Anti-TGF-β antibodies and their therapeutic uses

Abstract

The present disclosure encompasses novel anti-TGFβ antibodies, antigen binding proteins, and polynucleotides encoding same. The disclosure further provides uses of the novel antibodies, antigen binding proteins, and / or nucleotides of the invention for the treatment and / or prevention of TGFβ-associated disorders, particularly for the management of fibrosis-related disorders in canines and felines.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 036,092, filed June 8, 2020, which is incorporated herein by reference in its entirety.

[0002] This application relates to monoclonal antibodies, methods for their production, and therapeutic uses of these antibodies. In certain embodiments, the monoclonal antibodies are directed against transforming growth factor beta (TGFβ, TGFB, or TGFbeta). In other embodiments, the antibodies are chimeric or speciated antibodies. In other embodiments, therapeutic methods involving the antibodies of the invention are disclosed. [Background technology]

[0003] Transforming growth factor beta (TGFB, TGFβ, or TGFbeta, used interchangeably herein) is a cytokine that controls many key cellular functions, including proliferation, differentiation, survival, migration, and epithelial-mesenchymal transition. It is a member of a superfamily of 38 cytokines that includes TGFβ, bone morphogenetic proteins (BMPs), growth differentiation factors, inhibitors, and activins. TGFβ proteins regulate diverse biological processes, such as extracellular matrix formation, wound healing, embryonic development, bone development, hematopoiesis, immune and inflammatory responses, and malignant transformation. Deregulation of TGFβ leads to pathological conditions, including congenital anomalies, cancer, chronic inflammation, autoimmune, and fibrotic diseases.

[0004] TGFβ has three known isoforms: TGFβ1, 2, and 3. All three isoforms are initially translated as propeptides. The isoforms are synthesized in the endoplasmic reticulum as large precursor proteins (pro-TGFβ) that form dimeric complexes and are subsequently cleaved near the carboxy terminus to yield a mature 112-amino acid polypeptide that shares 60–80% conservation across the three TGFβ isoforms. The mature TGFβ dimer remains associated with the cleaved latent peptide portion of the precursor as an inactive latent complex. Newly synthesized TGFβ bound to the latency-associated peptide (LAP), forming the small latent complex (SLC), is biologically inactive and unable to bind to its receptor, TGFβRII. Through disulfide bond formation, this complex loosely binds to the latent TGFβ-binding protein (LTBP) to form the large latent complex (LLC). TGFβ is then secreted in a latent state and stored in the extracellular matrix (ECM). Activation of TGFβ involves its release from a latent complex following exposure to a number of different factors, including integrins, proteases, metalloproteases, reactive oxygen species (ROS), plasmin, and acid, which allows it to bind to its cell surface receptors for the initiation of TGFβ signaling.

[0005] TGFβ1, 2, and 3 are pleiotropic in their functions and are expressed in distinct patterns across cell and tissue types. Although they have similar in vitro activity, individual knockouts in specific cell types suggest nonidentical roles in vivo, despite their shared ability to bind to the same receptor (Akhurst et al., Nat Rev Drug Discov (2012) 11(10):790-811). Upon binding of TGFβ to TGFβRII, the receptor's constitutive kinase activity phosphorylates and activates TGFβRI, which then phosphorylates SMAD2 / 3, enabling its association with SMAD4. This complex localizes to the nucleus and functions as a transcription factor for TGFβ-responsive genes. In addition to this canonical signaling cascade, noncanonical pathways signal through other factors, including p38, MAPK, PI3K, AKT, JUN, JNK, and NK-KB. The net result is crosstalk between all these signaling pathways, integrating cellular context and the environment.

[0006] Many serious diseases are associated with dysfunction of the TGFβ-induced signaling pathway. The present invention is directed to potential treatments for chronic kidney disease (CKD) in both canines and felines. CKD involves the loss of functional kidney tissue due to a long-term, progressive process. While renal structural and functional changes are only loosely correlated, dramatic changes in kidney structure can be observed. The disease usually exists for months or years before it becomes clinically apparent and is invariably irreversible. Congenital disease results in a transient increase in prevalence in animals over the age of 3, but prevalence increases with advancing age after 5-6 years of age. In the elderly population, CKD affects 10% of dogs and 40-80% of cats. There is a clear unmet need in the veterinary field to treat CKD in both dogs and cats, a condition suggested to be affected by overproduction of TGFβ protein. Summary of the Invention

[0007] The present invention provides novel anti-transforming growth factor beta (TGFB, TGF beta or TGFβ) antigen binding proteins (antibodies, antibody fragments, antagonist antibodies as defined herein and used interchangeably) that bind to TGFβ1. In all embodiments, the present invention provides antibodies that bind to TGFβ1. In some embodiments, the present invention provides antigen binding proteins that additionally bind to TGFβ3. In some embodiments, the present invention provides antigen binding proteins that bind to TGFβ1, TGFβ2, and TGFβ3. The antigen binding proteins of the present invention block the biological activity of TGFβ1, 2, and / or 3 by preventing the binding of TGFβ1, 2, and / or 3 to their receptors, preventing activation of pathways associated with binding. Additionally, the present invention provides that the antagonistic action of the antibodies of the present invention prevents and / or treats TGFβ-related disorders as defined herein. The present invention further provides nucleotides encoding the antigen binding proteins of the present invention, as well as vectors and host cells for production. The invention further provides methods of making and using such antibodies / antigen binding proteins, as well as methods of treatment for treating TGFβ disorders in canines, felines and humans by administering antibodies of the invention.

[0008] In one aspect, the invention provides antibodies / antigen binding proteins (used interchangeably herein) that bind to TGFβ1. In one or more embodiments, the antigen binding protein also specifically binds TGFβ3. In one or more embodiments, the antigen binding protein also specifically binds TGFβ2 and TGFβ3. In one or more embodiments, the antigen binding proteins of the invention bind to canine TGFβ1. In one or more embodiments, the antigen binding proteins of the invention bind to feline TGFβ1. In one or more embodiments, the antigen binding proteins of the invention are administered as a pharmaceutical composition further comprising an excipient to treat a TGFβ-associated disorder. In one or more embodiments, the TGFβ-associated disorder comprises renal fibrosis / chronic kidney disease.

[0009] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline transforming growth factor beta-1 (TGFβ1). In one embodiment, the antigen binding protein of the invention specifically binds to canine TGFβ1 comprising an amino acid sequence comprising SEQ ID NO: 220. In one embodiment, the antigen binding protein of the invention specifically binds to canine TGFβ1 comprising an amino acid sequence comprising SEQ ID NO: 222. In one embodiment, the antigen binding protein is capable of binding to an epitope region on TGFβ1 comprising amino acids 91-104 of SEQ ID NO: 223. In one embodiment, the antigen binding protein is further capable of binding to amino acids 60-64 of SEQ ID NO: 223.

[0010] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, wherein the antigen binding protein has a Complementary Determining Region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:41, and an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:42. and a complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 224 [Threonine (T)-Glycine (G)-Glutamic acid (E)-Tyrosine (Y)-Serine (S)-Glycine (G)-Tyrosine (Y)-Aspartic acid (D)-Threonine (T)-(X1)-(X2)-(X3)-(X4)-(X5)], wherein X1 comprises lysine (K) or arginine (R), X2 comprises threonine (T) or alanine (A), and X3 comprises glutamic acid. and a light chain variable region (VL) comprising: a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 44; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 45; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 46; and any variant thereof having one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0011] In one or more embodiments, the present invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, the antigen binding protein comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:41; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:42; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:43, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO: and a light chain variable region (VL) comprising: a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 44; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 45; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 46; and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the present invention include variants that are not conservative amino acid substitutions.

[0012] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but does not bind to TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 43, or any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding protein of the invention includes variants that are not conservative amino acid substitutions.

[0013] In one or more embodiments, antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprise a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 141, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0014] In one or more embodiments, antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprise a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 142, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0015] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 149, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0016] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 150, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0017] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 153, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0018] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 154, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0019] In one or more embodiments, antigen binding proteins of the invention that specifically bind to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprise a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 155, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0020] In one or more embodiments, antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprise a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 156, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0021] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 162, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0022] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 166, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0023] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 167, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0024] In one or more embodiments, antigen binding proteins of the invention that specifically bind to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprise a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 169, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0025] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 170, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0026] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 171, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0027] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 172, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0028] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3 comprises a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 41, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 42, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 178, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0029] In one or more embodiments, antigen binding proteins of the invention that specifically bind to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3 include caninized, felineized, humanized, or chimeric binding proteins. In one or more embodiments, the antigen binding protein is a caninized antigen binding protein. In one or more embodiments, the antigen binding protein is a felineized antigen binding protein. In one or more embodiments, the antigen binding protein is a humanized antigen binding protein. In one or more embodiments, the antigen binding protein is a chimeric antigen binding protein.

[0030] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:252, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, and SEQ ID NO:268; and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53; and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0031] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 38, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 49, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0032] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 38, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0033] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 38, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 53, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0034] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 47, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 53, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0035] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 56, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0036] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 231, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0037] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 232, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0038] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 239, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding protein of the invention comprises a variant that is not a conservative amino acid substitution. In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 240, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0039] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 243, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0040] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 244, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0041] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 245, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0042] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 246, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0043] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 252, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0044] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 256, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0045] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 257, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0046] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 259, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0047] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 260, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0048] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 261, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0049] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 262, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0050] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the antigen binding protein comprising a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 268, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 51, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0051] In one or more embodiments, the invention provides caninized antigen binding proteins of the invention that specifically bind to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, wherein the caninized antigen binding protein further comprises a canine heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 127, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0052] In one or more embodiments, the invention provides that caninized antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3 further comprise a canine light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 129. In one embodiment, the antigen binding proteins of the invention comprise a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 55, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 49; a constant region for the heavy chain (SEQ ID NO: 127), and a constant region for the light chain (SEQ ID NO: 129); and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0053] In one embodiment, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, including felineized antigen binding proteins. In one embodiment, the antigen binding protein comprises a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 38 or SEQ ID NO: 59, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 40 or SEQ ID NO: 61, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0054] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not TGFβ2 or TGFβ3, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least about 95% sequence identity to the amino acid sequence SEQ ID NO: 59, and a light chain variable region (VL) having at least about 95% sequence identity to amino acids comprising SEQ ID NO: 61, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0055] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, wherein the antigen binding protein further comprises a feline heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 131, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0056] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, wherein the antigen binding protein further comprises a feline light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 133, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0057] In one embodiment, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, including humanized antigen binding proteins. In one embodiment, the invention provides antigen binding proteins, including chimeric antigen binding proteins and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0058] In one embodiment, the invention provides antigen binding proteins that specifically bind canine or feline TGFβ1 but do not bind TGFβ2 or TGFβ3, including chimeric antigen binding proteins. In one embodiment, the chimeric antigen binding protein comprises a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 38, and a light chain variable region (VL) having at least about 95% sequence identity to amino acids comprising SEQ ID NO: 40 and SEQ ID NO: 61, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the invention include variants that are not conservative amino acid substitutions.

[0059] In one or more embodiments, the present invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, wherein the protein is selected from the group consisting of a monoclonal antigen binding protein, a single-chain antigen binding protein, a tetrameric antigen binding protein, a tetravalent antigen binding protein, a multispecific antigen binding protein, a domain-specific antigen binding protein, a domain-deleted antigen binding protein, a fusion protein, an ScFc fusion protein, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, an ScFv fragment, an Fd fragment, a single-domain antigen binding protein, a dAb fragment, a small modular immunopharmaceutical (SMIP) nanobody, and an IgNAR molecule. In one embodiment, the antigen binding protein is a monoclonal antigen binding protein.

[0060] In one or more embodiments, antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3 are for use in treating canines for TGFβ-related disorders. In one or more embodiments, antigen binding proteins of the invention are for use in treating felines for TGFβ-related disorders. In one or more embodiments, antigen binding proteins of the invention are for use in treating humans for TGFβ-related disorders.

[0061] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, which binding proteins are used to reduce or eliminate a TGFβ-associated disorder. In one embodiment, the TGFβ-associated disorder is selected from the group consisting of a fibrotic disorder, a connective tissue disorder, a bone disorder, and a cell proliferation disorder. In one embodiment, the TGFβ-associated disorder comprises a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, liver cirrhosis, glial scarring, and systemic sclerosis / scleroderma. In one embodiment, the TGFβ disorder is renal fibrosis / chronic kidney disease.

[0062] In one or more embodiments, the present invention provides a pharmaceutical composition that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3, the pharmaceutical composition comprising a therapeutically effective amount of an antigen binding protein of the invention and a pharmaceutically acceptable carrier.

[0063] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3 provides a method of treating a subject for a TGFβ-related disorder by administering to the subject a therapeutic amount of a pharmaceutical composition of the invention. In one or more embodiments, the invention provides a method of treating a canine for a TGFβ-related disorder. In one embodiment, the invention provides a method for treating a feline for a TGFβ-related disorder. In one embodiment, the invention provides a method for treating a human for a TGFβ-related disorder. In one embodiment, the method provides for administering a therapeutically effective amount of a pharmaceutical composition comprising an antigen binding protein of the invention. In one or more embodiments, the invention provides that the TGFβ-related disorder is selected from the group consisting of a fibrotic disorder, a connective tissue disorder, a bone disorder, and a cell proliferation disorder. In one embodiment, the TGFβ-related disorder comprises a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, liver cirrhosis, glial scarring, and systemic sclerosis / scleroderma. In one embodiment, the TGFβ disorder is renal fibrosis / chronic kidney disease.

