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OSMR, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-betaR1 (ALK-5), TGF-betaR11, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFR p75NTR), TNFRSF9 (4-1BB CD137, ILA), VEFGR-1 (flt-1), VEGF, VEGFR, or VEGFR-3 (flt-4).

[0239] In some embodiments, the therapeutic protein is any protein described herein. In one embodiment, the bispecific antibody or Fc construct further comprises a modified canine IgG CH2 domain, IgG CH3 domain, or IgG Fc region described herein. In another embodiment, the bispecific antibody or Fc construct further comprises a modified feline IgG CH2 domain, IgG CH3 domain, or IgG Fc region described herein. The modified canine or feline IgG CH2 domain, IgG CH3 domain, or IgG Fc region can improve the half-life of the therapeutic protein in vivo.

[0240] Canine Fc domain Dogs have four types of IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, designated IgGA, IgGB, IgGC, and IgGD. The amino acid and DNA sequences of these heavy chains are available from Tang et al., 2001, Vet. Immunol. Immunopathol., 80:259-270, and the GENBANK database. For example, the amino acid sequence of the IgGA heavy chain has GENBANK accession number AAL35301.1, IgGB has GENBANK accession number AAL35302.1, IgGC has GENBANK accession number AAL35303.1, and IgGD has GENBANK accession number AAL35304.1. Canine antibodies also contain two types of light chains: kappa and lambda. The DNA and amino acid sequences of these light chains can also be obtained from the GENBANK database. For example, the canine kappa light chain amino acid sequence has accession number ABY57289.1, and the canine lambda light chain has accession number ABY55569.1.

[0241] CH2 region of canine Fc domain: The CH2 region of a canine antibody comprises or consists of amino acids 237-340 (according to EU numbering) of a canine IgG antibody. It should be understood that the CH2 region can include 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) additional amino acids or deletions at the N- and / or C-terminus.

[0242] The amino acid sequence of the CH2 region of canine IgGA is shown below: GPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISWFVDGK EVHTAKTQSR EQQFNGTYRV VSVLPIEHQD WLTGKEFKCR VNHIDLPSPI ERTISKAR (SEQ ID NO: 1)

[0243] The amino acid sequence of the CH2 domain of canine IgGB is shown below: GPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKAR (SEQ ID NO: 2)

[0244] The amino acid sequence of the CH2 domain of canine IgGC is shown below: GPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTP (SEQ ID NO: 3)

[0245] The amino acid sequence of the CH2 domain of canine IgGD is shown below: GPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWFVD GKEVHTAKTQ PREQQFNSTY RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR (SEQ ID NO: 4)

[0246] CH3 region of canine Fc domain: The CH3 region of a canine antibody comprises or consists of amino acids 345 to 447 (according to EU numbering) of a canine IgG antibody. It should be understood that the CH3 region can include 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) additional amino acids or deletions at the N- and / or C-terminus.

[0247] The amino acid sequence of the CH3 domain of canine IgGA is shown below: KPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK (SEQ ID NO: 5)

[0248] The amino acid sequence of the CH3 domain of canine IgGB is shown below: QP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK (SEQ ID NO: 6)

[0249] The amino acid sequence of the CH3 domain of canine IgGC is shown below: Q PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK (SEQ ID NO: 7)

[0250] The amino acid sequence of the CH3 domain of canine IgGD is shown below: QPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV EWQSNGQPEP ESKYHTTAPQ LDEDGSYFLY SKLSVDKSRW QQGDTFTCAV MHEALQNHYT DLSLSHSPGK (SEQ ID NO: 8)

[0251] Canine Fc region sequence: The Fc region of a canine IgG antibody comprises or consists of amino acids 231 to 447 (according to EU numbering) of a canine IgG antibody.

[0252] The amino acid sequence of the Fc domain of canine IgGA is shown below: VPEPLGGPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISWFVDGK EVHTAKTQSR EQQFNGTYRV VSVLPIEHQD WLTGKEFKCR VNHIDLPSPI ERTISKARGR AHKPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK (SEQ ID NO: 9)

[0253] The amino acid sequence of the Fc domain of canine IgGB is shown below: APEMLGGPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKARGQAHQP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK (SEQ ID NO: 10)

[0254] The amino acid sequence of the Fc domain of canine IgGC is shown below: GCGLLGGPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTPGQAHQ PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK (SEQ ID NO: 11)

[0255] The amino acid sequence of the Fc domain of canine IgGD is shown below: VPESLGGPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWFVD GKEVHTakTQ PREQQFNSTY RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR GQAHQPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV EWQSNGQPEP ESKYHTTAPQ LDEDGSYFLY SKLSVDKSRW QQGDTFTCAV MHEALQNHYT DLSLSHSPGK (SEQ ID NO: 12)

[0256] Feline Fc region Cats typically have three types of IgG heavy chains, designated IgG1a, IgG1b, and IgG2. These heavy chains represent three distinct subclasses of feline IgG. The amino acid and DNA sequences of these heavy chains are available from Tang et al., 2001, Vet. Immunol. Immunopathol., 80:259-270, and the GENBANK database. For example, the amino acid sequence of a feline IgG1a heavy chain has GENBANK accession number BAA32229.1, the feline IgG1b heavy chain has GENBANK accession number BAA32230.1, and the feline IgG2 heavy chain has GENBANK accession number KF811175.1. Feline antibodies also contain two types of light chains: kappa and lambda. The DNA and amino acid sequences of these light chains can also be obtained from the GENBANK database. For example, the feline kappa light chain amino acid sequence has accession number AF198257.1, and the feline lamda light chain has accession number E07339.1.

[0257] CH2 region of feline Fc domain: The CH2 region of a feline antibody comprises or consists of amino acids 231 to 340 (according to EU numbering) of a feline IgG antibody. It should be understood that the CH2 region can include 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) additional amino acids or deletions at the N- and / or C-terminus.

[0258] The amino acid sequence of the CH2 region of feline IgG1a is shown below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAK (SEQ ID NO: 13)

[0259] The amino acid sequence of the CH2 domain of feline IgG1b is shown below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDK (SEQ ID NO: 14)

[0260] The amino acid sequence of the CH2 domain of feline IgG2 is shown below: VPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAK (SEQ ID NO: 15)

[0261] CH3 region of feline Fc domain: The CH3 region of a feline antibody comprises or consists of amino acids 341 to 447 (according to EU numbering) of a feline IgG antibody. It should be understood that the CH3 region can include 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at the N- and / or C-terminus.

[0262] The amino acid sequence of the CH3 domain of feline IgG1a is shown below: GQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 16)

[0263] The amino acid sequence of the CH3 domain of feline IgG1b is shown below: GQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 17)

[0264] The amino acid sequence of the CH3 domain of feline IgG2 is shown below: GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 18)

[0265] Feline Fc region sequence: The Fc region of a feline IgG antibody comprises or consists of amino acids 231 to 447 (according to EU numbering) of a feline IgG antibody.

[0266] The amino acid sequence of the Fc domain of feline IgG1a is shown below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 19)

[0267] The amino acid sequence of the Fc domain of feline IgG1b is shown below: PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 20)

[0268] The amino acid sequence of the Fc domain of feline IgG2 is shown below: VPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 21)

[0269] Other substitutions that can be included in the bispecific binding agents The development of therapeutic polypeptides or proteins (e.g., bispecific binding agents such as bispecific antibodies or Fc constructs) is a complex process involving the coordination of a complex series of tasks to produce the desired polypeptide or protein. These include optimization of specificity, affinity, functional activity, expression levels in engineered cell lines, long-term stability, elimination or enhancement of effector functions, and development of commercially viable production and purification methods. The present disclosure encompasses substitutions at one or more additional amino acid positions of canine or feline Fc region variants that facilitate any one or more of the above goals.

[0270] In some embodiments, the canine Fc region variants described herein comprise amino acid substitutions at one or more additional amino acid positions that increase or decrease effector function and / or improve product heterogeneity.

[0271] In some embodiments, substitutions are introduced to reduce effector function of the canine Fc region. Such substitutions may be present at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions (numbered according to EU numbering) of a canine IgG: 238, 265, 297, 298, 299, 327, and 329. The substitution(s) may be for any of the other 19 amino acids. In some embodiments, the substitutions are conservative. In certain non-limiting embodiments, the substituted amino acid at position 238 is Ala, the substituted amino acid at position 265 is Ala, the substituted amino acid at position 297 is Ala or Gln, the substituted amino acid at position 298 is Pro, the substituted amino acid at position 299 is Ala, the substituted amino acid at position 327 is Gly, and the substituted amino acid at position 329 is Ala. In some embodiments, the variant Fc region is derived from a canine IgGB or IgGC antibody. In some embodiments, the variant Fc region is derived from a canine IgGB antibody.

[0272] In some embodiments, substitutions are introduced into a wild-type canine IgG Fc region to improve binding to Protein A and facilitate purification by Protein A chromatography. Such substitutions may be present at one or both (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following positions of canine IgG (numbered according to EU numbering): 252 and 254. The substitution(s) may be for any of the other 19 amino acids. In some embodiments, the substitutions are conservative substitutions. In certain non-limiting embodiments, the substituted amino acid at position 252 is Met and the substituted amino acid at position 254 is Ser.

[0273] In some embodiments, the feline Fc region variants described herein comprise amino acid substitutions at one or more additional amino acid positions that increase or decrease effector function and / or improve product heterogeneity.

[0274] In some embodiments, substitutions are introduced to reduce the effector function of the feline Fc region. Such substitutions are well known to those skilled in the art and may occur at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) positions of feline IgG. Illustrative examples of such substitutions include those disclosed in WO2019 / 035010A1. In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution may occur at amino acid position 297 (numbered according to EU numbering) of feline IgG. In some embodiments, the substituted amino acid at position 297 is Ala or Gln.

[0275] In some embodiments, substitutions are introduced into the wild-type feline IgG Fc region to improve binding to Protein A and facilitate purification by Protein A chromatography. Such substitutions may be present at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) positions of the feline IgG. Illustrative examples of such substitutions include those disclosed in WO2019 / 035010A1.

[0276] In some embodiments, substitutions are made to alter the binding affinity of the canine or feline Fc region variants described herein to FcRn (e.g., increase or decrease binding affinity to FcRn) compared to a wild-type canine or feline Fc. In some variations, the modifications may be one, two, three, or four modifications selected from the group consisting of 308F, 428L, 434M, and 434S, where numbering is according to EU numbering. In some embodiments, the Fc variants comprise one or more modifications selected from the group consisting of 252Y / 428L, 428L / 434H, 428L / 434F, 428L / 434Y, 428L / 434A, 428L / 434M, and 428L / 434S, where numbering is according to EU numbering. In some embodiments, the Fc variant comprises one or more modifications selected from the group consisting of 428L / 434S, 308F / 428L / 434S, where numbering is according to EU numbering. In some embodiments, the Fc variant comprises one or more modifications selected from the group consisting of 259I / 434S, 308F / 434S, 308F / 428L / 434S, 259I / 308F / 434S, 307Q / 308F / 434S, 250I / 308F / 434S, and 308F / 319L / 434S, where numbering is according to EU numbering. A detailed description of these modifications is described, for example, in US8883973B2, which is incorporated herein by reference in its entirety.

[0277] In some embodiments, the bispecific binding agent comprises a canine or feline antibody hinge region. In some embodiments, modifications can be made to the canine or feline antibody hinge region to increase half-life. In some embodiments, the modification is 228P according to EU numbering.

[0278] In some embodiments, binding to FcRn is pH-dependent. H310 and H435 (EU numbering) may be important for pH-dependent binding. Thus, in some embodiments, the amino acid at position 310 (EU numbering) is histidine. In some embodiments, the amino acid at position 435 (EU numbering) is histidine. In some embodiments, the amino acids at both positions are histidine.

[0279] In some embodiments, the Fc region has a MALA mutation (M234A and L235A mutations according to EU numbering) or a MALA-PG mutation (M234A, L235A, P329G mutations according to EU numbering). In some embodiments, the Fc region has a P234A, M234A, S234A, or I234A mutation. In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residues at positions 234 and 235 (EU numbering) are Ala.

[0280] Pharmaceutical Composition In one aspect, the invention features a pharmaceutical composition including (i) any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein; and (ii) a pharmaceutically acceptable carrier.

[0281] To prepare pharmaceutical or sterile compositions of the bispecific antibodies or Fc constructs described herein, the bispecific antibodies or Fc constructs can be combined with a pharmaceutically acceptable carrier or excipient (see, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984)).

[0282] Formulations of therapeutic and diagnostic agents can be prepared, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension by mixing with an acceptable carrier, excipient, or stabilizer (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: (See Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, the bispecific antibody or Fc construct of the invention is diluted to an appropriate concentration in sodium acetate solution at pH 5-6, with NaCl or sucrose added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to improve stability.

[0283] The toxicity and therapeutic efficacy of the composition administered alone or in combination with another agent can be determined, for example, by the LD 50 (lethal dose for 50% of the population) and ED 50The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is known as the therapeutic index (LD 50 / ED 50 In certain embodiments, bispecific antibodies or Fc constructs that exhibit high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used to formulate a range of dosages for use in canines or felines. The dosage of such compounds is preferably within the ED400 with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form and route of administration used.

[0284] Any suitable mode of administration can be used. Exemplary suitable routes of administration include oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial. In some embodiments, the bispecific antibody or Fc construct may be administered by an invasive route, for example, by injection. In further embodiments, the bispecific antibody or Fc construct is administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation or aerosol delivery.

[0285] The pharmaceutical compositions disclosed herein can also be administered by infusion. Examples of well-known implantable device and module forms for administering pharmaceutical compositions include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments. Many other such implantable devices, delivery systems, and modules are known to those skilled in the art.

[0286] Alternatively, bispecific antibodies or Fc constructs may be administered locally rather than systemically, for example, by directly injecting the antibody into an arthritic joint or a pathogen-induced lesion characterized by an immunopathology, often in a depot or sustained-release formulation. Furthermore, bispecific antibodies or Fc constructs may be administered in a targeted drug delivery system, such as liposomes coated with tissue-specific antibodies that target, for example, arthritic joints or pathogen-induced lesions characterized by an immunopathology. The liposomes are targeted to and selectively taken up by the affected tissue.

[0287] The dosing regimen will depend on several factors, including, but not limited to, the age, weight, and health of the canine or feline being treated, the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic bispecific antibody or Fc construct, and the accessibility of target cells in the biological matrix. Preferably, the dosing regimen will deliver enough therapeutic bispecific antibody or Fc construct to result in improvement of the target disease state while simultaneously minimizing undesirable side effects. Thus, the amount of biologic delivered will depend, in part, on the particular therapeutic bispecific antibody or Fc construct and the severity of the condition being treated. Guidance regarding the selection of appropriate doses of therapeutic antibodies is available (see, e.g., Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)).

[0288] The determination of the appropriate dose of a bispecific antibody or Fc construct can be performed by those skilled in the art, for example, using parameters or factors known or believed to affect treatment in the art. Generally, the dose is started at a dose slightly lower than the optimal dose and then increased by small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include, for example, symptoms of inflammation or measures of the level of inflammatory cytokines produced.

[0289] Nucleic acids, vectors, host cells, and methods of production The present disclosure also encompasses one or more nucleic acids encoding the bispecific binding agents (e.g., bispecific antibodies or Fc constructs) described herein, one or more vectors comprising said one or more nucleic acids, and host cells comprising said one or more nucleic acids or said one or more vectors.

[0290] In one aspect, the invention features one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein.

[0291] In another aspect, the invention features one or more expression vectors including one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein.

[0292] In another aspect, the invention features a host cell containing one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein, or one or more expression vectors containing one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein.

[0293] In another aspect, the present invention provides a method of making a bispecific antibody or Fc construct, comprising the steps of: (a) providing one or more nucleic acids encoding any one of the bispecific antibodies disclosed herein or any one of the Fc constructs disclosed herein; (b) expressing the one or more nucleic acids in a host cell culture, thereby producing a bispecific antibody or Fc construct; and optionally (c) recovering the bispecific antibody or Fc construct produced in (ii) from the host cell culture; The present invention is characterized by a method comprising:

[0294] In some embodiments, the host cell culture comprises (i) one host cell population expressing both a first companion animal Fc region variant and a second companion animal Fc region variant, or (ii) two host cell populations comprising a first population expressing a first companion animal Fc region variant and a second population expressing a second companion animal Fc region variant.

[0295] The bispecific antibodies or Fc constructs described herein can be produced in bacterial cells or eukaryotic cells. Some polypeptide components of the bispecific antibodies or Fc constructs, such as Fab, can be produced in bacterial cells, such as E. coli cells. Polypeptides can also be produced in eukaryotic cells, such as transformed cell lines (e.g., CHO, 293E, COS, 293T, Hela). In addition, polypeptides (e.g., scFv) can be expressed in yeast cells, such as Pichia (see, e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces. To produce the desired bispecific antibodies or Fc constructs, one or more polynucleotides encoding the bispecific antibodies or Fc constructs are constructed, introduced into one or more expression vectors, and then expressed in a suitable host cell. To improve expression, the nucleotide sequence of the gene can be recoded without changing the amino acid sequence (or with minimal changes, such as removal of the C-terminal residue of the heavy or light chain). Regions that can be recoded include regions associated with translation initiation, codon usage, and potential unintended mRNA splicing. Polynucleotides encoding the Fc region variants described herein can be readily envisioned by those skilled in the art.