[0064] In one or more embodiments, the invention provides methods of inhibiting TGFβ activity in a subject by administering a pharmaceutical composition comprising an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3. In one embodiment, the subject comprises a canine, feline, or human. In one embodiment, the subject comprises a canine. In one embodiment, the subject comprises a feline. In one embodiment, the subject comprises a human.

[0065] In one or more embodiments, the present invention provides isolated nucleic acid sequences having at least about 95% sequence identity to a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds canine or feline TGFβ1 but not TGFβ2 or TGFβ3, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions. In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen binding proteins of the invention, including a nucleotide sequence encoding a VH that has 95% sequence identity to SEQ ID NO: 54, and a nucleotide sequence encoding a VL that has 95% sequence identity to SEQ ID NO: 293, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0066] In one or more embodiments, the invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 54, a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 293, a nucleotide sequence encoding a canine heavy chain constant region having 95% sequence identity to SEQ ID NO: 128, and a nucleotide sequence encoding a canine light chain constant region having 95% sequence identity to SEQ ID NO: 130, and any variants thereof with one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0067] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 58 and a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 60, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0068] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 58 and a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 39, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0069] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 37 and a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 60, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0070] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 58, a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 60, a nucleotide sequence encoding a feline heavy chain constant region having 95% sequence identity to SEQ ID NO: 132, and a nucleotide sequence encoding a feline light chain constant region having 95% sequence identity to SEQ ID NO: 134, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0071] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 58, a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 39, a nucleotide sequence encoding a feline heavy chain constant region having 95% sequence identity to SEQ ID NO: 132, and a nucleotide sequence encoding a feline light chain constant region having 95% sequence identity to SEQ ID NO: 134, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0072] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 but not TGFβ2 or TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 37, a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 60, a nucleotide sequence encoding a feline heavy chain constant region having 95% sequence identity to SEQ ID NO: 132, and a nucleotide sequence encoding a feline light chain constant region having 95% sequence identity to SEQ ID NO: 134, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0073] In one or more embodiments, the present invention provides a vector comprising a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 but not to TGFβ2 or TGFβ3.

[0074] In one embodiment, the invention provides a host cell comprising a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds canine or feline TGFβ1 but not TGFβ2 or TGFβ3. In one embodiment, the invention provides a host cell comprising a vector comprising a nucleic acid encoding an antigen binding protein of the invention. In one embodiment, the invention provides a host cell that produces the antigen binding protein of the invention.

[0075] In one or more embodiments, the invention provides a method of producing an antigen binding protein of the invention that specifically binds canine or feline TGFβ1 but not TGFβ2 or TGFβ3, the method comprising culturing a host cell of the invention under conditions that result in the production of the antigen binding protein, and isolating the antigen binding protein from the host cell or from the host cell culture medium.

[0076] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline transforming growth factor beta-1 (TGFβ1) and transforming growth factor-3 (TGFβ3). In one embodiment, the antigen binding proteins of the invention are capable of binding to an epitope region on TGFβ1 comprising amino acids 82-91 of SEQ ID NO:223. In one embodiment, the antigen binding proteins of the invention are further capable of binding to amino acids 60-64 of SEQ ID NO:223.

[0077] In one or more embodiments, the invention provides antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence that comprises at least about 95% sequence identity to an amino acid sequence that comprises SEQ ID NO:5, a complementarity determining region 2 (CDR2) comprising an amino acid sequence that comprises at least about 95% sequence identity to an amino acid sequence that comprises SEQ ID NO:6, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence that comprises at least about 95% sequence identity to an amino acid sequence that comprises SEQ ID NO:7. and a light chain variable region (VL) comprising: a complementarity determining region 1 (CDR1) comprising an amino acid sequence comprising at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 8; a complementarity determining region 2 (CDR2) comprising an amino acid sequence comprising at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 9; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence comprising at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 10; and any variants thereof having one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the present invention do not comprise conservative amino acid substitutions.

[0078] In one or more embodiments, the invention provides antigen binding proteins of the invention that specifically bind to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding proteins comprise a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO:5, a complementarity determining region 2 (CDR2) comprising the amino acid sequence SEQ ID NO:6, and a complementarity determining region 3 (CDR3) comprising the amino acid sequence SEQ ID NO:7; and a light chain variable region (VL) comprising a complementarity determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO:8, a complementarity determining region 2 (CDR2) comprising the amino acid sequence SEQ ID NO:9, and a complementarity determining region 3 (CDR3) comprising the amino acid sequence SEQ ID NO:10; and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0079] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding proteins comprise caninized, feline, humanized, murine, or chimeric antigen binding proteins. In one embodiment, the antigen binding protein comprises a caninized antigen binding protein. In one embodiment, the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16; and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24; and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, the antigen binding proteins of the present invention include variants that are not conservative amino acid substitutions.

[0080] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding proteins comprise caninized, feline, humanized, murine, or chimeric antigen binding proteins. In one embodiment, the antigen binding protein comprises a caninized antigen binding protein. In one embodiment, the antigen binding protein comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16; and a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24; and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the present invention do not have conservative amino acid substitutions.

[0081] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 12, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 24, and any variants thereof with one or more conservative amino acid substitutions; in one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0082] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 14, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 24, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0083] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 13, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 23, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0084] In one or more embodiments, the present invention further provides antigen binding proteins that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a canine heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 127. In one or more embodiments, the canine heavy chain constant region comprises conservative amino acid substitutions. In one or more embodiments, the amino acid substitutions are not conservative.

[0085] In one or more embodiments, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a canine heavy chain constant region comprising the amino acid sequence SEQ ID NO: 127. In one or more embodiments, the canine heavy chain constant region comprises conservative amino acid substitutions. In one or more embodiments, the amino acid substitutions are not conservative.

[0086] In one or more embodiments, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a canine light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 129. In one or more embodiments, the invention provides antigen binding proteins comprising a canine light chain constant region comprising the amino acid sequence SEQ ID NO: 129. In one or more embodiments, the canine light chain constant region comprises conservative amino acid substitutions. In one or more embodiments, the amino acid substitutions are not conservative.

[0087] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3, but not TGFβ2, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 12, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 24, a canine heavy chain constant region comprising an amino acid sequence that has at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 127, and a canine light chain constant region comprising an amino acid sequence that has at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 129, and any variants thereof with one or more conservative amino acid substitutions; in one or more embodiments, a variant of the antigen binding protein of the invention is not a conservative amino acid substitution.

[0088] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a heavy chain variable region (VH) comprising the amino acid sequence SEQ ID NO: 12, and a light chain variable region (VL) comprising the amino acid sequence SEQ ID NO: 24, a canine heavy chain constant region comprising an amino acid sequence comprising SEQ ID NO: 127, and a canine light chain constant region comprising an amino acid sequence comprising SEQ ID NO: 129, and any variants thereof with one or more conservative amino acid substitutions; in one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0089] In one embodiment, the invention provides antigen binding proteins that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, including felineized antigen binding proteins. In one embodiment, the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 32 or SEQ ID NO: 34, and SEQ ID NO: 36, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0090] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3, but not TGFβ2, comprising a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 26, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 32, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0091] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding proteins further comprise a feline heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 131, and any variants thereof having one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0092] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding proteins comprise a feline light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 133, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0093] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 26, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 32, a feline heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 131, and a feline light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 133, and any variants thereof with one or more conservative amino acid substitutions. In one or more embodiments, variants of the antigen binding proteins of the invention are not conservative amino acid substitutions.

[0094] In one embodiment, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, including humanized antigen binding proteins, hi one embodiment, the invention provides antigen binding proteins, including chimeric antigen binding proteins.

[0095] In one or more embodiments, antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3, but not TGFβ2, are for use in treating canines for TGFβ-related disorders. In one or more embodiments, antigen binding proteins of the invention are for use in treating felines for TGFβ-related disorders. In one or more embodiments, antigen binding proteins of the invention are for use in treating humans for TGFβ-related disorders.

[0096] In one or more embodiments, the present invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1 and TGFβ3 but not TGFβ2, wherein the protein is selected from the group consisting of a monoclonal antigen binding protein, a single-chain antigen binding protein, a tetrameric antigen binding protein, a tetravalent antigen binding protein, a multispecific antigen binding protein, a domain-specific antigen binding protein, a domain-deleted antigen binding protein, a fusion protein, an ScFc fusion protein, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, an ScFv fragment, an Fd fragment, a single-domain antigen binding protein, a dAb fragment, a small modular immunopharmaceutical (SMIP) nanobody, and an IgNAR molecule. In one embodiment, the antigen binding protein is a monoclonal antigen binding protein.

[0097] In one or more embodiments, the present invention provides an antigen binding protein that specifically binds to TGFβ1 and TGFβ3, but not to TGFβ2, for use in treating a TGFβ-associated disorder in a canine or feline, wherein the binding protein reduces or eliminates the TGFβ-associated disorder. In one embodiment, the TGFβ-associated disorder is selected from the group consisting of a fibrotic disorder, a connective tissue disorder, a bone disorder, and a cell proliferation disorder. In one embodiment, the TGFβ-associated disorder comprises a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, liver cirrhosis, glial scarring, and systemic sclerosis / scleroderma. In one embodiment, the TGFβ disorder is renal fibrosis / chronic kidney disease.

[0098] In one or more embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 and TGFβ3, but not TGFβ2, and a pharmaceutically acceptable carrier.

[0099] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 and TGFβ3, but not TGFβ2, provides a method of treating a subject for a TGFβ-related disorder by administering to the subject a therapeutic amount of a pharmaceutical composition of the invention. In one or more embodiments, the invention provides a method of treating a canine for a TGFβ-related disorder. In one embodiment, the invention provides a method for treating a feline for a TGFβ-related disorder. In one embodiment, the invention provides a method for treating a human for a TGFβ-related disorder. In one embodiment, the method provides for administering a therapeutically effective amount of a pharmaceutical composition comprising an antigen binding protein of the invention. In one or more embodiments, the invention provides that the TGFβ-related disorder is selected from the group consisting of a fibrotic disorder, a connective tissue disorder, a bone disorder, and a cell proliferation disorder. In one embodiment, the TGFβ-related disorder comprises a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, liver cirrhosis, glial scarring, and systemic sclerosis / scleroderma. In one embodiment, the TGFβ disorder is renal fibrosis / chronic kidney disease.

[0100] In one or more embodiments, the present invention provides methods of inhibiting TGFβ activity in a subject by administering a pharmaceutical composition comprising an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 and TGFβ3, but not TGFβ2. In one embodiment, the subject comprises a canine, feline, or human. In one embodiment, the subject comprises a canine. In one embodiment, the subject comprises a feline. In one embodiment, the subject comprises a human.

[0101] In one or more embodiments, the present invention provides isolated nucleic acid sequences having at least about 95% sequence identity to a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds canine or feline TGFβ1 and TGFβ3, but not TGFβ2, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0102] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 11 and a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 23, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0103] In one or more embodiments, the invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 11, a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 23, a nucleotide sequence encoding a canine heavy chain constant region having 95% sequence identity to SEQ ID NO: 128, and a nucleotide sequence encoding a canine light chain constant region having 95% sequence identity to SEQ ID NO: 130, and any variants thereof with one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0104] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, including a nucleotide sequence encoding a VH that has 95% sequence identity to SEQ ID NO: 13 and a nucleotide sequence encoding a VL that has 95% sequence identity to SEQ ID NO: 23, and any variants thereof with one or more nucleic acid substitutions that result in conservative amino acid substitutions.

[0105] In one or more embodiments, the invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 15, and a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 23, and any variants thereof with one or more nucleic acid substitutions that result in conservative amino acid substitutions. In one or more embodiments, the invention further provides isolated nucleic acid sequences encoding a canine heavy chain constant region having 95% sequence identity to SEQ ID NO: 128, and a nucleotide sequence encoding a canine light chain constant region having 95% sequence identity to SEQ ID NO: 130, and any variants thereof with one or more nucleic acid substitutions that result in conservative amino acid substitutions.

[0106] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 25 and a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 32, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0107] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1 and TGFβ3 but not TGFβ2, the nucleic acid sequences further comprising a nucleotide sequence encoding a feline heavy chain constant region having 95% sequence identity to SEQ ID NO: 132, and a nucleotide sequence encoding a feline light chain constant region having 95% sequence identity to SEQ ID NO: 134, and any variants thereof having one or more nucleic acid substitutions that result in conservative amino acid substitutions.

[0108] In one or more embodiments, the present invention provides a vector comprising a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1 and TGFβ3, but not TGFβ2.

[0109] In one embodiment, the invention provides a host cell comprising a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds canine or feline TGFβ1 and TGFβ3 but not TGFβ2. In one embodiment, the invention provides a host cell comprising a vector comprising a nucleic acid encoding an antigen binding protein of the invention. In one embodiment, the invention provides a host cell that produces the antigen binding protein of the invention.