[0296] Standard molecular biology techniques can be used to prepare the recombinant expression vector(s), transfect the host cells, select for transformants, culture the host cells, and recover the polypeptide (e.g., bispecific antibody or Fc construct).

[0297] When a bispecific binding agent (e.g., a bispecific antibody or an Fc construct) is expressed in a bacterial cell (e.g., E. coli), the expression vector may have features that allow for amplification of the vector in the bacterial cell. Additionally, when E. coli such as JM109, DH5α, HB101, or XL1-Blue is used as a host, the vector may have a promoter, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter, which can enable efficient expression in E. coli. Examples of such vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21, which expresses T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. When produced in the periplasm of E. coli, the pelB signal sequence (Lei et al., 2004) is used. al., J. Bacteriol., 169:4379 (1987)) may be used as a signal sequence for antibody secretion. For bacterial expression, the calcium chloride method or electroporation may be used to introduce the expression vector into bacterial cells.

[0298] When a bispecific binding agent (e.g., a bispecific antibody or Fc construct) is expressed in animal cells, such as CHO, COS, and NIH3T3 cells, the expression vector may comprise a promoter for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)) (e.g., the early simian virus 40 promoter), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter (e.g., the human cytomegalovirus immediate-early promoter). In addition to the nucleic acid sequence encoding the Fc region variant, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Examples of vectors having a selectable marker include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0299] In some embodiments, bispecific antibodies or Fc constructs are produced in mammalian cells. Exemplary mammalian host cells for expressing one or more polypeptides (e.g., bispecific antibodies or Fc constructs) include Chinese hamster ovary (CHO) cells (e.g., dhfr-CHO cells described in Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA, 77:4216-4220, used with the DHFR selection marker described in Kaufman and Sharp (1982), Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphoid cell lines such as NS0 myeloma cells and SP2 cells, and cells derived from transgenic animals, e.g., transgenic mammals. For example, the cells are mammary epithelial cells.

[0300] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy and light chains of an antibody is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to enhancer / promoter regulatory elements (e.g., those derived from SV40, CMV, adenovirus, etc., e.g., the CMV enhancer / AdMLP promoter regulatory element or the SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of gene transcription. The recombinant expression vector also contains a DHFR gene, which allows CHO cells transfected with the vector to be selected using methotrexate selection / amplification. The selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0301] Treatment method The bispecific binding agents (e.g., bispecific antibodies or Fc constructs) disclosed herein can be used to treat or prevent any disease or disorder in companion animals (e.g., dogs or cats) in need thereof.

[0302] In one aspect, the invention features a method of treating or preventing a disease or disorder in a companion animal in need thereof, comprising administering to the companion animal an effective amount of a composition comprising any one of the bispecific antibodies disclosed herein, any one of the Fc constructs disclosed herein, or a pharmaceutical composition comprising same.

[0303] In one aspect, the invention features a method of treating or preventing a canine disease or disorder in a dog in need thereof, comprising administering to the dog an effective amount of a composition comprising any one of the bispecific antibodies comprising a canine Fc region variant disclosed herein, any one of the Fc constructs comprising a canine Fc region variant disclosed herein, or a pharmaceutical composition comprising same.

[0304] In some embodiments, the canine disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer. In some embodiments, the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0305] In another aspect, the invention features any one of the bispecific antibodies comprising a canine Fc region variant disclosed herein, any one of the Fc constructs comprising a canine Fc region variant disclosed herein, or a pharmaceutical composition comprising same, for use in treating or preventing a canine disease or disorder in a dog in need thereof.

[0306] In some embodiments, the canine disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer. In some embodiments, the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0307] In another aspect, the invention features a method of treating or preventing a feline disease or disorder in a cat in need thereof, comprising administering to the cat an effective amount of a composition comprising any one of the bispecific antibodies comprising a feline Fc region variant disclosed herein, any one of the Fc constructs comprising a feline Fc region variant disclosed herein, or a pharmaceutical composition comprising same.

[0308] In some embodiments, the feline disease or disorder is an allergic disease, chronic pain, acute pain, an inflammatory disease, an autoimmune disease, an endocrine disease, a gastrointestinal disease, a cardiovascular disease, a renal disease, a reproductive-related disorder, an infectious disease, or cancer. In some embodiments, the feline disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0309] In another aspect, the invention features any one of the bispecific antibodies comprising a feline Fc region variant disclosed herein, any one of the Fc constructs comprising a feline Fc region variant disclosed herein, or a pharmaceutical composition comprising same, for use in treating or preventing a feline disease or disorder in a cat in need thereof.

[0310] In some embodiments, the feline disease or disorder is an allergic disease, chronic pain, acute pain, an inflammatory disease, an autoimmune disease, an endocrine disease, a gastrointestinal disease, a cardiovascular disease, a renal disease, a reproductive-related disorder, an infectious disease, or cancer. In some embodiments, the feline disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0311] Any suitable companion animal (e.g., canine or feline) disease or disorder can be treated. In some embodiments, the canine or feline disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disorder, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer.

[0312] In other embodiments, the canine or feline disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

[0313] In some embodiments, the disease, disorder, condition, or symptom being treated or prevented is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, skeletal / musculoskeletal disease, cardiovascular disease, neurological disease, renal disease, metabolic disease, immunological disease, genetic / hereditary disease, reproductive function-related disorder, infectious disease, or cancer. In certain embodiments, the disease or disorder being treated or prevented is atopic dermatitis, allergic dermatitis, food allergy, osteoarthritis pain, perioperative pain, dental pain, cancer pain, arthritis, anemia, obesity, or diabetes.

[0314] Antibodies can be used not only to treat or prevent disease, but also to regulate normal biological functions, for example, to control fertility or behavior.

[0315] In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered parenterally by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarterially, intrasynovially, intrathecally, or by inhalation.

[0316] In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered in an amount ranging from 0.01 mg / kg to 50 mg / kg body weight per dose. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered at 0.01-55 mg / kg, 0.01-50 mg / kg, 0.01-45 mg / kg, 0.01-40 mg / kg, 0.01-35 mg / kg, 0.01-30 mg / kg, 0.01-25 mg / kg, 0.01-20 mg / kg, 0.01-15 mg / kg, 0.01-10 mg / kg, 0.01-5 mg / kg, or 0.01-1 mg / kg, e.g., daily, weekly, monthly, every two months, every three months, every four months, every five months, or every six months. An exemplary dosage of an antibody in canines can be in the range of 0.01 mg / kg to 15 mg / kg. Thus, one or more doses of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 10 mg / kg, or 15 mg / kg (or any combination thereof) may be administered to the animal. One exemplary dosage of an antibody in a feline may be in the range of 0.01 mg / kg to 10 mg / kg. Thus, one or more doses of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the animal. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered at 2 mg / kg body weight per dose.

[0317] In some embodiments, a bispecific binding agent (e.g., a bispecific antibody or Fc construct) disclosed herein, or a pharmaceutical composition comprising a bispecific binding agent (e.g., a bispecific antibody or Fc construct) disclosed herein, is administered within 1, 2, 3, 4, 5, or 6 months, or within 1, 2, or 3 weeks of each other. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered weekly. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered every two weeks. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered every three weeks. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered monthly. In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered every two months. In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered every three months. In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered every four months. In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered every five months. In some embodiments, the bispecific antibodies or Fc constructs disclosed herein, or pharmaceutical compositions comprising the bispecific antibodies or Fc constructs disclosed herein, are administered every six months.In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered to a dog or cat at one time or over a series of treatments. In some embodiments, a dose is administered once a week for at least two or three consecutive weeks, and in some embodiments, this treatment cycle is repeated two or more times, optionally separated by a treatment-free period of one or more weeks.

[0318] In some embodiments, a bispecific binding agent (e.g., a bispecific antibody or Fc construct) disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered in combination, parallel, sequentially, or concomitantly with one or more additional therapeutic agents. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered in combination with one or more additional therapeutic agents. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered in parallel with one or more additional therapeutic agents. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered sequentially with one or more additional therapeutic agents. In some embodiments, a bispecific antibody or Fc construct disclosed herein, or a pharmaceutical composition comprising a bispecific antibody or Fc construct disclosed herein, is administered in combination with one or more additional therapeutic agents. Any suitable additional therapeutic agent may be used.

[0319] diagnosis The bispecific binding agents (e.g., bispecific antibodies or Fc constructs) disclosed herein can also be used for various diagnostic purposes, for example, to determine whether a dog or cat has a particular disease or disorder. In some embodiments, the bispecific antibodies or Fc constructs can comprise a binding domain. The binding domain can specifically bind to a protein, subunit, domain, motif, and / or epitope (e.g., a marker of a cancer cell) described herein. In some embodiments, the bispecific antibodies or Fc constructs further comprise a group for labeling. Generally, labeling groups are divided into various classes depending on the assay in which they are detected: a) isotopic labels, which can be radioisotopes or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox-active moieties; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylation groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags, etc.). In some embodiments, the labeling group is attached to the antibody via a spacer arm of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used in the practice of the present invention.

[0320] In some embodiments, the labeling group is a probe, a dye (e.g., a fluorescent dye), or a radioisotope (e.g., 3 H, 14 C. 22 Na, 36 Cl, 35 S, 33 P, or 125 I).

[0321] Specific labels can also include optical dyes, such as, but not limited to, chromophores, fluorophores, and fluorophores, the latter being specific in many embodiments. Fluorophores can be either "small molecule" fluores or proteinaceous fluores.

[0322] The fluorescent label can be any molecule that can be detected through its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Suitable optical dyes, including fluorophores, include Molecular Probes Handbook by Richard P. Haugland, which is incorporated by reference in its entirety.

[0323] Suitable proteinaceous fluorescent labels include, but are not limited to, green fluorescent proteins including GFPs of Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent proteins (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), improved yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al. al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA. 85:2603-2607) and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / 49019, U.S. Patent Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, 5,925,558). All of the references cited above in this paragraph are expressly incorporated herein by reference in their entirety.

[0324] Assay Fc γ RI and FcγRIII binding: Binding to FcγRI and FcγRIII is a measure of the ability of an antibody to mediate ADCC. To assess this property of an antibody, assays that measure antibody binding to FcγRI and FcγRIII can be performed using methods known in the art.

[0325] C1q binding: Binding to C1q, the first component of complement, is a measure of the ability of an antibody to mediate complement-dependent cytotoxicity (CDC). To assess this property of an antibody, an assay that measures the binding of the antibody to C1q can be performed using methods known in the art.

[0326] Half-life: Methods for measuring the half-life of antibodies are well known in the art. See, for example, Booth et al., MAbs, 10(7):1098-1110 (2018). As an example, the half-life of an antibody (e.g., a feline antibody) can be measured by injecting the antibody into an animal model (e.g., a canine model) and measuring the level of the antibody in the serum over a period of time. Exemplary animal models include non-human primate models and transgenic mouse models. The transgenic mouse model is deficient in the mouse FcRn alpha chain and can express a canine FcRn alpha transgene (e.g., under the control of a constitutive promoter). The canine FcRn alpha chain can pair with the mouse β2-microglobulin protein in vivo to form a functional chimeric FcRn heterodimer. As an example, the half-life of a canine antibody can be measured by injecting the antibody into a canine model and measuring the level of the antibody in the serum over a period of time. [Example]

[0327] Example 1. Evaluation of Knob-in-Hole (KiH) Mutations in Canine IgGB Constant Domains In a first attempt to evaluate whether knob-in-hole mutations could be used to create canine IgG heterodimers, we introduced the following mutations into canine IgGB: T366W and S354C in the "knob" region. The following mutations were introduced into the "hole" region: T366S, L368A, Y407V, and Y349C (as described in the human Fc region, e.g., U.S. Pat. No. 5,731,168 and Schaefer et al., 2011, Proc. Natl. Acad. Sci. USA, 108:11187-11192). These mutations were in a constant domain that already contained half-life-extending mutations (A426Y + T286L, as described in U.S. Pat. No. 11,434,276, incorporated herein by reference in its entirety) and MALA mutations (M234A and L235A) that reduce effector function. For expression purposes, the Protein A binding site was knocked out of the "hole" strand by introducing the mutations H435R and Y436F to allow preferential purification of the heterodimer (described in U.S. Patent No. 8,586,713, which is incorporated herein by reference in its entirety). Additionally, the C-terminal lysine was removed from the sequence (see Table 4). [Table 4]

[0328] Evaluation of canine knob-in-hole mutations was performed using two test target variable regions and light chains. Both antibodies were evaluated for both knob and hole mutations. Bispecific antibodies (biAbs) were expressed in Chinese hamster ovary (CHO) cells at a 1:1:1:1 ratio and purified using Protein A resin. The resulting biAbs were analyzed for binding, aggregation, purity, and correct chain pairing.

[0329] Concentrations were measured using A280 in a NANODROP™ OneC instrument (Thermo Fisher). Transient yields were within the standard range for in-house canine antibody expression.

[0330] Size exclusion was performed on a Waters ALLIANCE™ e2796 Bioseparations Module using a Sepax ZENIX™ SEC-150 (7.8 x 200 mm, 3 μm) column to assess the presence and proportion of high molecular weight species (HMWS) and low molecular weight species (LMWS). The mobile phase was 20 mM sodium phosphate, 0.3 M NaCl, pH 6.8, with an isocratic flow rate of 1.0 mL / min for 20 min. Both knob-in-hole antibody configurations contained no HMWS and had a main peak with a purity of 92–93%. As shown in the chromatogram in Figure 1, 7–8% LMWS was present as a shoulder of the main peak.

[0331] Strand pairing was assessed by intact mass using liquid chromatography-mass spectrometry (LC-MS).

[0332] Samples were deglycosylated with PNGase F and run on a PLRP-S (2.1 x 50 mm, 8 μm) column in either reduced or non-reduced conditions on an Agilent 1290 Infinity II uPLC, followed by analysis on an Agilent 6530 electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-QToF). Measured mass assignments were determined using BioConfirm 10.0 software and are summarized in Table 5, and deconvoluted subunit mass spectra are shown in Figure 2. [Table 5]

[0333] The amino acid sequence of each chain was confirmed by both non-reduced and reduced mass spectrometry. Non-reduced intact mass analysis confirmed the assembly of the knobs-in-hole construct. However, the major species observed in both configurations was HC1 + HC2 + 2 × LC1 + 16 × disulfide bonds, rather than the expected HC1 + HC2 + LC1 + LC2 + 16 × disulfide bonds. The percentage of deconvoluted peaks was 30% for LC1 + LC2, compared with 70% for 2 × LC1. As expected, light chain mispairing was not prevented by the current mutations, but knobs-in-holes are present in both species (schematic representation of confirmed species in Figure 3).

[0334] The affinity of the bispecific antibodies for different targets was evaluated by surface plasmon resonance (SPR) on a BIACORE™ T200. Antibodies were captured using a Protein A Series S chip. Antigen binding was then evaluated at multiple concentrations starting from 50 nM (target 1) or 100 nM (target 2) using PBSP + running buffer (Cytiva) at a flow rate of 30 μL / min. The association time was 120 seconds, and the dissociation time was run for 600 seconds. The chip surface was regenerated with 10 mM glycine. Reference-subtracted sensorgrams were fitted to a 1:1 binding model using the BIACORE™ T200 evaluation software. The data are shown in Table 6 below, and the sensorgrams are shown in Figure 4. Affinity for target 2 was retained in both configurations. However, there was a significant decrease in binding to target 1 with the 006 knob + 225 hole configuration. [Table 6]

[0335] Example 2. Evaluation of VHH-Fc with knob-in-hole mutations in the canine IgGB constant domain To continue the knobs-in-holes evaluation using canine IgG, we tested VHH constructs instead of variable regions and light chains. This allowed us to create bispecific antibodies without concerns about light chain mispairing. To generate VHHs, we immunized llamas with either target 2 or target 3. Peripheral blood mononuclear cells (PBMCs) from llamas with high titers against the immunizing antigen were isolated, and RNA was isolated. cDNA was generated from the RNA, and a phage display library containing VHH domains was generated. The VHH phage display library was then subjected to selection and screening using either canine target 2 or target 3. ELISA-positive VHHs were sequenced, and unique VHHs with a C-terminal 8xHis tag were synthesized. A subset of VHHs was expressed in E. coli and purified by nickel chromatography. The purified VHHs were then screened for affinity, and the top binders were used in the following examples. Target 2 VHH, 02F09R3, was formatted with an intact hinge (PKRENGRVPRPPDCPKCP, SEQ ID NO: 363; or VPKRENGRVPRPPDCPKCP, SEQ ID NO: 364) followed by the CH2 and CH3 of canine IgGB (sequences in Table 7). In some cases, the construct contained a linker (GPGGQ, SEQ ID NO: 38) between the VHH and hinge, and in other cases no linker was added. Both were expressed without knobs-in-holes mutations for evaluation of the VHH-Fc as a standalone molecule. [Table 7]

[0336] In a separate construct, VHH 02F09R3-Fc, the constant canine IgGB sequence was modified in the "knob" side with the following mutations: T366W and S354C. For the "hole" side mutations, target 3 VHH 01E03R3 was selected and the following mutations were introduced: T366S, L368A, Y407V, and Y349C. The mutations were in the constant domain, which already contained half-life-extending mutations (A426Y + T286L) and MALA mutations (M234A and L235A) that reduce effector function. For expression purposes, the Protein A binding site was knocked out of the "hole" chain by introducing the mutations H435R and Y436F, allowing preferential purification of the heterodimer. Additionally, the C-terminal lysine was removed from the sequence.