[0110] In one or more embodiments, the invention provides a method of producing an antigen binding protein of the invention that specifically binds canine or feline TGFβ1 and TGFβ3 but not TGFβ2, the method comprising culturing a host cell of the invention under conditions that result in the production of the antigen binding protein, and isolating the antigen binding protein from the host cell or from the host cell culture medium.

[0111] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline transforming growth factor beta-1 (TGFβ1), transforming growth factor 2 (TGFβ2), and transforming growth factor 3 (TGFβ3). In one embodiment, the antigen binding protein is capable of binding to an epitope region on TGFβ1 comprising amino acids 57-66 and amino acids 90-103 of SEQ ID NO:223. In one embodiment, the antigen binding protein is further capable of binding to amino acids 25-43 of SEQ ID NO:223. In one embodiment, the antigen binding protein is further capable of binding to amino acids 67-89 of SEQ ID NO:223.

[0112] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the antigen binding protein comprises an amino acid sequence with at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 270 [glycine(G)-tyrosine(Y)-(X1)-phenylalanine(F)-(X2)-(X3)-tyrosine(Y)], where (X1) comprises threonine (T) or isoleucine (I), (X2) comprises isoleucine (I) or methionine (M), and (X3) comprises threonine (T) or lysine (K), and a complementarity determining region 1 (CDR1) that has at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 271 [phenylalanine(F)-proline(P)-(X4)-(X5)-glycine(G)-(X6)]. a heavy chain variable region (VH) comprising: a complementarity determining region 2 (CDR2) having an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 272 [glycine(G)-(X7)-glycine(G)-asparagine(N)-tyrosine(Y)-alanine(A)-leucine(L)-aspartic acid(D)-alanine(A)-methionine(M)-aspartic acid(D)-tyrosine(Y), wherein (X7) comprises aspartic acid (D) or tyrosine (Y);and a light chain variable region (VL) comprising: a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 69; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 70; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 273 [glutamine (Q)-glutamine (Q)-asparagine (N)-(X8)-glutamic acid (E)-aspartic acid (D)-proline (P)-leucine (L)-(X9)), wherein (X8) comprises asparagine (N) or aspartic acid (D), and (X9) comprises threonine (T) or serine (S); and any variants thereof having one or more conservative amino acid substitutions. In some embodiments, the variant is not a conservative amino acid substitution. ;

[0113] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, the antigen binding proteins comprising a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, and SEQ ID NO:277; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:67, SEQ ID NO:278, SEQ ID NO:279, and SEQ ID NO:280; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:68, or SEQ ID NO:281. and a light chain variable region (VL) comprising: a heavy chain variable region (VH) comprising: a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 69; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 70; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 282, and SEQ ID NO: 283; and any variant thereof having one or more conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino acid substitution.

[0114] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the antigen binding protein comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence SEQ ID NO: 66, a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 67, and a complementarity determining region 3 (CDR4) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 68. and a light chain variable region (VL) comprising: a complementarity determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 69; a complementarity determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 70; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 71; and any variants thereof having one or more conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino acid substitution.

[0115] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the antigen binding proteins comprise caninized, feline, humanized, or chimeric antigen binding proteins. In one embodiment, the antigen binding protein comprises a caninized antigen binding protein.

[0116] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3, the antigen binding protein being selected from the group consisting of SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:216, and SEQ ID NO:218; and any variant thereof having one or more conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino acid substitution.

[0117] In one embodiment, the present invention provides a. a heavy chain variable region (VH) comprising SEQ ID NO: 181, and a light chain variable region (VL) comprising SEQ ID NO: 85; b. a heavy chain variable region (VH) comprising SEQ ID NO: 181, and a light chain variable region (VL) comprising SEQ ID NO: 216; c. a heavy chain variable region (VH) comprising SEQ ID NO: 183, and a light chain variable region (VL) comprising SEQ ID NO: 85; d. a heavy chain variable region (VH) comprising SEQ ID NO: 185, and a light chain variable region (VL) comprising SEQ ID NO: 85; e. a heavy chain variable region (VH) comprising SEQ ID NO: 187, and a light chain variable region (VL) comprising SEQ ID NO: 85; f. a heavy chain variable region (VH) comprising SEQ ID NO: 187, and a light chain variable region (VL) comprising SEQ ID NO: 218; g. A heavy chain variable region (VH) comprising SEQ ID NO: 185, and a light chain variable region (VL) comprising SEQ ID NO: 218; h. a heavy chain variable region (VH) comprising SEQ ID NO: 189, and a light chain variable region (VL) comprising SEQ ID NO: 218; i. a heavy chain variable region (VH) comprising SEQ ID NO: 191, and a light chain variable region (VL) comprising SEQ ID NO: 218; j. a heavy chain variable region (VH) comprising SEQ ID NO: 193, and a light chain variable region (VL) comprising SEQ ID NO: 218; k. A heavy chain variable region (VH) comprising SEQ ID NO: 195, and a light chain variable region (VL) comprising SEQ ID NO: 218; l. A heavy chain variable region (VH) comprising SEQ ID NO: 197, and a light chain variable region (VL) comprising SEQ ID NO: 218; m. a heavy chain variable region (VH) comprising SEQ ID NO: 199, and a light chain variable region (VL) comprising SEQ ID NO: 218; n. A heavy chain variable region (VH) comprising SEQ ID NO: 199, and a light chain variable region (VL) comprising SEQ ID NO: 85; o. a heavy chain variable region (VH) comprising SEQ ID NO: 193, and a light chain variable region (VL) comprising SEQ ID NO: 85; p. a heavy chain variable region (VH) comprising SEQ ID NO: 195, and a light chain variable region (VL) comprising SEQ ID NO: 85; q. a heavy chain variable region (VH) comprising SEQ ID NO: 191, and a light chain variable region (VL) comprising SEQ ID NO: 85; r. a heavy chain variable region (VH) comprising SEQ ID NO: 197, and a light chain variable region (VL) comprising SEQ ID NO: 85; s. a heavy chain variable region (VH) comprising SEQ ID NO: 189, and a light chain variable region (VL) comprising SEQ ID NO: 85; t. A heavy chain variable region (VH) comprising SEQ ID NO: 201, and a light chain variable region (VL) comprising SEQ ID NO: 218. u. a heavy chain variable region (VH) comprising SEQ ID NO: 203, and a light chain variable region (VL) comprising SEQ ID NO: 218; v. A heavy chain variable region (VH) comprising SEQ ID NO: 205, and a light chain variable region (VL) comprising SEQ ID NO: 218 w. a heavy chain variable region (VH) comprising SEQ ID NO: 201, and a light chain variable region (VL) comprising SEQ ID NO: 85; x. A heavy chain variable region (VH) comprising SEQ ID NO: 205, and a light chain variable region (VL) comprising SEQ ID NO: 85 y. A heavy chain variable region (VH) paired with a light chain variable region (VL) that has at least 95% sequence identity to the amino acids comprising the heavy chain variable region (VH) comprising SEQ ID NO: 215, and the light chain variable region (VL) comprising SEQ ID NO: 218; and Antigen binding proteins are provided, including any variants thereof having one or more conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino acid substitution.

[0118] In one or more embodiments, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the antigen binding proteins comprise a canine heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 127. In one or more embodiments, the invention provides antigen binding proteins comprising a canine heavy chain constant region comprising the amino acid sequence SEQ ID NO: 127. In one or more embodiments, the canine heavy chain constant region comprises conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino acid substitution.

[0119] In one or more embodiments, the invention provides antigen binding proteins comprising a canine light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 129. In one or more embodiments, the invention provides antigen binding proteins comprising a canine light chain constant region comprising the amino acid sequence SEQ ID NO: 129. In one or more embodiments, the canine light chain constant region comprises conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino acid substitution.

[0120] In one embodiment, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, including felineized antigen binding proteins. In one embodiment, the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NO: 120 or SEQ ID NO: 122, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 99, and any variant thereof with one or more conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino acid substitution.

[0121] In one embodiment, the invention provides an antigen binding protein that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 73, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 85, and any variant thereof with one or more conservative amino acid substitutions.

[0122] In one or more embodiments an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3, further comprising a heavy chain constant region having at least 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 127, and a light chain constant region having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 129, and any variant thereof having one or more conservative amino acid substitutions. In one or more embodiments the variant is not a conservative amino acid substitution.

[0123] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the antigen binding proteins comprise a feline heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 131. In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the antigen binding proteins comprise a feline light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 133.

[0124] In one embodiment, the invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, including humanized antigen binding proteins, hi one embodiment, the invention provides antigen binding proteins, including chimeric antigen binding proteins.

[0125] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, the proteins being selected from the group consisting of monoclonal antigen binding proteins, single-chain antigen binding proteins, tetrameric antigen binding proteins, tetravalent antigen binding proteins, multispecific antigen binding proteins, domain-specific antigen binding proteins, domain-deleted antigen binding proteins, fusion proteins, ScFc fusion proteins, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, ScFv fragments, Fd fragments, single-domain antigen binding proteins, dAb fragments, small modular immunopharmaceutical (SMIP) nanobodies, and IgNAR molecules. In one embodiment, the antigen binding protein is a monoclonal antigen binding protein.

[0126] In one or more embodiments, the present invention provides antigen binding proteins that specifically bind to canine or feline TGFβ1, TGFβ2, and TGFβ3, wherein the binding proteins are for use in reducing or eliminating a TGFβ-associated disorder. In one embodiment, the TGFβ-associated disorder is selected from the group consisting of a fibrotic disorder, a connective tissue disorder, a bone disorder, and a cell proliferation disorder. In one embodiment, the TGFβ-associated disorder comprises a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, liver cirrhosis, glial scarring, and systemic sclerosis / scleroderma. In one embodiment, the TGFβ disorder is renal fibrosis / chronic kidney disease.

[0127] In one or more embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3, and a pharmaceutically acceptable carrier.

[0128] In one or more embodiments, an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3 in a canine or feline provides a method of treating a subject for a TGFβ-related disorder by administering to the subject a therapeutic amount of a pharmaceutical composition of the invention. In one or more embodiments, the invention provides a method of treating a canine for a TGFβ-related disorder. In one embodiment, the invention provides a method for treating a feline for a TGFβ-related disorder. In one embodiment, the invention provides a method for treating a human for a TGFβ-related disorder. In one embodiment, the method provides for administering a therapeutically effective amount of a pharmaceutical composition comprising an antigen binding protein of the invention. In one or more embodiments, the invention provides that the TGFβ-related disorder is selected from the group consisting of a fibrotic disorder, a connective tissue disorder, a bone disorder, and a cell proliferation disorder. In one embodiment, the TGFβ-related disorder comprises a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, liver cirrhosis, glial scarring, and systemic sclerosis / scleroderma. In one embodiment, the TGFβ disorder is renal fibrosis / chronic kidney disease.

[0129] In one or more embodiments, the present invention provides methods of inhibiting TGFβ activity in a subject by administering a pharmaceutical composition comprising an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the subject comprises a canine, feline, or human. In one embodiment, the subject comprises a canine. In one embodiment, the subject comprises a feline. In one embodiment, the subject comprises a human.

[0130] In one or more embodiments, the present invention provides isolated nucleic acid sequences having at least about 95% sequence identity to a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds canine or feline TGFβ1, TGFβ2, and TGFβ3, and any variants thereof having one or more nucleic acid substitutions that result in conservative amino acid substitutions.

[0131] In one or more embodiments, the invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1, TGFβ2, and TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 72 and a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 84, and any variants thereof having one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0132] In one or more embodiments, the invention provides isolated nucleic acid sequences encoding antigen binding proteins of the invention that specifically bind canine or feline TGFβ1, TGFβ2, and TGFβ3, including a nucleotide sequence encoding a VH having 95% sequence identity to SEQ ID NO: 72, a nucleotide sequence encoding a VL having 95% sequence identity to SEQ ID NO: 84, a nucleotide sequence encoding a canine heavy chain constant region having 95% sequence identity to SEQ ID NO: 128, and a nucleotide sequence encoding a canine light chain constant region having 95% sequence identity to SEQ ID NO: 130, and any variant thereof with one or more nucleic acid substitutions resulting in conservative amino acid substitutions.

[0133] In one or more embodiments, the present invention provides vectors comprising a nucleic acid sequence encoding an antigen binding protein of the invention that specifically binds to canine or feline TGFβ1, TGFβ2, and TGFβ3.

[0134] In one embodiment, the invention provides a host cell comprising a nucleic acid sequence encoding a present antigen binding protein that specifically binds canine or feline TGFβ1, TGFβ2, and TGFβ3. In one embodiment, the invention provides a host cell comprising a vector comprising a nucleic acid encoding an antigen binding protein of the invention. In one embodiment, the invention provides a host cell that produces an antigen binding protein of the invention.