[0337] VHH-Fc was expressed in CHO cells and purified using Protein A resin. The resulting VHH-Fc was analyzed for binding, aggregation, purity, and correct chain pairing.

[0338] Concentrations were measured using A280 in a NANODROP™ OneC instrument (Thermo Fisher). Transient yields of VHH-Fc (with or without linkers) were higher than those observed with VHH knob-in-hole constructs, the latter within the standard range for in-house canine antibody expression.

[0339] Size exclusion was performed on a Waters ALLIANCE™ e2796 column using a YMC-Pack-Diol-200 (300 x 8 mm IDS-5 μm, 20 nm) or Sepax ZENIX™ SEC-150 (7.8 x 200 mm, 3 μm) column to assess the presence and proportion of high-molecular-weight (HMWS) and low-molecular-weight (LMWS). The mobile phase was 20 mM sodium phosphate, 0.3 M NaCl, pH 6.8, with an isocratic flow rate of 1.0 mL / min for 20 min. The column retention times were significantly different. Both knob-in-hole antibody configurations contained no HMWS and had a main peak with a purity of 92–93%. As shown in the chromatogram in Figure 1, 7–8% LMWS was present as a shoulder of the main peak. One of the VHH-Fc constructs contained a small amount of HMWS (0.2%), and all constructs had a main peak with purity ranging from 92 to 99%. As shown in the chromatograms in Figure 5, 0.6 to 7.2% LMWS was present as a shoulder of the main peak.

[0340] Chain pairing was assessed by intact mass analysis using liquid chromatography-mass spectrometry as described above. Measured mass assignments were determined using BioConfirm 10.0 software and are summarized in Table 8, and deconvoluted subunit mass spectra are shown in Figure 6. The amino acid sequence of each chain was confirmed by both non-reduced and reduced mass analysis. Non-reduced intact mass analysis confirmed the assembly of the VHH-Fc and VHH knob-in-hole constructs (schematic representation of confirmed species in Figure 7). [Table 8]

[0341] Example 3. Evaluation of mAb / VHH-Fc Knob-in-Hole This monovalent bispecific antibody format consists of one side as a half antibody and the other side as a VHH-Fc. One side contains the knob mutation while the other side maintains the hole mutation, thus combining the constructs of Example 1 and Example 2 (sequences in Table 4 and Table 7). Light chain mispairing is not an issue because pairing can only occur on the half antibody side. Two configurations were evaluated: (1) 006-knob and 01E03R3-hole, and (2) 02F09R3-knob and 006-hole.

[0342] Bispecific mAb / VHHs were expressed in CHO cells at a 1:1:1 ratio (HC1:HC2:LC1) and purified using Protein A resin. The resulting biAbs were analyzed for binding, aggregation, purity, and correct chain pairing.

[0343] Concentrations were measured using A280 in a NANODROP™ OneC instrument (Thermo Fisher). Transient yields were within the standard range for in-house canine antibody expression.

[0344] Size exclusion was performed as described above using Sepax ZENIX™ SEC-150. Both knob-in-hole antibody configurations contained small amounts of HMWS (1-2.5%) and had a main peak of 89-91% purity. As shown in the chromatograms in Figure 8, 7-8% LMWS was present as a shoulder to the main peak. As expected, the retention times were longer than standard IgG but shorter than the VHH-Fc constructs, and the molecular weights were between the two.

[0345] Strand pairing was assessed by intact mass analysis using liquid chromatography-mass spectrometry as described above. Measured mass assignments were determined using BioConfirm 10.0 software and are summarized in Table 9, and deconvoluted subunit mass spectra are shown in Figure 9. The amino acid sequence of each strand was confirmed by both non-reduced and reduced mass analysis. Non-reduced intact mass analysis confirmed the assembly of the knobs-in-hole construct (schematic representation of confirmed species in Figure 10). [Table 9]

[0346] The affinity of the bispecifics for Target 1 and Target 2 was assessed by SPR on a BIACORE™ T200. Antibodies were captured using an anti-canine-conjugated CM5 chip. Antigen binding was then assessed as described above. Reference-subtracted sensorgrams were fitted to a 1:1 binding model using the BIACORE™ T200 evaluation software. The data are shown in Table 10 below, and the sensorgrams are shown in Figure 11. Affinity was maintained for both targets. [Table 10]

[0347] Example 4. Evaluation of fully monoclonal canine IgGB antibodies with a C-terminal linker followed by a VHH This bivalent, bispecific antibody format consists of a monoclonal antibody with a C-terminal linker followed by a VHH. Because there is only one heavy chain construct, no knobs-in-holes sequence was required. The construct contains only one light chain against target 1, so mismatching is not an issue. The sequence of this construct is in Table 11. [Table 11]

[0348] IgG / VHH constructs were expressed in CHO cells at a 1:1 ratio (HC:LC) and purified using Protein A resin. The resulting biAbs were analyzed for binding, aggregation, purity, and correct chain pairing.

[0349] Concentrations were measured using A280 in a NANODROP™ OneC instrument (Thermo Fisher). Transient yields were within the standard range for in-house canine antibody expression.

[0350] Size exclusion was performed as described above using Sepax ZENIX™ SEC-150. Both knob-in-hole antibody configurations contained no HMWS and had a main peak of 94% purity. As shown in the chromatogram in Figure 12, 6% LMWS was present as a shoulder on the main peak. As expected, due to the larger size of this construct, the retention time was shorter than that of standard IgG.

[0351] Strand pairing was assessed by intact mass analysis using liquid chromatography-mass spectrometry as described above. Measured mass assignments were determined using BioConfirm 10.0 software and are summarized in Table 12, and deconvoluted subunit mass spectra are shown in Figure 13. The amino acid sequence of each strand was confirmed by both non-reduced and reduced mass analysis. Non-reduced intact mass analysis confirmed the correct assembly of the construct (schematic representation of confirmed species in Figure 14). [Table 12]

[0352] The affinity of the bispecific antibodies to different targets was evaluated by SPR in a BIACORE™ T200. The antibodies were captured using an anti-canine-conjugated CM5 chip, and antigen binding was evaluated as described above. The reference-subtracted sensorgrams were fitted to a 1:1 binding model using the BIACORE™ T200 evaluation software. The data are shown in Table 13 below, and the sensorgrams are shown in Figure 15. Affinity was maintained for both targets. [Table 13]

[0353] Example 5. Evaluation of knob-in-hole mutations in feline IgG1a constant domains To evaluate whether knob-in-hole mutations could be used to create heterodimers of feline IgG, feline IgG1a was mutated at the "knob" side with T366W. At the "hole" side, mutations T366S, L368A, and Y407V (described as human Fc regions in U.S. Patent No. 5,731,168, incorporated herein by reference in its entirety) were made. For some sequences, cysteine ​​mutations were not added because A354 is not conserved compared to its human counterpart, S354 (Y349 is conserved in the feline sequence). However, these were tested in other instances to determine whether the nonconserved A354 could be mutated to a cysteine.

[0354] These mutations were in the constant domain, which already contained half-life-extending mutations (T286E+Q311V+S428Y; described in U.S. Patent Application Publication No. US2022 / 0259282, incorporated herein by reference in its entirety) and MALA mutations (M234A and L235A) that reduce effector function. For expression purposes, the Protein A binding site was knocked out of the "hole" strand by introducing the mutations H435R and Y436F, allowing for preferential purification of the heterodimer (described in U.S. Pat. No. 8,586,713, incorporated herein by reference in its entirety). Additionally, the C-terminal lysine was removed from the sequence (see Table 14). [Table 14]

[0355] Evaluation of feline IgGs with knob-into-hole mutations was performed using two test target variable regions and light chains. Both antibodies were evaluated with both knob and hole mutations. Bispecific antibodies were expressed in CHO cells at a 1:1:1:1 ratio and purified using Protein A resin. The resulting biAbs were analyzed for binding, aggregation, purity, and correct chain pairing.

[0356] Concentrations were measured using A280 in a NANODROP™ OneC instrument (Thermo Fisher). Transient yields were within the standard range for in-house feline antibody expression.

[0357] Size exclusion was performed on a Waters Alliance e2796 column using a YMC-Pack-Diol-200 (300 x 8 mm ID, S-5 μm, 20 nm) to assess the presence and proportion of high-molecular-weight species (HMWS) and low-molecular-weight species (LMWS). The mobile phase was 20 mM sodium phosphate, 0.3 M NaCl, pH 6.8, with an isocratic flow rate of 1.0 mL / min for 18 min. All constructs contained two major species: one with a retention time of 6.21 min ranging from 42 to 48% of the peak area, and the other, equivalent to the monoclonal mAb, with a retention time of 7.4 min ranging from 50 to 57% of the peak area. As shown in Figure 16, no LMWS were observed in the chromatograms.