[0135] In one or more embodiments, the invention provides a method of producing an antigen binding protein of the invention that specifically binds canine or feline TGFβ1, TGFβ2, and TGFβ3, the method comprising culturing a host cell of the invention under conditions that result in the production of the antigen binding protein, and isolating the antigen binding protein from the host cell or from the host cell culture medium. [Brief explanation of the drawings]

[0136] [Figure 1] 1 presents epitope mapping data for anti-TGFβ antibodies of the present invention that bind to TGFβ1. [Figure 2] 1 depicts the general structure of native mouse immunoglobulin G (IgG) highlighting the antigen-binding site. [Figure 3] 1 is a schematic representation of the general structure of one embodiment of mouse:canine IgG. [Figure 4] Depicts heterochimeric molecules. [Figure 5] Represents speciation or caninization of mouse IgG. [Figure 6] 1 is a graphical representation of ZTS-426 SMAD3 inhibition curves for TGFβ1 and TGFβ3. [Figure 7] 1 shows a model of the secondary structure of the mature TGFβ1 dimer with the bound Fab fragment of ZTS-426. [Figure 8] 1 depicts a model of the superposition of the ZTS-426 Fab complex with the TGFβ1-bound TGFβRI / TGFβRII complex. [Figure 9] 1 is a graphical representation of modulation of disease progression in Alport dogs treated with ZTS-426 at different doses. [Figure 10] 1 depicts a phase 2 scenario plan for research related to the treatment of CKD. [Figure 11A] 1 is a graphical representation of survival curves in relation to time to sCr doubling after treatment with ZTS-426. [Figure 11B]Graphical representation of survival curve / time to stage 4 (sCr>5.0) after treatment with ZTS-426. [Figure 11C] 1 is a graphical representation of survival curves / time to Phos elevation (>4.5 g / dl) after treatment with ZTS-426. [Figure 11D] Graphical representation of survival curves / death due to CKD / time to discontinuation after treatment with ZTS-426. [Figure 11E] 1 is a graphical representation of survival curves / time to end of study following treatment with ZTS-426. [Figure 11F] 1 is a graphical representation of survival curves / time to uremic crisis after treatment with ZTS-426. [Figure 12] 1 is a graphical representation of mean dog serum ZTS-4155 concentration-time graphs. DETAILED DESCRIPTION OF THE INVENTION

[0137] A brief description of arrays SEQ ID NO: 1 contains the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the murine 04H09 monoclonal antibody. SEQ ID NO: 2 contains the amino acid sequence of the variable region of the heavy chain (VH) of the murine 04H09 monoclonal antibody. SEQ ID NO:3 contains the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the murine 04H09 monoclonal antibody. SEQ ID NO: 4 contains the amino acid sequence of the variable region of the light chain (VL) of the murine 04H09 monoclonal antibody. SEQ ID NO: 5 comprises the amino acid sequence of the first CDR in the heavy chain of the 04H09 monoclonal antibody, referred to herein as 04H09 CDR-H1. SEQ ID NO: 6 comprises the amino acid sequence of the second CDR in the heavy chain of the 04H09 monoclonal antibody, referred to herein as 04H09 CDR-H2. SEQ ID NO: 7 contains the amino acid sequence of the third CDR in the heavy chain of the 04H09 monoclonal antibody, referred to herein as 04H09 CDR-H3. SEQ ID NO: 8 comprises the amino acid sequence of the first CDR in the light chain of the 04H09 monoclonal antibody, designated herein as 04H09 CDR-K1. SEQ ID NO: 9 contains the amino acid sequence of the second CDR in the light chain of the 04H09 monoclonal antibody, designated herein as 04H09 CDR-K2. SEQ ID NO: 10 comprises the amino acid sequence of the third CDR in the light chain of the 04H09 monoclonal antibody, designated herein as 04H09 CDR-K3. SEQ ID NO: 11 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the caninized 04H09 antibody, designated herein as Can04H09-VH1. SEQ ID NO: 12 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized 04H09 antibody, designated herein as Can04H09-VH1. SEQ ID NO: 13 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the caninized 04H09 antibody, designated herein as Can04H09-VH2. SEQ ID NO: 14 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized 04H09 antibody, designated herein as Can04H09-VH2. SEQ ID NO: 15 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the caninized 04H09 antibody, designated herein as Can04H09-VH3. SEQ ID NO: 16 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized 04H09 antibody, designated herein as Can04H09-VH3. SEQ ID NO: 17 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL1. SEQ ID NO: 18 contains the amino acid sequence of the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL1. SEQ ID NO: 19 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL2. SEQ ID NO: 20 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL2. SEQ ID NO: 21 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL3. SEQ ID NO: 22 contains the amino acid sequence of the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL3. SEQ ID NO: 23 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL4. SEQ ID NO: 24 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized 04H09 antibody, designated herein as Can04H09-VL4. SEQ ID NO: 25 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the felinized 04H09 antibody, designated herein as Fel04H09-H636. SEQ ID NO: 26 contains the amino acid sequence of the variable region of the heavy chain (VH) of the felinized 04H09 antibody, referred to herein as Fel04H09-H636. SEQ ID NO: 27 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the felinized 04H09 antibody, designated herein as Fel04H09-H1-2. SEQ ID NO: 28 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the felinized 04H09 antibody, designated herein as Fel04H09-H1-2. SEQ ID NO: 29 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the felinized 04H09 antibody, designated herein as Fel04H09-H618. SEQ ID NO: 30 contains the amino acid sequence of the variable region of the heavy chain (VH) of the felinized 04H09 antibody, designated herein as Fel04H09-H618. SEQ ID NO: 31 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized 04H09 antibody, designated herein as Fel04H09-K4-1. SEQ ID NO: 32 contains the amino acid sequence of the variable region of the light chain (VL) of the felinized 04H09 antibody, designated herein as Fel04H09-K4-1. SEQ ID NO: 33 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized 04H09 antibody, designated herein as Fel04H09-K36. SEQ ID NO: 34 contains the amino acid sequence of the variable region of the light chain (VL) of the felinized 04H09 antibody, designated herein as Fel04H09-K36. SEQ ID NO: 35 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized 04H09 antibody, designated herein as Fel04H09-K1-1. SEQ ID NO: 36 comprises the amino acid sequence of the variable region of the light chain (VL) of the felinized 04H09 antibody, designated herein as Fel04H09-K1-1. SEQ ID NO: 37 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the murine SL501 monoclonal antibody. SEQ ID NO: 38 contains the amino acid sequence of the variable region of the heavy chain (VH) of the murine SL501 monoclonal antibody. SEQ ID NO: 39 contains the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the murine SL501 monoclonal antibody. SEQ ID NO: 40 contains the amino acid sequence of the variable region of the light chain (VL) of the murine SL501 monoclonal antibody. SEQ ID NO: 41 comprises the amino acid sequence of the first CDR in the heavy chain of the SL501 monoclonal antibody, referred to herein as SL501 CDR-H1. SEQ ID NO: 42 comprises the amino acid sequence of the second CDR in the heavy chain of the SL501 monoclonal antibody, referred to herein as SL501 CDR-H2. SEQ ID NO: 43 comprises the amino acid sequence of the third CDR in the heavy chain of the SL501 monoclonal antibody, referred to herein as SL501 CDR-H3. SEQ ID NO: 44 comprises the amino acid sequence of the first CDR in the light chain of the SL501 monoclonal antibody, designated herein as SL501 CDR-K1. SEQ ID NO: 45 comprises the amino acid sequence of the second CDR in the light chain of the SL501 monoclonal antibody, designated herein as SL501 CDR-K2. SEQ ID NO: 46 comprises the amino acid sequence of the third CDR in the light chain of the SL501 monoclonal antibody, designated herein as SL501 CDR-K3. SEQ ID NO: 47 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody, referred to herein as Can SL501-VH1. SEQ ID NO: 48 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody, designated herein as Can SL501-VH2. SEQ ID NO: 49 contains the amino acid sequence of the variable region of the light chain (VL) of the caninized SL501 antibody, designated herein as Can SL501-VL1. SEQ ID NO: 50 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized SL501 antibody, designated herein as Can SL501-VL2. SEQ ID NO: 51 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized SL501 antibody, designated herein as Can SL501-VL2. SEQ ID NO: 52 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the partially caninized SL501 antibody, referred to herein as Can SL501-VL-hybrid. SEQ ID NO: 53 contains the amino acid sequence of the variable region of the light chain (VL) of the partially caninized SL501 antibody, referred to herein as Can SL501-VL-hybrid. SEQ ID NO: 54 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the caninized SL501 antibody, designated herein as Can SL501-VH3. SEQ ID NO: 55 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody, designated herein as Can SL501-VH3. SEQ ID NO: 56 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the can SL501-VH3 antibody, which has nucleotide substitutions resulting in amino acid substitutions at positions 44 and 46 within the framework 2 region of can SL501-VH3, referred to herein as can SL501-VH3-FW2. SEQ ID NO: 57 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the can SL501-VH3 antibody, which has amino acid substitutions at positions 44 and 46 within the framework 2 region of can SL501-VH3, referred to herein as can SL501-VH3-FW2. SEQ ID NO: 58 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the felinized SL501 antibody, designated herein as Fel SL501-VH3-9. SEQ ID NO: 59 contains the amino acid sequence of the variable region of the heavy chain (VH) of the felinized SL501 antibody, referred to herein as Fel SL501-VH3-9. SEQ ID NO: 60 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized SL501 antibody, designated herein as Fel SL501-VL1-1. SEQ ID NO: 61 comprises the amino acid sequence of the variable region of the light chain (VL) of the felinized SL501 antibody, referred to herein as Fel SL501-VL1-1. SEQ ID NO: 62 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the mHcLb monoclonal antibody. SEQ ID NO: 63 contains the amino acid sequence of the variable region of the heavy chain (VH) of the mHcLb monoclonal antibody. SEQ ID NO: 64 contains the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the mHcLb monoclonal antibody. SEQ ID NO: 65 contains the amino acid sequence of the variable region of the light chain (VL) of the mHcLb monoclonal antibody. SEQ ID NO: 66 comprises the amino acid sequence of the first CDR in the heavy chain of the mHcLb monoclonal antibody, referred to herein as mHcLb CDR-H1. SEQ ID NO: 67 comprises the amino acid sequence of the second CDR in the heavy chain of the mHcLb monoclonal antibody, referred to herein as mHcLb CDR-H2. SEQ ID NO: 68 comprises the amino acid sequence of the third CDR in the heavy chain of the mHcLb monoclonal antibody, referred to herein as mHcLb CDR-H3. SEQ ID NO: 69 comprises the amino acid sequence of the first CDR in the light chain of the mHcLb monoclonal antibody, designated herein as mHcLb CDR-K1. SEQ ID NO: 70 comprises the amino acid sequence of the second CDR in the light chain of the mHcLb monoclonal antibody, designated herein as mHcLb CDR-K2. SEQ ID NO: 71 comprises the amino acid sequence of the third CDR in the light chain of the mHcLb monoclonal antibody, designated herein as mHcLb CDR-K3. SEQ ID NO: 72 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the caninized HcLb antibody, designated herein as CanHcLb-VH1. SEQ ID NO: 73 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized HcLb antibody, designated herein as CanHcLb-VH1. SEQ ID NO: 74 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the caninized HcLb antibody, designated herein as CanHcLb-VH2. SEQ ID NO: 75 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized HcLb antibody, designated herein as CanHcLb-VH2. SEQ ID NO: 76 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the caninized HcLb antibody, designated herein as CanHcLb-VH3. SEQ ID NO: 77 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized HcLb antibody, designated herein as CanHcLb-VH3. SEQ ID NO: 78 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL1. SEQ ID NO: 79 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL1. SEQ ID NO: 80 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL2. SEQ ID NO: 81 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL2. SEQ ID NO: 82 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL3. SEQ ID NO: 83 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL3. SEQ ID NO: 84 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL4. SEQ ID NO: 85 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL4. SEQ ID NO: 86 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL5. SEQ ID NO: 87 comprises the amino acid sequence of the variable region of the light chain (VL) of the caninized HcLb antibody, designated herein as CanHcLb-VL5. SEQ ID NO: 88 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the canHcLb-VL4 antibody, having a nucleotide substitution resulting in an amino acid substitution at position 71 within the framework 2 region of canHcLb-VL4, referred to herein as canHcLb-VL4-S71P. SEQ ID NO: 89 comprises the amino acid sequence of the variable region of the light chain (VH) of the canHcLb-VL4 antibody, with an amino acid substitution at position 71 within the framework 2 region of canHcLb-VL4, referred to herein as canHcLb-VL4-S71P. SEQ ID NO: 90 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the canHcLb-VL4 antibody, having a nucleotide substitution resulting in an amino acid substitution at position 73 within the framework 2 region of canHcLb-VL4, referred to herein as canHcLb-VL4-Q73K. SEQ ID NO: 91 comprises the amino acid sequence of the variable region of the light chain (VH) of the canHcLb-VL4 antibody, with an amino acid substitution at position 73 within the framework 2 region of canHcLb-VL4, referred to herein as canHcLb-VL4-Q73K. SEQ ID NO: 92 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the canHcLb-VL4 antibody, having nucleotide substitutions resulting in amino acid substitutions at positions 71 and 73 within the framework 2 region of canHcLb-VL4, referred to herein as canHcLb-VL4-S71P. SEQ ID NO: 93 comprises the amino acid sequence of the variable region of the light chain (VH) of the canHcLb-VL4 antibody, with amino acid substitutions at positions 71 and 73 within the framework 2 region of canHcLb-VL4, referred to herein as canHcLb-VL4-S71P-Q73K. SEQ ID NO: 94 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the felinized HcLb antibody, designated herein as FelHcLb-H636. SEQ ID NO: 95 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the felinized HcLb antibody, designated herein as FelHcLb-H636. SEQ ID NO: 96 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the felinized HcLb antibody, designated herein as FelHcLb-H1-1. SEQ ID NO: 97 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the felinized HcLb antibody, designated herein as FelHcLb-H1-1. SEQ ID NO: 98 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K1-1. SEQ ID NO: 99 comprises the amino acid sequence of the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K1-1. SEQ ID NO: 100 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K36. SEQ ID NO: 101 comprises the amino acid sequence of the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K36. SEQ ID NO: 102 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K4-1. SEQ ID NO: 103 comprises the amino acid sequence of the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K2D-2. SEQ ID NO: 104 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K1-1. SEQ ID NO: 105 comprises the amino acid sequence of the variable region of the light chain (VL) of the felinized HcLb antibody, designated herein as FelHcLb-K2D-2. SEQ ID NO: 106 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the felinized HcLb antibody, designated herein as FelHcLb-H618s. SEQ ID NO: 107 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the felinized HcLb antibody, referred to herein as FelHcLb-H618s. SEQ ID NO: 108 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H636x. SEQ ID NO: 109 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H636x. SEQ ID NO: 110 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H1-1x. SEQ ID NO: 111 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H1-1x. SEQ ID NO: 112 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H618x. SEQ ID NO: 113 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H618x. SEQ ID NO: 114 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H634x. SEQ ID NO: 115 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H2 region, referred to herein as FelHcLb-H634x. SEQ ID NO: 116 comprises the amino acid sequence of a modified version of the second CDR in the heavy chain of the mHcLb monoclonal antibody, referred to herein as mHcLb CDR-H2x. SEQ ID NO: 117 comprises the amino acid sequence of a modified version of the first CDR in the heavy chain of the mHcLb monoclonal antibody, referred to herein as mHcLb CDR-H1-WMN. SEQ ID NO: 118 comprises the amino acid sequence of a modified version of the first CDR in the heavy chain of the mHcLb monoclonal antibody, referred to herein as mHcLb CDR-H1-MN. SEQ ID NO: 119 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H1 region, referred to herein as FelHcLb-H1-1WMN. SEQ ID NO: 120 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H1 region, referred to herein as FelHcLb-H1-1WMN. SEQ ID NO: 121 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody having modified CDR-H1 and CDR-H2 regions, referred to herein as FelHcLb-H1-1xWMN. SEQ ID NO: 122 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with modified CDR-H1 and CDR-H2 regions, referred to herein as FelHcLb-H1-1xWMN. SEQ ID NO: 123 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H1 region, referred to herein as FelHcLb-H1-1WM. SEQ ID NO: 124 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with a modified CDR-H1 region, referred to herein as FelHcLb-H1-1WM. SEQ ID NO: 125 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of a felinized HcLb antibody having modified CDR-H1 and CDR-H2 regions, referred to herein as FelHcLb-H1-1xWM. SEQ ID NO: 126 comprises the amino acid sequence of the variable region of the heavy chain (VH) of a felinized HcLb antibody with modified CDR-H1 and CDR-H2 regions, referred to herein as FelHcLb-H1-1xWM. SEQ ID NO: 127 comprises the amino acid sequence of the canine heavy chain constant region of a caninized antibody of the invention. SEQ ID NO: 128 comprises the nucleotide sequence of DNA encoding the canine heavy chain constant region of a caninized antibody of the invention. SEQ ID NO: 129 contains the amino acid sequence of the canine light (kappa) chain constant region of a caninized antibody of the invention. SEQ ID NO: 130 comprises the nucleotide sequence encoding the canine light chain constant region of a caninized antibody of the invention. SEQ ID NO: 131 comprises the amino acid sequence of the feline heavy chain constant region of a felineized antibody of the invention. SEQ ID NO: 132 comprises the nucleotide sequence encoding the feline heavy chain constant region of a felineized antibody of the invention. SEQ ID NO: 133 comprises the amino acid sequence of the feline light chain constant region of a felineized antibody of the invention. SEQ ID NO: 134 comprises the nucleotide sequence encoding the feline light chain constant region of a felineized antibody of the invention. SEQ ID NO: 135 comprises the amino acid sequence of a single substitution (D108E) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 136 comprises the amino acid sequence of a single substitution (D108P) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 137 comprises the amino acid sequence of a single substitution (D108Q) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 138 comprises the amino acid sequence of a single substitution (D108N) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 139 comprises the amino acid sequence of a single substitution (D108S) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 140 comprises the amino acid sequence of a single substitution (D108T) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 141 comprises the amino acid sequence of a single substitution (D108K) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 142 comprises the amino acid sequence of a single substitution (D108R) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 143 comprises the amino acid sequence of a single substitution (D108H) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 144 comprises the amino acid sequence of a single substitution (P109S) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 145 comprises the amino acid sequence of a single substitution (P109H) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 146 comprises the amino acid sequence of a single substitution (P109Y) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 147 comprises the amino acid sequence of a single substitution (P109W) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 148 comprises the amino acid sequence of a single substitution (P109F) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 149 comprises the amino acid sequence of a single substitution (P109T) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 150 comprises the amino acid sequence of a single substitution (P109A) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 151 comprises the amino acid sequence of a single substitution (P109G) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 152 comprises the amino acid sequence of a single substitution (Q110S) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 153 comprises the amino acid sequence of a single substitution (Q110N) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 154 comprises the amino acid sequence of a single substitution (Q110D) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 155 comprises the amino acid sequence of a single substitution (Q110E) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 156 comprises the amino acid sequence of a single substitution (Q110K) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 157 comprises the amino acid sequence of a single substitution (Q110R) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 158 comprises the amino acid sequence of a single substitution (Q110H) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 159 comprises the amino acid sequence of a single substitution (Q110T) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 160 comprises the amino acid sequence of a single substitution (Q110V) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 161 comprises the amino acid sequence of a single substitution (Y111P) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 162 comprises the amino acid sequence of a single substitution (Y111F) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 163 comprises the amino acid sequence of a single substitution (Y111W) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 164 comprises the amino acid sequence of a single substitution (Y111H) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 165 comprises the amino acid sequence of a single substitution (Y111M) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 166 comprises the amino acid sequence of a single substitution (Y111I) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 167 comprises the amino acid sequence of a single substitution (Y111L) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 168 comprises the amino acid sequence of a single substitution (Y111V) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 169 comprises the amino acid sequence of a single substitution (Y111T) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 170 comprises the amino acid sequence of a single substitution (Y111E) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 171 comprises the amino acid sequence of a single substitution (S112E) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 172 comprises the amino acid sequence of a single substitution (S112Q) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 173 comprises the amino acid sequence of a single substitution (S112N) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 174 comprises the amino acid sequence of a single substitution (S112T) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 175 comprises the amino acid sequence of a single substitution (S112A) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 176 comprises the amino acid sequence of a single substitution (S112G) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 177 comprises the amino acid sequence of a single substitution (S112P) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 178 comprises the amino acid sequence of a single substitution (S112D) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 179 comprises the amino acid sequence of a single substitution (S112L) within CDR H3 in the heavy chain variable region (VH) of Can SL501-VH3. SEQ ID NO: 180 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as HcLb / mat / P / H3 / 100 / DY. SEQ ID NO: 181 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as HcLb / mat / P / H3 / 100 / DY. SEQ ID NO: 182 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as HcLb / mat / P / H2 / 55 / SW. SEQ ID NO: 183 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as HcLb / mat / P / H2 / 55 / SW. SEQ ID NO: 184 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 54AG / 100DY. SEQ ID NO: 185 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 54AG / 100DY. SEQ ID NO: 186 contains the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY. SEQ ID NO: 187 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY. SEQ ID NO: 188 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 28 / TI. SEQ ID NO: 189 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 28 / TI. SEQ ID NO: 190 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 30 / IM. SEQ ID NO: 191 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 30 / IM. SEQ ID NO: 192 contains the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as CanHcLb / 55SW / 100DY / 57 / SM. SEQ ID NO: 193 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as CanHcLb / 55SW / 100DY / 57 / SM. SEQ ID NO: 194 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 57 / SV. SEQ ID NO: 195 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 57 / SV. SEQ ID NO: 196 contains the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 31 / TK. SEQ ID NO: 197 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 31 / TK. SEQ ID NO: 198 contains the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK. SEQ ID NO: 199 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK. SEQ ID NO:200 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK / 28TI. SEQ ID NO: 201 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK / 28TI. SEQ ID NO:202 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK / 57SM. SEQ ID NO: 203 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK / 57SM. SEQ ID NO:204 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK / 28TI / 57SM. SEQ ID NO: 205 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 30IM / 31TK / 28TI / 57SM. SEQ ID NO: 206 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 57SM / 30IM. SEQ ID NO: 207 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 57SM / 30IM. SEQ ID NO: 208 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 57SV / 30IM. SEQ ID NO: 209 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 55SW / 100DY / 57SV / 30IM. SEQ ID NO: 210 comprises the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 57SM / 30IM / 31TF. SEQ ID NO: 211 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 57SM / 30IM / 31TF. SEQ ID NO: 212 contains the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 57SM / 30IM / 31TF / 28TK. SEQ ID NO: 213 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 57SM / 30IM / 31TF / 28TK. SEQ ID NO: 214 contains the nucleotide sequence of DNA encoding the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 57SM / 30IW / 31TF / 28TK. SEQ ID NO: 215 contains the amino acid sequence of the variable region of the heavy chain (VH) of the affinity matured caninized Hclb H1K4 antibody designated herein as canHcLb / 55SW / 100DY / 57SM / 30IW / 31TF / 28TK. SEQ ID NO: 216 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the affinity matured caninized Hclb H1K4 antibody, designated herein as HcLb / mat / K3 / 237 / ND. SEQ ID NO: 217 contains the amino acid sequence of the variable region of the light chain (VL) of the affinity matured caninized Hclb H1K4 antibody, designated herein as HcLb / mat / K3 / 237 / ND. SEQ ID NO: 218 comprises the nucleotide sequence of DNA encoding the variable region of the light chain (VL) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 237ND / 242TS. SEQ ID NO: 219 contains the amino acid sequence of the variable region of the light chain (VL) of the affinity matured caninized Hclb H1K4 antibody, designated herein as canHcLb / 237ND / 242TS. SEQ ID NO: 220 comprises the amino acid sequence of canine TGFβ1. SEQ ID NO: 221 comprises the amino acid sequence of human TGFβ1. SEQ ID NO: 222 comprises the amino acid sequence of feline TGFβ1. SEQ ID NO: 223 comprises the amino acid sequence of a TGFβ1 fragment containing an epitope for an anti-TGF antibody of the invention, as shown in FIG. SEQ ID NO: 224 comprises the amino acid sequence of the VH CDR3 of the SL501 antigen binding protein, wherein (X1) can be (K or R), (X2) can be (T or A), (X3) can be (Q, N, D, E, or K), and X4 can be (S, E, Q, or D). SEQ ID NO: 225 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108E point mutation. SEQ ID NO: 226 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108P point mutation. SEQ ID NO: 227 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108Q point mutation. SEQ ID NO: 228 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108N point mutation. SEQ ID NO: 229 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108S point mutation. SEQ ID NO: 230 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108T point mutation. SEQ ID NO: 231 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108K point mutation. SEQ ID NO: 232 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108R point mutation. SEQ ID NO: 233 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the D108H point mutation. SEQ ID NO: 234 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109S point mutation. SEQ ID NO: 235 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109H point mutation. SEQ ID NO: 236 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109Y point mutation. SEQ ID NO: 237 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109W point mutation. SEQ ID NO: 238 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109F point mutation. SEQ ID NO: 239 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109T point mutation. SEQ ID NO: 240 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109A point mutation. SEQ ID NO: 241 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the P109G point mutation. SEQ ID NO: 242 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110S point mutation. SEQ ID NO: 243 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110N point mutation. SEQ ID NO: 244 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110D point mutation. SEQ ID NO: 245 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110E point mutation. SEQ ID NO: 246 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110K point mutation. SEQ ID NO: 247 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110R point mutation. SEQ ID NO: 248 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110H point mutation. SEQ ID NO: 249 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110T point mutation. SEQ ID NO: 250 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Q110V point mutation. SEQ ID NO: 251 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111P point mutation. SEQ ID NO: 252 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111F point mutation. SEQ ID NO: 253 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111W point mutation. SEQ ID NO: 254 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111H point mutation. SEQ ID NO: 255 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111M point mutation. SEQ ID NO: 256 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111I point mutation. SEQ ID NO: 257 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111L point mutation. SEQ ID NO: 258 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111V point mutation. SEQ ID NO: 259 contains the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111T point mutation. SEQ ID NO: 260 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the Y111E point mutation. SEQ ID NO: 261 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112E point mutation. SEQ ID NO: 262 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112Q point mutation. SEQ ID NO: 263 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112N point mutation. SEQ ID NO: 264 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112T point mutation. SEQ ID NO: 265 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112A point mutation. SEQ ID NO: 266 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112G point mutation. SEQ ID NO: 267 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112P point mutation. SEQ ID NO: 268 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112D point mutation. SEQ ID NO: 269 comprises the amino acid sequence of the variable region of the heavy chain (VH) of the caninized SL501 antibody containing the S112L point mutation. SEQ ID NO: 270 comprises the amino acid sequence of GY-(X1)-F-(X2)-(X3)-Y, where (X1) comprises T or I, (X2) comprises I or M, and (X3) comprises T or K. SEQ ID NO: 271 comprises the amino acid sequence of FP-(X4)-(X5)-G-(X6), where (X4) comprises A or G, (X5) comprises S or W, and (X6) comprises S, M, or V, the amino acid sequence of the variable region of the heavy chain (VH) CDR2 of HcLB. SEQ ID NO: 272 comprises the amino acid sequence of the variable region of the heavy chain (VH) CDR3 of HcLB, G-(X7)-GNYALDAMDY, where (X7) comprises D or Y. SEQ ID NO: 273 comprises the amino acid sequence of QQN-(X8)-EDPL-(X9), where (X8) comprises N or D, and (X9) comprises T or S, and comprises the amino acid sequence of the variable region of the light chain (VL) CDR3 of HcLB. SEQ ID NO: 274 comprises the amino acid sequence of CDR1 of the variable region of the heavy chain (VH) of the HcLB antibody containing the T28I point mutation. SEQ ID NO: 275 comprises the amino acid sequence of CDR1 of the variable region of the heavy chain (VH) of the HcLB antibody containing the I30M point mutation. SEQ ID NO: 276 comprises the amino acid sequence of CDR1 of the variable region of the heavy chain (VH) of the HcLB antibody containing the T31K point mutation. SEQ ID NO: 277 comprises the amino acid sequence of CDR1 of the variable region of the heavy chain (VH) of the HcLB antibody, comprising the I30M and T31K point mutations. SEQ ID NO: 278 contains the amino acid sequence of CDR1 of the variable region of the heavy chain (VH) of the HcLB antibody, containing the T28I, I30M, and T31K point mutations. SEQ ID NO: 279 comprises the amino acid sequence of CDR2 of the variable region of the heavy chain (VH) of the HcLB antibody containing the A54G point mutation. SEQ ID NO: 280 comprises the amino acid sequence of CDR2 of the variable region of the heavy chain (VH) of the HcLB antibody containing the S55W point mutation. SEQ ID NO: 281 comprises the amino acid sequence of CDR2 of the variable region of the heavy chain (VH) of the HcLB antibody containing the S57M point mutation. SEQ ID NO: 282 comprises the amino acid sequence of CDR2 of the variable region of the heavy chain (VH) of the HcLB antibody, comprising the S55W and S57M point mutations. SEQ ID NO: 283 comprises the amino acid sequence of the CDR3 of the variable region of the heavy chain (VH) of the HcLB antibody containing the D100Y point mutation. SEQ ID NO: 284 comprises the amino acid sequence of the CDR3 of the variable region of the light chain (VL) of the HcLB antibody containing the N237D point mutation. SEQ ID NO: 285 comprises the amino acid sequence of the CDR3 of the variable region of the light chain (VH) of the HcLB antibody containing the T242S point mutation. SEQ ID NO: 286 comprises the amino acid sequence of the CDR3 of the variable region of the light chain (VL) of the HcLB antibody, comprising the N237D and T242S point mutations. SEQ ID NO: 287 contains the nucleic acid sequence of the HC-65e canine heavy chain constant region. SEQ ID NO: 288 contains the amino acid sequence of the HC-65e canine heavy chain constant region. SEQ ID NO: 289 contains the nucleotide sequence of the feline heavy chain constant region. SEQ ID NO: 290 comprises the amino acid sequence of the feline heavy chain constant region. SEQ ID NO: 291 contains the nucleic acid sequence of the HC-65 canine heavy chain constant region. SEQ ID NO: 292 contains the amino acid sequence of the HC-65 canine heavy chain constant region. SEQ ID NO:293 contains the nucleotide sequence encoding the amino acid sequence of the variable region of the light chain (VL) of the caninized SL501 antibody, designated herein as Can SL501-VL1.