[0358] Strand pairing was assessed by intact mass analysis using liquid chromatography mass spectrometry.

[0359] Samples were either non-deglycosylated or deglycosylated with PNGase F, and run on a PLRP-S (2.1 x 50 mm, 8 μm) column on an Agilent 1290 Infinity II uPLC, either reduced or non-reduced, followed by analysis on an Agilent 6530 ESI-QToF. Measured mass assignments were determined using BioConfirm 10.0 software and are summarized in Table 15. [Table 15-1] [Table 15-2]

[0360] The amino acid sequence of each chain was confirmed by reduced intact mass analysis. However, only one light chain appeared to be the major species. The percentage of deconvoluted peaks was higher in LC1 (approximately 74–92%) compared to LC2 (approximately 8–26%). Non-reduced intact mass analysis confirmed the assembly of the knobs-in-hole construct. However, a knob-knob species was observed in the 076 knob + 023 hole construct. The hole-in-hole construct could not be purified due to the Protein A knockout for that chain. Additionally, the major species observed in the construct without the additional cysteine ​​was "HC1 + HC2 + 2 × LC1." With the additional cysteine, the major construct was "HC1 + HC2 + 2 × LC1 + 4 × cysteinylation" (Figure 17). The presence of this cysteinylation suggests that the disulfide bond was not properly formed and that the strategy of introducing a stability-increasing cysteine ​​at the nonconserved A354 position was not as effective as expected. As expected, light chain mispairing was not prevented by the current mutations, but knob-in-hole is the predominant species in all constructs.

[0361] Finally, the affinities of the bispecific antibody constructs for different targets were evaluated by surface plasmon resonance (SPR) on a BIACORE™ T200. Antibodies were captured using an anti-feline-conjugated CM5 chip. Antigen binding was then evaluated at multiple concentrations starting from 50 nM using PBSP + running buffer (Cytiva) at a flow rate of 30 μL / min. The association time was 120 s, and the dissociation time was 600 s. The chip surface was regenerated with 10 mM glycine. Reference-subtracted sensorgrams were fitted to a 1:1 binding model using the BIACORE™ T200 evaluation software. The data are shown in Table 16 below. Affinity for Target 1 was retained in both configurations. However, there was a significant decrease in binding to Target 2 in all constructs. This can be explained by the absence of the Target 2 light chain in the constructs, as observed in the intact mass data. The knob-in-hole mutations introduced did not completely prevent light chain mispairing. [Table 16]

Claims

1. A bispecific antibody, (a) a first binding domain that binds to a first antigen, said first binding domain being linked to a first companion animal Fc region variant; (b) a second binding domain that binds to a second antigen, the second binding domain being linked to a second companion animal Fc region variant; wherein at least one of the first binding domain and the second binding domain comprises a single domain antibody.

2. 2. The bispecific antibody of claim 1, wherein the first binding domain and the second binding domain each specifically bind to an antigen independently selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-31R, IL-33, CD3, CD20, CD47, CD52, and complexes of the complement system.

3. 3. The bispecific antibody of claim 1 or 2, wherein the first binding domain and / or the second binding domain comprises a ligand-binding portion of an antibody, an antibody fragment, or a receptor.

4. The antibody fragment may be Fab, single chain variable fragment (scFv), Fv, Fab', Fab'-SH, F(ab') ２ 4. The bispecific antibody of claim 3, wherein the antibody is selected from the group consisting of:

5. 5. The bispecific antibody of claim 1 , wherein the single domain antibody is linked to the first companion animal Fc region variant or the second companion animal Fc region variant directly or via a peptide linker.

6. 6. The bispecific antibody of claim 1 , wherein the single domain antibody is a VHH domain.

7. (i) the VHH domain comprises a C-terminal residue, and the first companion animal Fc region variant or the second companion animal Fc region variant comprises an N-terminal residue, and the C-terminal residue of the VHH domain is linked to the N-terminal residue of the first companion animal Fc region variant or the second companion animal Fc region variant directly or via a peptide linker; or (ii) the VHH domain comprises an N-terminal residue, and the first companion animal Fc region variant or the second companion animal Fc region variant comprises a C-terminal residue, and the N-terminal residue of the VHH domain is linked to the C-terminal residue of the first companion animal Fc region variant or the second companion animal Fc region variant directly or via a peptide linker; The bispecific antibody of claim 6.

8. The peptide linker is (a) GPGGQ (SEQ ID NO: 38); (b) PKRENGRVPRPPDCPKCP (SEQ ID NO: 363); (c) VPKRENGRVPRPPDCPKCP (SEQ ID NO: 364); (d) FNECRCTDTPPCPVPEP (SEQ ID NO: 22); (e) PKRENGRVPRPPDCPKCPAPEM (SEQ ID NO: 23); (f) AKECECKCNCNNCPCPGCGL (SEQ ID NO: 24); (g) PKESTCKCISPCPVPES (SEQ ID NO: 25); (h) PKESTCKCIPPCPVPES (SEQ ID NO: 26); (i) KTDHPPGPKPCDCPKCP (SEQ ID NO: 27), and (j) KTASTIESKTGEGPKCP (SEQ ID NO: 29) 8. The bispecific antibody of claim 5, comprising an amino acid sequence selected from the group consisting of:

9. The bispecific antibody of claim 1 , wherein the first companion animal Fc region variant and the second companion animal Fc region variant are canine Fc region variants.

10. 10. The bispecific antibody of claim 9, wherein the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12.

11. 11. The bispecific antibody of claim 9 or 10, wherein the first canine Fc region variant and the second canine Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first canine Fc region variant and the second canine Fc region variant.

12. 12. The bispecific antibody of claim 11, wherein the first canine Fc region variant and the second canine Fc region variant each comprise a protrusion or a recess, wherein if the first canine Fc region comprises a protrusion, the second canine Fc region comprises a recess, and if the first canine Fc region comprises a recess, the second canine Fc region comprises a protrusion.

13. the first canine Fc region variant and the second canine Fc region variant are (a) S354C and T366W in the first canine Fc region variant and Y349C, T366S, L368A, and Y407V in the second canine Fc region variant; (b) T366W in the first canine Fc region variant and T366S, L368A, and Y407V in the second canine Fc region variant; (c) R392D and K409D in the first canine Fc region variant and E356K and D399K in the second canine Fc region variant; (d) S364H and F405A in the first canine Fc region variant and Y349T and T394F in the second canine Fc region variant; (e) F405L in the first canine Fc region variant and K409R in the second canine Fc region variant; (f) T366L, R392L, and T394W in the first canine Fc region variant and L351Y, F405A, and Y407V in the second canine Fc region variant; (g) K360E and K409W in the first canine Fc region variant and S347R, D399V, and F405T in the second canine Fc region variant; (h) Y349C, K360E, and K409W in the first canine Fc region variant and S347R, S354C, D399V, and F405T in the second canine Fc region variant; (i) K370E and K409W in the first canine Fc region variant and E357N, D399V, and F405T in the second canine Fc region variant; (j) K360D, D399M, and Y407A in the first canine Fc region variant and Q345R, S347R, T366V, and K409V in the second canine Fc region variant; (k) Y349S, T366M, K370Y, and K409V in the first canine Fc region variant and E356G, E357D, S364Q, and Y407A in the second canine Fc region variant; (l) L351D and L368E in the first canine Fc region variant and L351K and T366K in the second canine Fc region variant; (m) L368D and K370S in the first canine Fc region variant and E356Q and S364K in the second canine Fc region variant; or (n) T366Y in the first canine Fc region variant and T366S, L368A, and Y407T in the second canine Fc region variant.

13. The bispecific antibody of claim 11 or 12, comprising an amino acid substitution selected from the group consisting of:

14. 14. The bispecific antibody of any one of claims 9 to 13, wherein the first canine Fc region variant comprises a first charged region and the second canine Fc region variant comprises a second charged region, the first charged region forming a charge pair with the second charged region.

15. 15. The bispecific antibody of claim 14, wherein the first charged region comprises a basic amino acid residue and the second charged region comprises an acidic amino acid residue.

16. the first canine Fc region variant and the second canine Fc region variant comprise the following amino acid substitutions: S183D in the first canine Fc region variant and S183K in the second canine Fc region variant.

16. The bispecific antibody of claim 15, further comprising a CH1 domain comprising:

17. the first canine Fc region variant and the second canine Fc region variant are (a) K409D in the first canine Fc region variant and D399K in the second canine Fc region variant; (b) K390D and K409D in the first canine Fc region variant and E356K and D399K in the second canine Fc region variant; (c) K390D and K409D in the first canine Fc region variant and E357K and D399K in the second canine Fc region variant; and (d) K370D and K409D in the first canine Fc region variant and E357K and D399K in the second canine Fc region variant.