[0138] The invention disclosed herein provides anti-TGFβ antigen binding proteins / antibodies / antibody fragments (the terms are used interchangeably) that bind to TGFβ1 and / or TGFβ2 and / or TGFβ proteins with high affinity and specificity. The invention further provides antigen binding proteins and polypeptides that are variants of the antigen binding proteins, which also bind to any one of the TGFβ proteins or polypeptides described herein, and methods of making and using the proteins. In some embodiments, the invention also provides polynucleotides encoding the antigen binding proteins and / or polypeptides. The invention disclosed herein also provides methods for preventing and / or treating a TGFβ-related disorder selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders, by administering a therapeutically effective amount of an anti-TGFβ antigen binding protein and respective variant of the invention described herein.

[0139] General Techniques and Definitions It is understood that the present invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Unless otherwise defined, scientific and technical terms used in connection with the invention described herein shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include the singular form. In general, the terminology and techniques utilized in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. It is well known in the art that different techniques may be substituted for those described.

[0140] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application.

[0141] Standard techniques are used for recombinant DNA, oligonucleotide and polynucleotide synthesis, tissue culture, cell transfection and transformation, among many other commonly used techniques well known to those of skill in the art. General techniques well known to those of skill in the art are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and as described, including but not limited to the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., MOLECULAR CLONING: LAB. MANUAL (3rded., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y., 2001), and Ausubel et al. Current Protocols in Molecular Biology (New York: Greene Publishing Association J Wiley Interscience), Oligonucleotide Synthesis (M.J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press, Animal Cell Culture (R.1. Freshney, ed. 1987), Introduction to Cell and Tissue Culture (1.P. Mather and P.E. Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993 - 1998) J.Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987), Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (E. Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (YTDeVita et al. al.,eds.,JBLippincott Company, 1993), as well as different and current protocols used by those skilled in the art. Before describing the invention in detail, several terms used in the context of the invention will be defined. In addition to these terms, other terms will be defined elsewhere herein as necessary. As used above and throughout this disclosure, the following terms and abbreviations shall be understood to have the following meanings unless otherwise indicated: Unless expressly defined herein, technical terms used herein have their art-recognized meanings.

[0142] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to an "antibody" includes a plurality of such antibodies.

[0143] It should be noted that in this disclosure, the terms "comprises," "comprised," "comprising," "contains," "containing," "consisting of," "consisted of," "consisted essentially of," "includes," "included," and the like are defined in accordance with standard U.S. and international patent law practice.

[0144] The term "about" is used herein to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term "at least about" is used herein to indicate the lower limit of a range. For example, it should be clear to one of ordinary skill in the art that the term "having at least about 95% sequence identity" includes 95% sequence identity through 100% sequence identity. The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive, although the present disclosure supports the definition referring to alternatives only and to "and / or."

[0145] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0146] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred to by their commonly accepted one-letter codes. Macromolecular structures, such as polypeptide structures, can be described in terms of various levels of organization. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered three-dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, or cytoplasmic tail domains. Domains are portions of polypeptides that form compact units of the polypeptide. Exemplary domains include domains with enzymatic activity. Domains may be composed of stretches of beta-sheet and smaller organized segments such as alpha helices. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units.

[0147] The term "conservative amino acid substitution" refers to any amino acid substitution for a given amino acid residue, where the substituted residue is chemically so similar to that of the given residue that it does not result in a substantial loss of polypeptide function (e.g., enzymatic activity). Conservative amino acid substitutions are generally known in the art, and examples are described, for example, in U.S. Pat. Nos. 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferred embodiment, the conservative amino acid substitution is any that occurs within one of the following six groups: small aliphatic substantially non-polar residues: Ala, Gly, Pro, Ser, and Thr; • Large aliphatic non-polar residues: Ile, Leu, and Val, Met, Polar negatively charged residues and their amides: Asp and Glu, Amides of polar negatively charged residues: Asn and Gln, His, Polar positively charged residues: Arg and Lys, His, and • Large aromatic residues: Trp and Tyr, Phe.

[0148] In preferred embodiments, the conservative amino acid substitution is any one of the following listed as naturally occurring residue (conservative substitution) pairs: Ala (Ser); Arg (Lys); Asn (Gln; His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn; Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gln; Glu); Met (Leu; Ile); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe), and Val (Ile; Leu).

[0149] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, it can optionally comprise modified amino acids, and it can be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides of the present invention are based on antibodies, the polypeptides can occur as single chains or associated chains.

[0150] As used herein, "antibody," "antigen-binding protein," and the like refer to a polypeptide comprising a region encoded by an immunoglobulin gene or antibody fragment thereof that specifically binds to and recognizes an antigen. An exemplary immunoglobulin (antibody) structural unit may comprise a tetramer, each tetramer consisting of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain and variable heavy chain refer to these light and heavy chains. Antibodies exist, for example, as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. While various antibody fragments are defined in terms of digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized either chemically or de novo using recombinant DNA methodologies. Accordingly, the term "antibody", as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies or identified using other methods known in the art.

[0151] As used herein, the light chains of intact antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. All light chains contain one variable domain (V L ) and one constant domain (C L ), and as described herein, there are several different types of heavy chains that define the class or isotype of the antibody. All heavy chains contain a series of immunoglobulin domains, usually three constant domains (C H1 , C H2 and C H3 ) and one variable domain (V) that is important for antigen binding. H ) and

[0152] The term "variable" region includes framework and CDRs (also known as "hypervariable regions"), and refers to the fact that certain portions of variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "complementarity-determining regions (CDRs)" or "hypervariable regions" in both the light-chain and heavy-chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain multiple FRs that primarily adopt a β-sheet configuration connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669, and Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, as described herein.

[0153] The characteristics of the four human IgG subclasses are well established, each with distinct characteristics and significantly different contributions to the immune system. Human IgG subclasses are differentiated by their binding affinity to immune effector proteins, including neonatal Fc receptors (FcRn), Fc gamma receptors (FcγR), and the complement protein C1q. These receptor proteins play roles in serum half-life, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC), respectively. Affinity for these receptors is often used to characterize the functional properties of antibodies (Bruggemann et al., 1987). Higher affinity for FcγR1 and FcγRIII indicates that an antibody has ADCC activity, whereas binding to the inhibitory receptor FcγRIIb contributes to less ADCC activity (Daeron, 1997; Armour et al., 1999; Clynes et al., 2000). Similarly, binding to C1q, the first protein in the complement cascade, activates phagocytes and signal complement activity, helping to destroy pathogens (Schifferli et al., 1986; Garred et al., 1989; Moore et al., 2010). FcRn binding is associated with antibody recycling and correlates with in vivo half-life (Ghetie et al., 1996; Israel et al., 1996; Praetor and Hunziker, 2002; Jefferis, 2007). The unique features of IgG subclasses aid in the design of antibody therapeutics.

[0154] In 1967, Johnson and Vaughan reported the existence of six canine immunoglobulins (Johnson and Vaughan, 1967; Johnson et al., 1967). Subsequent studies focused on IgG, and Mazza et al. (1993) isolated four fractions from IgG-rich canine serum and separated each by gel filtration, protein A / G binding, and electrophoretic mobility. These fractions were used to obtain antibody reagents specific for canine IgG (Mazza et al., 1994). This work initiated a series of studies investigating canine IgG in various disease states, but the functionality of canine immunoglobulins and how they interact with immune effector proteins remained unclear. In 2001, Tang et al. (2001) provided the canine IgG sequences necessary to begin to answer these questions. Like human IgG, canine IgG consists of four subclasses. Based on the order of accession numbers [AF354264, AF354265, AF354266, and AF354267], Bergeron et al. (Veterinary Immunology and Immunopathology 157 (2014) 31-41) designated these canine IgG subclasses A, B, C, and D, respectively. The alphabetical nomenclature associated with canine IgG sequences is based on their prevalence in the body. Bergeron et al. provided a functional analysis of each subclass.

[0155] Little was known about feline IgG until 2014, when Strietzel et al. (Veterinary Immunology and Immunopathology 158 (2014) 214-223) disclosed functional properties related to two previously known sequences isolated from feline spleen cDNA libraries. These two IgG sequences, IgG1a and IgG1b, had been isolated but uncharacterized (Kanai, TH, et al., 2000 Vet Immunol, Immunopathol. 73 (1), 53-62). Strietzel et al. reported a third feline IgG sequence, designated IgG2, and described the interactions of three feline IgGs with identified feline FcγRI, FcγRIII, FcRn, and C1q. Feline kappa and lambda light chain regions were additionally isolated.

[0156] A "functional Fc region" possesses at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, neonatal receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require the Fc region to be in combination with a binding domain (e.g., an antibody variable domain), and may be assessed using a variety of assays known in the art for assessing such antibody effector functions.

[0157] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Non-limiting examples of sequences for native Fc region sequences include an amino acid sequence having about 80-99% sequence identity to SEQ ID NO: 292. In one embodiment, an antibody of the invention comprises a native Fc region comprising SEQ ID NO: 292. A native Fc region herein preferably has at least about 80% sequence identity to a native sequence Fc region and / or the Fc region of a parent polypeptide, most preferably at least about 90% sequence identity thereto, and more preferably at least about 95% sequence identity thereto. A "variant Fc region," or "mutated" or "mutant" Fc region, comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification and may or may not retain at least one effector function of a native sequence Fc region compared to the native Fc region sequence. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, compared to a native-sequence Fc region or the Fc region of a parent polypeptide, and preferably about one to about five amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein preferably has at least about 80% sequence identity with the native-sequence Fc region and / or the Fc region of a parent polypeptide, most preferably at least about 90% sequence identity, and more preferably at least about 95% sequence identity. A variant or mutated Fc region may also essentially eliminate the function of an antibody Fc region. A variant or mutated Fc region may also add or enhance the function of an antibody Fc region. For example, an Fc region mutation may eliminate an antibody effector function. In another example, a mutated Fc region may enhance an antibody effector function. In yet another example, a mutated Fc region may alter the half-life or affect binding of other intracellular factors that may determine antibody properties. In one embodiment, an antibody of the invention comprises a mutated Fc region. In one embodiment, an antibody of the invention comprises a variant or mutated Fc region comprising an amino acid sequence comprising about 80-99% sequence identity to SEQ ID NO: 288.In one embodiment, an antibody of the invention comprises a variant or mutated Fc region comprising an amino acid sequence comprising SEQ ID NO:288.

[0158] As used herein, "Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. Preferred FcRs are native-sequence FcRs. Further, preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587, and Kim et al., 1994, J. Immunol., 24:249).

[0159] As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, e.g., as described in U.S. Pat. Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.

[0160] "Complement-dependent cytotoxicity" and "CDC" refer to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996).

[0161] Many techniques known in the art can be used to prepare antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies. Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be used. Random combination of heavy and light chain gene products generates a large pool of antibodies with different antigen specificities. Techniques for producing single-chain or recombinant antibodies are known in the art and can be adapted to produce antibodies against the polypeptides of the present invention. Phage display technology can also be used to identify antibodies and heteromeric fragments that specifically bind to a selected antigen. Antibodies can also be bispecific, i.e., capable of recognizing two different antigens, or heteroconjugates, e.g., two covalently linked antibodies, or immunotoxins can be created.

[0162] "Native antibodies" and "native immunoglobulins" are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (I) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the variable domain of the light chain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. Figure 2 shows an example of the general structure of native mouse immunoglobulin G (IgG), highlighting the antigen-binding site.

[0163] As used herein, the terms "antigen-binding protein," "antibody," "antagonist antibody," "antigen-binding fragment," and the like, which may be used interchangeably herein, refer to a polypeptide or fragment thereof that comprises an antigen-binding site. Thus, the isolated antibody or fragment can be a polyclonal antibody, a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a heterochimeric antibody, a caninized antibody, a feline antibody, a humanized antibody, a fully canine antibody, a fully feline antibody, or a fully human antibody.

[0164] In some embodiments, the terms "antigen-binding protein," "antibody," "antagonist antibody," and the like refer preferably to monoclonal antibodies and fragments thereof, and immunologically binding equivalents thereof, capable of binding to TGFβ proteins and fragments thereof. Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies, antigen-recognizing fragments thereof, small modular immunopharmaceuticals (SMIPs), nanobodies, IgNAR molecules, and equivalents recognized by those skilled in the art as antigen-binding proteins or antibody fragments, as well as any of the foregoing fragments and their chemically or genetically engineered counterparts, as well as other antibody fragments and variants thereof, fusion proteins comprising antibody portions, and any other modified configurations of immunoglobulin molecules comprising an antigen recognition site. Antibodies and antigen-binding proteins can be produced, for example, but not limited to, conventional hybridoma techniques (Kohler et al., Nature 256:495-499 (1975)), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display techniques using antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)), or other techniques used and known by those of skill in the art.

[0165] As defined herein, a "monoclonal antibody" is a single, pure, homogeneous type of antibody. All monoclonal antibodies produced are identical and have the same antigen specificity. Monoclonal antibodies are homogeneous antibody populations in which the monoclonal antibody is composed of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an antigen. A population of monoclonal antibodies is highly specific and directed against a single antigenic site. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies, antigen-recognizing fragments thereof, small modular immunopharmaceuticals (SMIPs), nanobodies, IgNAR molecules, etc.), variants thereof, fusion proteins containing antibody portions, and any other modified configuration of an immunoglobulin molecule that contains an antigen-recognition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (eg, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).

[0166] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0167] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. The three hypervariable regions of each variable domain interact to form the V H -V LIt is in this configuration that an antigen-binding site on the surface of the dimer is defined. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site. Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0168] The monoclonal antibodies described herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species, while the remaining chains are identical or homologous to corresponding sequences in antibodies from another species, and also include fragments of such antibodies, so long as they exhibit the desired biological activity. Typically, chimeric antibodies are antibodies in which the light and heavy chain genes are constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of genes from a murine monoclonal antibody (or any other species of antibody, including human and feline) can be linked to canine constant segments, e.g., the amino acid sequence of the HC-65e canine heavy chain constant region represented herein by SEQ ID NO: 288. Additionally, chimeric feline antibodies are produced in the same manner, except that the amino acid sequence comprising SEQ ID NO: 290, the feline heavy chain constant region, is linked to the variable segment of an antibody from another species (murine, canine, human, etc.). Figure 3 is a schematic diagram of the general structure of one embodiment of a mouse:canine IgG. In this embodiment, the antigen-binding site is murine, while the Fc portion is canine. This illustration does not limit the claimed invention to only mouse / canine chimeras, but may also apply to antibody combinations of any species: canine, feline, murine, and human, to name a few, as described herein.

[0169] As defined herein, the term "heterochimeric" refers to an antibody in which one of the antibody chains (heavy or light) is speciesized (i.e., caninized or feline), while the other is chimeric. Figure 4 shows one embodiment of a heterochimeric molecule. In this embodiment, a caninized variable heavy chain (in which all of the CDRs are murine and all of the FRs are canine) is paired with a chimeric variable light chain (in which all of the CDRs are murine and all of the FRs are murine). In this embodiment, both the variable heavy and variable light chains are fused to canine constant regions. As with chimeric antibodies, there are no limitations on the species and moiety combinations of antibodies.

[0170] The terms "canine antibody," "feline antibody," "human antibody," and the like, as used herein, refer to antibodies (antigen-binding proteins) generated against a target and isolated from lymphocytes from within the target species. These antibodies have been recombinantly modified in vitro to contain target species-specific constant regions, as described herein. Additionally, antibodies as described herein have been identified, isolated, and modified to alter the antibody constant region, followed by expression and isolation from in vitro cell culture systems known and routinely used by those of skill in the art.

[0171] The phrases "recombinant canine antibody," "recombinant feline antibody," "recombinant human antibody," and the like all include speciation antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial canine (or feline, human, etc.) antibody libraries, antibodies isolated from animals (e.g., mice) transgenic for canine immunoglobulin genes (see, e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving recombining canine (or feline, human, etc.) immunoglobulin gene sequences with other DNA sequences.

[0172] For the sake of brevity, the following describes "caninized" antibodies; however, it can apply to felineized, humanized, or any other "speciesized" antigen-binding proteins. As an example, "caninization" is defined as a method for transferring non-canine antigen-binding regions from a donor antibody to a less immunogenic canine antibody acceptor, resulting in a treatment useful as a therapeutic in dogs. Caninized antibodies are canine antibody sequences in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (donor antibody), such as mouse, rat, rabbit, cat, dog, goat, chicken, cow, horse, llama, camel, dromedary, shark, non-human primate, human, humanized, recombinant, or engineered sequence with desired properties, specificity, affinity, and capacity. Furthermore, caninized antibodies may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. As described herein, modifications to the hypervariable and / or framework regions cannot be predicted prior to such experiments, but are determined for each independently engineered, species-specific (caninized) antibody based on experiments known to those skilled in the art. A caninized antibody may optionally comprise all or at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin. Figure 5 is a diagram of one embodiment showing the speciesization or caninization of a mouse IgG. In this embodiment, mouse CDRs are grafted onto a canine framework. In some cases, the mouse framework or its residues outside the hypervariable regions are maintained. All descriptions of caninization of antigen-binding proteins and all descriptions of caninized antigen-binding proteins may conceptually be applicable to any "specialized" antibody, whether caninized, feline, humanized, etc.

[0173] A "parent" antibody, as described herein, is an antibody encoded by an amino acid sequence used to prepare a variant. Preferably, for caninized or canine antibodies, the parent antibody has canine framework regions and, if present, canine antibody constant regions. For example, the parent antibody can be a caninized or canine antibody. The same is true for felineized, humanized, equine, or bovine antibodies.

[0174] The term "backmutation" refers to a process in which some or all of the somatically mutated amino acids of a canine antibody are replaced with the corresponding germline residues from a homologous germline antibody sequence. The heavy and light chain sequences of the canine antibody of the invention are separately aligned with the germline sequence to identify the sequences with the highest homology. Differences in the canine antibody of the invention are restored to the germline sequence by mutating the defined nucleotide positions that encode those different amino acids. The role of each amino acid thus identified as a candidate for backmutation should be investigated for its direct or indirect role in antigen binding, and any amino acids found after mutation that affect any desirable characteristics of the canine antibody should not be included in the final canine antibody; for example, activity-enhancing amino acids identified by selective mutagenesis approaches are not subjected to backmutation. To minimize the number of amino acids subjected to backmutation, those amino acid positions that differ from the closest germline sequence but are found to be identical to the corresponding amino acid in a second germline sequence can be retained, as long as the second germline sequence is identical and colinear to the sequence of a canine antibody of the invention. Backmutation of selected target framework residues to the corresponding donor residues may be required to restore and / or improve affinity.

[0175] An "antigen" is a molecule, or portion of a molecule, capable of being bound by an antibody. Generally, an epitope consists of a chemically active surface grouping of a molecule, e.g., amino acids or sugar side chains, with specific three-dimensional structural and charge characteristics. An epitope is an antigenic determinant on a protein that is recognized by the immune system. The epitope-recognizing components of the immune system are antibodies, T cells, and B cells. T cell epitopes are displayed on the surface of antigen-presenting cells (APCs) and are typically 8-11 (MHC class I) or 15+ (MHC class II) amino acids in length. Recognition of displayed MHC-peptide complexes by T cells is critical for their activation. These mechanisms enable the proper recognition of "self" versus "non-self" proteins, such as bacteria and viruses. Individual, not necessarily adjacent, amino acid residues contribute to interaction with the APC binding groove and subsequent recognition by the T cell receptor (Janeway, Travers, Walport, Immunobiology: The Immune System in Health and Disease. 5 th edition New York: Garland Science; 2001). Epitopes recognized by soluble antibodies and cell surface-associated B cell receptors vary greatly in length and degree of continuity (Sivalingam and Shepherd, Immunol. 2012;51(3-4):304-309). Again, linear epitopes, or even epitopes found in continuous stretches of protein sequence, often have discontinuous amino acids that represent key points of contact with the antibody paratope or B cell receptor. Epitopes recognized by antibodies and B cells may be amino acids and conformations that include common contact regions on the protein in three-dimensional space, depending on the tertiary and quaternary structural features of the protein. These residues are often found in spatially distinct regions of the primary amino acid sequence.

[0176] As used herein, the terms "TGF beta," "TGFβ," and "TGFB," when used interchangeably herein, refer to transforming growth factor beta protein 1 (TGFβ1), transforming growth factor beta protein 2 (TGFβ2), and transforming growth factor beta protein 3 (TGFβ3). TGFβ proteins are part of a superfamily of related growth factors that exert pleiotropic effects on wound healing by regulating cell proliferation and migration, cell differentiation, apoptosis, ECM (extracellular matrix) production, and immune regulation. As used herein, inhibition of TGFβ proteins by use of the antigen binding proteins of the invention is used to treat TGFβ-associated disorders such as fibrotic disorders, bone disorders, and cell proliferation disorders.

[0177] As used herein, "anti-TGFβ antigen binding protein," which may be referred to interchangeably as "anti-TGFβ antibody" and "anti-TGFβ antagonist antibody," "anti-TGFβ antigen binding fragment," "anti-TGFβ antigen binding portion," etc., describes any functional molecule that inhibits the binding of TGFβ1 and / or TGFβ2 and / or TGFβ3 proteins to their specific receptors, and thus inhibits the biological function of their associated respective TGFβ signaling pathways. In some embodiments, the invention provides that an anti-TGFβ antigen binding protein binds to TGFβ1 protein. In some embodiments, an antigen binding protein of the invention may have stronger binding and functional inhibition of TGFβ1, but still weaker binding and functional inhibition of TGFβ2 and / or TGFβ3. In some embodiments, the invention provides that an anti-TGFβ antigen binding protein binds to TGFβ2 protein. In some embodiments, the invention provides that an anti-TGFβ antigen binding protein binds to TGFβ3 protein. In some embodiments, the invention provides that an anti-TGFβ antigen binding protein binds to TGFβ1, 2, and 3 proteins. In some embodiments, anti-TGFβ antigen binding proteins bind to TGFβ1 and TGFβ2. In some embodiments, TGFβ antigen binding proteins of the invention bind to TGFβ1 and TGFβ3 proteins. Anti-TGFβ antigen binding proteins of the invention include binding proteins and antibodies that block, antagonize, suppress, or reduce (including significantly reduce) TGFβ biological activity, including downstream pathways mediated by TGFβ1 and / or TGFβ2 and / or TGFβ3 signaling, or any combination thereof, and / or inhibit TGFβ protein binding to the TGFR2 receptor, such as receptor binding and / or elicitation of a cellular response to TGFβ1 and / or TGFβ2 and / or TGFβ3 proteins.For purposes of the present invention, the term "anti-TGFβ antigen binding protein" or "anti-TGFβ antagonist antibody" or "TGFβ antibody" is expressly understood to encompass all previously identified terms, titles, and functional states and characteristics whereby the biological activity of TGFβ itself, or the consequences of biological activity, is substantially abolished, reduced, or neutralized to any meaningful degree, including, but not limited to, its ability to mediate any aspect of the development or treatment of a TGFβ-associated disorder, such as a fibrotic disorder, a bone disorder, and / or a cell proliferation disorder. Examples of anti-TGFβ antigen binding proteins are provided herein.