17. The bispecific antibody of claim 15 or 16, comprising a CH3 domain comprising an amino acid substitution selected from the group consisting of:

18. the first canine Fc region variant and the second canine Fc region variant comprise the following amino acid substitutions: S176K in the first canine Fc region variant and S176D in the second canine Fc region variant.

18. The bispecific antibody of claim 15, further comprising a CL domain comprising:

19. the first canid Fc region variant and / or the second canid Fc region variant comprises the following amino acid substitution: (a) 252Y and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (b) 252M and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 256D or 256F; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (c) 434R, (d) 426Y and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (e) 426H and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (f) 426F and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 426H; and (g) 434R and, optionally, at least one amino acid substitution selected from the group consisting of 286L; 286Y; 312P; and 436H. wherein the amino acid positions are based on EU numbering.

20. The bispecific antibody of claim 1 , wherein the first companion animal Fc region variant and the second companion animal Fc region variant are feline Fc region variants.

21. 21. The bispecific antibody of claim 20, wherein the first feline Fc region variant or the second feline Fc region variant comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21.

22. 22. The bispecific antibody of claim 20 or 21, wherein the first feline Fc region variant and the second feline Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first feline Fc region variant and the second feline Fc region variant.

23. 23. The bispecific antibody of claim 22, wherein the first feline Fc region variant and the second feline Fc region variant each comprise a protrusion or a recess, and wherein if the first feline Fc region comprises a protrusion, the second feline Fc region comprises a recess, and if the first feline Fc region comprises a recess, the second feline Fc region comprises a protrusion.

24. the first feline Fc region variant and the second feline Fc region variant are (a) T366W in the first feline Fc region variant and T366S, L368A, and Y407V in the second feline Fc region variant; (b) T366W in the first feline Fc region variant and T366S, L368A, and Y398T in the second feline Fc region variant; (c) A354C and T366W in the first feline Fc region variant and Y349C, T366S, L368A, and Y407V in the second feline Fc region variant; (d) R392D and K409D in the first feline Fc region variant and E356K and D399K in the second feline Fc region variant; (e) S364H and F405A in the first feline Fc region variant and Y349T and T394F in the second feline Fc region variant; (f) F405L in the first feline Fc region variant and K409R in the second feline Fc region variant; (g) T366L, R392L, and T394W in the first feline Fc region variant and L351Y, F405A, and Y407V in the second feline Fc region variant; (h) R360E and K409W in the first feline Fc region variant and Q347R, D399V, and F405T in the second feline Fc region variant; (i) Y349C, R360E, and K409W in the first feline Fc region variant and Q347R, A354C, D399V, F405T in the second feline Fc region variant; (j) K370E and K409W in the first feline Fc region variant and E357N, D399V, and F405T in the second feline Fc region variant; (k) R360D, D399M, and Y407A in the first feline Fc region variant and E345R, Q347R, T366V, and K409V in the second feline Fc region variant; (l) Y349S, K370Y, T366M, and K409V in the first feline Fc region variant and E356G, E357D, S364Q, and Y407A in the second feline Fc region variant; (m) L351D and L368E in the first feline Fc region variant and L351K and T366K in the second feline Fc region variant; (n) L368D and K370S in the first feline Fc region variant and E356Q and S364K in the second feline Fc region variant; and (o) T366Y in the first feline Fc region variant and T366S, L368A, and Y407T in the second feline Fc region variant.

24. The bispecific antibody of claim 22 or 23, comprising an amino acid substitution selected from the group consisting of:

25. 25. The bispecific antibody of any one of claims 20 to 24, wherein the first feline Fc region variant comprises a first charged region and the second feline Fc region variant comprises a second charged region, the first charged region forming a charge pair with the second charged region.

26. 26. The bispecific antibody of claim 25, wherein the first charged region comprises a basic amino acid residue and the second charged region comprises an acidic amino acid residue.

27. The first feline Fc region variant and the second feline Fc region variant comprise the following amino acid substitutions: S183D in the first feline Fc region variant and S183K in the second feline Fc region variant.

27. The bispecific antibody of claim 26, further comprising a CH1 domain comprising:

28. the first feline Fc region variant and the second feline Fc region variant are (a) K409D in the first feline Fc region variant and D399K in the second feline Fc region variant; and (b) K370D and K409D in the first feline Fc region variant and E357K and D399K in the second feline Fc region variant.

28. The bispecific antibody of claim 26 or 27, comprising a CH3 domain comprising an amino acid substitution selected from the group consisting of:

29. The first feline Fc region variant and the second feline Fc region variant comprise the following amino acid substitutions: S176K in the first feline Fc region variant and S176D in the second feline Fc region variant.

29. The bispecific antibody of any one of claims 26 to 28, further comprising a CL domain comprising:

30. The first feline Fc region variant and / or the second feline Fc region variant may comprise the following amino acid substitutions: (a) at least one amino acid substitution selected from the group consisting of 286E, 311V, and 428Y; (b) two or more amino acid substitutions selected from the group consisting of 286E, 311V, and 428Y; and (c) 286E, 311V, and 428Y wherein the amino acid positions are based on EU numbering.

31. (a) a first polypeptide comprising a first companion animal Fc region variant; (b) a second polypeptide comprising a second companion animal Fc region variant; and 1. An Fc construct comprising: the first companion animal Fc region variant and the second companion animal Fc region variant comprise complementary dimerization selectivity modules that promote dimerization between the first companion animal Fc region variant and the second companion animal Fc region variant; the first polypeptide or the second polypeptide does not comprise an antibody; The Fc construct.

32. 32. The Fc construct of claim 31, further comprising a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1, VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, and a thrombopoietin binding peptide.

33. 33. The Fc construct of claim 31 or 32, wherein the first companion animal Fc region variant and the second companion animal Fc region variant are canine Fc region variants.

34. 34. The Fc construct of claim 33, wherein the first canine Fc region variant or the second canine Fc region variant comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12.

35. 35. The Fc construct of claim 34, wherein the first canid Fc region variant and the second canid Fc region variant each comprise a protrusion or a recess, wherein if the first canid Fc region comprises a protrusion, the second canid Fc region comprises a recess, and if the first canid Fc region comprises a recess, the second canid Fc region comprises a protrusion.

36. the first canine Fc region variant and the second canine Fc region variant each comprise: (a) S354C and T366W in the first canine Fc region variant and T366S, L368A, Y407V, and Y349C in the second canine Fc region variant; (b) T366W in the first canine Fc region variant and T366S, L368A, and Y407V in the second canine Fc region variant; (c) R392D and K409D in the first canine Fc region variant and E356K and D399K in the second canine Fc region variant; (d) S364H and F405A in the first canine Fc region variant and Y349T and T394F in the second canine Fc region variant; (e) F405L in the first canine Fc region variant and K409R in the second canine Fc region variant; (f) T366L, R392L, and T394W in the first canine Fc region variant and L351Y, F405A, and Y407V in the second canine Fc region variant; (g) K360E and K409W in the first canine Fc region variant and S347R, D399V, and F405T in the second canine Fc region variant; (h) Y349C, K360E, and K409W in the first canine Fc region variant and S347R, S354C, D399V, and F405T in the second canine Fc region variant; (i) K370E and K409W in the first canine Fc region variant and E357N, D399V, and F405T in the second canine Fc region variant; (j) K360D, D399M, and Y407A in the first canine Fc region variant and Q345R, S347R, T366V, and K409V in the second canine Fc region variant; (k) Y349S, T366M, K370Y, and K409V in the first canine Fc region variant and E356G, E357D, S364Q, and Y407A in the second canine Fc region variant; (l) L351D and L368E in the first canine Fc region variant and L351K and T366K in the second canine Fc region variant; (m) L368D and K370S in the first canine Fc region variant and E356Q and S364K in the second canine Fc region variant; or (n) T366Y in the first canine Fc region variant and T366S, L368A, and Y407T in the second canine Fc region variant.

36. The Fc construct of claim 34 or 35, comprising an amino acid substitution selected from the group consisting of: wherein the amino acid positions are based on EU numbering.

37. 37. The Fc construct of any one of claims 33 to 36, wherein the first canine Fc region variant comprises a first charged region and the second canine Fc region variant comprises a second charged region, the first charged region forming a charge pair with the second charged region.

38. 38. The Fc construct of claim 37, wherein the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

39. the first canine Fc region variant and the second canine Fc region variant comprise the following amino acid substitutions: S183D in the first canine Fc region variant and S183K in the second canine Fc region variant.

39. The Fc construct of claim 38, further comprising a CH1 domain comprising:

40. the first canine Fc region variant and the second canine Fc region variant are (a) K409D in the first canine Fc region variant and D399K in the second canine Fc region variant; (b) K390D and K409D in the first canine Fc region variant and E356K and D399K in the second canine Fc region variant; (c) K390D and K409D in the first canine Fc region variant and E357K and D399K in the second canine Fc region variant; and (d) K370D and K409D in the first canine Fc region variant and E357K and D399K in the second canine Fc region variant.

40. The Fc construct of claim 38 or 39, comprising a CH3 domain comprising an amino acid substitution selected from the group consisting of:

41. the first canine Fc region variant and the second canine Fc region variant comprise the following amino acid substitutions: S176K in the first canine Fc region variant and S176D in the second canine Fc region variant.

41. The Fc construct of any one of claims 38 to 40, further comprising a CL domain comprising:

42. the first canine Fc region variant and the second canine Fc region variant comprise the following amino acid substitutions: (a) 252Y and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (b) 252M and, optionally, at least one amino acid substitution selected from the group consisting of: 251D or 251E; 256D or 256F; 285N or 285D; 286D; 307Q; 308P; 315D; 430A or 430K; 433K; 435Y; and 436H; (c) 434R, (d) 426Y and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (e) 426H and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 436H; (f) 426F and, optionally, at least one amino acid substitution selected from the group consisting of 286F, 286W, 286L, or 286Y; 312P; 434R; and 426H; and (g) 434R and, optionally, at least one amino acid substitution selected from the group consisting of 286L; 286Y; 312P; and 436H.

42. The Fc construct of any one of claims 33 to 41, further comprising at least one of: wherein the amino acid positions are based on EU numbering.

43. 33. The Fc construct of claim 31 or 32, wherein the first companion animal Fc region variant and the second companion animal Fc region variant are feline Fc region variants.

44. 44. The Fc construct of claim 43, wherein said first feline Fc region variant or said second feline Fc region variant comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-21.

45. 45. The Fc construct of claim 44, wherein the first feline Fc region variant and the second feline Fc region variant each comprise a protrusion or a recess, and wherein if the first feline Fc region comprises a protrusion, the second feline Fc region comprises a recess, and if the first feline Fc region comprises a recess, the second feline Fc region comprises a protrusion.

46. the first feline Fc region variant and the second feline Fc region variant are (a) T366W in the first feline Fc region variant and T366S, L368A, and Y407V in the second feline Fc region variant; (b) T366W in the first feline Fc region variant and T366S, L368A, and Y398T in the second feline Fc region variant; (c) A354C and T366W in the first feline Fc region variant and Y349C, T366S, L368A, and Y407V in the second feline Fc region variant; (d) R392D and K409D in the first feline Fc region variant and E356K and D399K in the second feline Fc region variant; (e) S364H and F405A in the first feline Fc region variant and Y349T and T394F in the second feline Fc region variant; (f) F405L in the first feline Fc region variant and K409R in the second feline Fc region variant; (g) T366L, R392L, and T394W in the first feline Fc region variant and L351Y, F405A, and Y407V in the second feline Fc region variant; (h) R360E and K409W in the first feline Fc region variant and Q347R, D399V, and F405T in the second feline Fc region variant; (i) Y349C, R360E, and K409W in the first feline Fc region variant and Q347R, A354C, D399V, F405T in the second feline Fc region variant; (j) K370E and K409W in the first feline Fc region variant and E357N, D399V, and F405T in the second feline Fc region variant; (k) R360D, D399M, and Y407A in the first feline Fc region variant and E345R, Q347R, T366V, and K409V in the second feline Fc region variant; (l) Y349S, K370Y, T366M, and K409V in the first feline Fc region variant and E356G, E357D, S364Q, and Y407A in the second feline Fc region variant; (m) L351D and L368E in the first feline Fc region variant and L351K and T366K in the second feline Fc region variant; (n) L368D and K370S in the first feline Fc region variant and E356Q and S364K in the second feline Fc region variant; and (o) T366Y in the first feline Fc region variant and T366S, L368A, and Y407T in the second feline Fc region variant.

46. ​​The Fc construct of claim 44 or 45, comprising an amino acid substitution selected from the group consisting of: wherein the amino acid positions are based on EU numbering.

47. 47. The Fc construct of any one of claims 43 to 46, wherein the first feline Fc region variant comprises a first charged region and the second feline Fc region variant comprises a second charged region, the first charged region forming a charge pair with the second charged region.

48. 48. The Fc construct of claim 47, wherein the first charged region comprises basic amino acid residues and the second charged region comprises acidic amino acid residues.

49. The first feline Fc region variant and the second feline Fc region variant comprise the following amino acid substitutions: S183D in the first feline Fc region variant and S183K in the second feline Fc region variant.

49. The Fc construct of claim 48, further comprising a CH1 domain comprising:

50. the first feline Fc region variant and the second feline Fc region variant are (a) K409D in the first feline Fc region variant and D399K in the second feline Fc region variant; and (b) K370D and K409D in the first feline Fc region variant and E357K and D399K in the second feline Fc region variant.

50. The Fc construct of claim 48 or 49, comprising a CH3 domain comprising an amino acid substitution selected from the group consisting of:

51. The first feline Fc region variant and the second feline Fc region variant comprise the following amino acid substitutions: S176K in the first feline Fc region variant and S176D in the second feline Fc region variant.

51. The Fc construct of any one of claims 48 to 50, further comprising a CL domain comprising:

52. The first feline Fc region variant and the second feline Fc region variant comprise the following amino acid substitutions: (a) at least one amino acid substitution selected from the group consisting of 286E, 311V, and 428Y; (d) two or more amino acid substitutions selected from the group consisting of 286E, 311V, and 428Y; and (c) 286E, 311V, and 428Y 52. The Fc construct of any one of claims 43 to 51, further comprising at least one of: wherein the amino acid positions are based on EU numbering.

53. 53. A pharmaceutical composition comprising (i) a bispecific antibody according to any one of claims 1 to 30 or an Fc construct according to any one of claims 31 to 52, and (ii) a pharmaceutically acceptable excipient.

54. 53. One or more nucleic acids encoding the bispecific antibody of any one of claims 1 to 30 or the Fc construct of any one of claims 31 to 52.

55. 55. One or more expression vectors comprising one or more nucleic acids of claim 54.

56. 56. A host cell comprising one or more nucleic acids of claim 54 or one or more expression vectors of claim 55.

57. 1. A method of making a bispecific antibody or Fc construct, comprising: (a) providing one or more nucleic acids of claim 54; (b) expressing said one or more nucleic acids in a host cell culture, thereby producing said bispecific antibody or said Fc construct; and optionally (c) recovering the bispecific antibody or the Fc construct produced in (ii) from the host cell culture; The method comprising:

58. The method of claim 57, wherein the host cell culture comprises (i) one host cell population expressing both the first companion animal Fc region variant and the second companion animal Fc region variant, or (ii) two host cell populations, including a first population expressing the first companion animal Fc region variant and a second population expressing the second companion animal Fc region variant.

59. 54. A method of treating or preventing a canine disease or disorder in a dog in need thereof, the method comprising administering to the dog an effective amount of a composition comprising a bispecific antibody described in any one of claims 9 to 19, an Fc construct described in any one of claims 33 to 42, or a pharmaceutical composition described in claim 53.

60. 60. The method of claim 59, wherein the canine disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer.

61. 60. The method of claim 59, wherein the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

62. 54. A bispecific antibody according to any one of claims 9 to 19, an Fc construct according to any one of claims 33 to 42, or a pharmaceutical composition according to claim 53, for use in treating or preventing a canine disease or disorder in a dog in need thereof.

63. 63. The bispecific antibody or Fc construct for use or pharmaceutical composition for use according to claim 62, wherein the canine disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer.

64. 63. The bispecific antibody or Fc construct for use or pharmaceutical composition for use according to claim 62, wherein the canine disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

65. 52. A method of treating or preventing a feline disease or disorder in a cat in need thereof, the method comprising administering to the cat an effective amount of a composition comprising a bispecific antibody of any one of claims 20 to 30, an Fc construct of any one of claims 43 to 52, or a pharmaceutical composition of claim 53.

66. 66. The method of claim 65, wherein the feline disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer.

67. 66. The method of claim 65, wherein the feline disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

68. 54. A bispecific antibody according to any one of claims 20 to 30, an Fc construct according to any one of claims 43 to 52, or a pharmaceutical composition according to claim 53, for use in the treatment or prevention of a feline disease or disorder in a cat in need thereof.

69. 69. The bispecific antibody or Fc construct for use or the pharmaceutical composition for use according to claim 68, wherein the feline disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, renal disease, reproductive function-related disorder, infectious disease, or cancer.

70. 69. The bispecific antibody or Fc construct for use or the pharmaceutical composition for use according to claim 68, wherein the feline disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthritic pain, arthritis, anemia, or obesity.

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