[0178] A "variant" anti-TGFβ antibody, as used herein, refers to a molecule that differs in amino acid sequence from the "parent" anti-TGFβ antibody amino acid sequence by the addition, deletion, and / or substitution of one or more amino acid residues in the parent antibody sequence, but retains at least one desired activity of the parent anti-TGFβ antibody. Desired activities may include the ability to specifically bind to an antigen, the ability to reduce, inhibit, or neutralize TGFβ activity in an animal, and the ability to inhibit TGFβ-mediated SMAD signaling in a cell-based assay. In one embodiment, a variant comprises one or more amino acid substitutions in one or more hypervariable and / or framework regions of the parent antibody. For example, a variant may comprise at least one, or from about one to about ten, or from about two to about five substitutions in one or more hypervariable and / or framework regions of the parent antibody. Typically, a variant has an amino acid sequence that has at least 50% amino acid sequence identity with the heavy or light chain variable domain sequence of the parent antibody, or between at least about 65%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the parent antibody. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the parent antibody residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Neither N-terminal, C-terminal, nor internal extensions, deletions, nor insertions into the antibody sequence shall be construed as affecting sequence identity or homology. The variants retain the ability to bind to TGFβ variants and may have stronger binding affinity, an enhanced ability to reduce, inhibit, or neutralize TGFβ activity in animals, and / or an enhanced ability to inhibit TGFβ-mediated SMAD signaling in cell-based assays.

[0179] "TGFβ receptor" refers to a polypeptide that is bound or activated by TGFβ protein. TGFβ receptors are single-pass serine / threonine kinase receptors that belong to the TGFβ receptor family. They exist in several different isoforms that can be homodimeric or heterodimeric. Three TGFβ receptors specific to TGFβ proteins can be distinguished by their structural and functional properties. TGFβR1 (ALK5) and TGFβR2 have similar ligand binding affinities. Both TGFβR1 and TGFβR2 have high affinity for TGFβ1 and low affinity for TGFβ2. TGFβR3 (β-glycan) has high affinity for both homodimeric TGFβ1 and TGFβ2, as well as heterodimeric TGFβ1 and TGFβ2. TGFβ receptors also bind to TGFβ3. Mechanistically, TGFβ protein first binds to the TGFβR2 receptor, which recruits and phosphorylates TGFβR1. TGFβR1 then phosphorylates receptor-regulated SMADs (R-SMADs), which can then bind to the co-SMAD SMAD4. The R-SMAD / co-SMAD complex functions as a transcription factor and accumulates in the nucleus where it is involved in regulating target gene expression.

[0180] The term "neutralizing," as used herein with respect to the activity of the monoclonal antibodies of the invention, refers to the ability to substantially antagonize, inhibit, prevent, limit, slow, destroy, eliminate, halt, reduce, or reverse the progression or severity of the inhibited biological activity or characteristic, disease, or condition, including, but not limited to, the biological activity or characteristic. Inhibition or neutralization is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. An antigen-binding protein is said to "neutralize" an antigen when antibody binding to the antigen results in partial or complete inhibition or reduction of the antigen's biological function. Neutralization of the biological activity of TGFβ protein is assessed by measuring partial or complete inhibition or reduction of one or more in vitro or in vivo indicators of TGFβ activity, such as differences in TGFβ receptor binding and signaling pathways. The ability to neutralize TGFβ activity is assessed as described herein by measuring inhibition of Smad2 phosphorylation, as described in the in vitro assays described herein. Neutralization of TGFβ in vivo can result in inhibition of TGFβ-induced cell phenotypic switching, cell proliferation, and cell survival in disease states.

[0181] As used herein, "immunospecific" binding of an antibody refers to an antigen-specific binding interaction that occurs between the antigen-combining site of the antibody and the specific antigen recognized by that antibody (i.e., the antibody reacts with the protein in an ELISA or other immunoassay and does not detectably react with unrelated proteins; additionally, it also means that an antibody of the invention will also bind to the target antigen at the epitope in vivo). An epitope that an antibody or polypeptide "specifically binds" or "preferentially binds" (used interchangeably herein) is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance containing the antigen more frequently, more rapidly, with a longer duration, and / or with a higher affinity than alternative cells or substances. An antibody "specifically binds" or "preferentially binds" a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, an antigen binding protein that specifically or preferentially binds to a TGFβ epitope is a protein that binds to this epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds to other epitopes or non-TGFB epitopes.

[0182] The term "specifically" in the context of antibody binding refers to high avidity and / or high affinity binding of an antibody to a particular antigen, i.e., polypeptide, or epitope. An antibody that specifically binds to an antigen is stronger than the binding of the same antibody to other antigens. An antibody that specifically binds to a polypeptide may be capable of binding to other polypeptides at a weak but detectable level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily distinguishable from the binding of the specific antibody to the polypeptide of interest, e.g., by the use of appropriate controls. Generally, a specific antibody binds to a polypeptide of interest at a level greater than 10%. -7 M or less, 10 -8 M or less 10-9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, or 10 -13 K such as M or less D The antigen binds with a binding affinity having a

[0183] As used herein, the term "affinity" refers to the strength of binding of a single antigen binding site to an antigenic determinant. Affinity depends on the tightness of the stereochemical fit between the binding site of an antibody or antigen-binding protein and the antigenic determinant, the size of the contact area between them, the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium analysis or surface plasmon resonance (SPR) methods (e.g., BIACORE™). SPR methods rely on the phenomenon of surface plasmon resonance (SPR), which occurs when surface plasmon waves are excited at a metal / liquid interface. Light is directed to the side of the surface not in contact with the sample and reflected from it, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. A bimolecular binding event causes a change in the refractive index at the surface layer, which is detected as a change in the SPR signal.

[0184] "K D The term " as used herein is intended to refer to the dissociation constant of an antibody-antigen interaction. The dissociation constant K D , and the association constant K a is a quantitative measure of affinity. At equilibrium, free antigen (Ag) and free antibody (Ab) are in equilibrium with the antigen-antibody complex (Ag-Ab), and the rate constant, k a and k d quantifies the rate of each reaction. At equilibrium, ka[Ab][Ag] = kd[Ag-Ab]. The dissociation constant, K d is K D = kd / ka = [Ag][Ab] / [Ag-Ab]. D has units of concentration, most typically M, mM, μM, nM, pM, etc. DWhen comparing antibody affinities, expressed as the association constant, K, a higher affinity for TGFB is indicated by a lower value. a is given by Ka = ka / kd = [Ag-Ab] / [Ag][Ab]. a is the unit of the reciprocal of concentration, most typically M -1 , mM -1 , μM -1 , nM -1 , pM -1 As used herein, the term "avidity" refers to the strength of antigen-antibody binding after the formation of a reversible complex. Anti-TGFB antibodies have a K D (higher K value) to about D Dissociation constant (K value) within the range D ) for their binding to the TGFB protein. D can be characterized in terms of

[0185] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," and the like, may be used interchangeably herein and refer to a series of nucleotide bases (also referred to as "nucleotides") in DNA and RNA. Nucleic acids can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. The term "nucleic acid" includes, for example, single-stranded and double-stranded molecules. A nucleic acid can be, for example, a gene or gene fragment, an exon, an intron, a DNA molecule (e.g., cDNA), an RNA molecule (e.g., mRNA), a recombinant nucleic acid, a plasmid, and other vectors, primers, and probes. Both 5' to 3' (sense) and 3' to 5' (antisense) polynucleotides are included. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. Other types of modifications include, for example, "capping," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing intercalators (e.g., acridine, psoralens, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides.Additionally, any of the hydroxyl groups normally present in the sugar may be replaced with, for example, a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or conjugated to a solid support. The 5'- and 3'-terminal OH may be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs, such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0186] As used herein, "vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. A vector has an expression control sequence, as described herein, which refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is "operably linked" to a nucleic acid sequence to be transcribed. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous.

[0187] Just as a polypeptide can contain conservative amino acid substitutions, the polynucleotide can contain conservative codon substitutions. A codon substitution is considered conservative if, when expressed, it produces a conservative amino acid substitution as described above. Degenerate codon substitutions that do not result in an amino acid substitution can also be useful in the polynucleotides of the present invention. Thus, for example, a polynucleotide encoding a selected polypeptide useful in one embodiment of the present invention can be mutated by degenerate codon substitutions to approximate the codon usage exhibited by the expression host cell transformed therewith, or to otherwise improve its expression.

[0188] A "variant" nucleic acid, as used herein, refers to a molecule that differs in sequence from a "parent" nucleic acid. Deviations in polynucleotide sequence can result from mutational changes, such as deletions, substitutions, or additions of one or more nucleotides. Each of these changes, alone or in combination, can occur one or more times in a given sequence.

[0189] The term "isolated" means that a material (e.g., an antigen-binding protein or nucleic acid as described herein) is separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the material, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. With respect to nucleic acids, isolated nucleic acids can include those separated from the 5' to 3' sequences with which they are normally associated in chromosomes. In preferred embodiments, the material is purified to greater than 95%, and most preferably greater than 99%, by weight of the material. Isolated material includes the material in situ within recombinant cells, since at least one component of the material's natural environment will not be present. Ordinarily, however, isolated material will be prepared by at least one purification step as used herein.

[0190] The terms "cell," "cell line," and "cell culture" can be used interchangeably. These terms also include all subsequent generations, that is, their progeny. It is understood that all progeny may not be identical due to deliberate or inadvertent mutation. In the context of expression of heterologous nucleic acid sequences, a "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial cell, yeast cell, mammalian cell, and insect cell) whether located in vitro or in vivo. For example, a host cell can be located in a transgenic animal. A host cell can be used as a recipient for vectors and can include any transformable organism capable of replicating the vector and / or expressing a heterologous nucleic acid encoded by the vector.

[0191] As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody or nucleic acid. The label may be itself detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0192] "Subject" or "patient" refers to an animal in need of treatment that can be affected by the molecules of the present invention. Animals that can be treated according to the present invention include vertebrates, and in particular mammals such as canines, felines, or humans are particularly preferred examples.

[0193] A "composition" is intended to mean a combination of an active agent, whether a chemical composition, a biological composition, or a biological treatment (particularly an antigen binding protein as described herein), with another compound or composition, which may be inert (e.g., a label) or active, such as an adjuvant.

[0194] As defined herein, "pharmaceutically acceptable carriers" suitable for use in the present invention are well known to those skilled in the art. Such carriers include, but are not limited to, water, saline, buffered saline, phosphate buffer, alcoholic / aqueous solutions, emulsions, or suspensions. Other conventionally used diluents, adjuvants, and excipients can be added according to conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusters may also be used. Buffers include, but are not limited to, salts prepared from organic acids or bases. Representative buffers include, but are not limited to, organic acid salts such as citric acid, citrate, ascorbic acid, gluconic acid, histidine-Hel, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, Tris, trimethamine hydrochloride, or phosphate buffers. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents (e.g., EDTA), inert gases, and the like, may also be provided in the pharmaceutical carrier. The present invention is not limited by the choice of carrier. The preparation of these pharmaceutically acceptable compositions from the above ingredients with appropriate pH, isotonicity, stability, and other conventional characteristics is within the skill of the art. See, for example, texts such as Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott Williams & Wilkins, publ., 2000, and The Handbook of Pharmaceutical Excipients, 4th ed., eds. R.C. Rowe et al., APhA Publications, 2003.

[0195] A "therapeutically effective amount" (or "effective amount") refers to an amount of an active ingredient, e.g., an agent according to the present invention, sufficient to produce a beneficial or desired result when administered to a subject or patient. An effective dose can be administered in one or more administrations. A therapeutically effective amount of a composition according to the present invention can be readily determined by one of ordinary skill in the art. In the context of the present invention, a "therapeutically effective amount" is one that produces an objectively measured change in one or more parameters associated with a TGFB-associated condition sufficient to produce a beneficial or desired result, including a clinical result such as alleviation or reduction in pain sensation. An effective dose can be administered in one or more administrations. For purposes of the present invention, an effective amount of a composition is an amount sufficient to prevent, treat, reduce, or eliminate a TGFβ-associated disorder, defined herein as a fibrotic disorder, a bone disorder, or a cell proliferation disorder. A therapeutically effective amount will vary depending on the particular subject and condition being treated, the weight and age of the subject, the severity of the condition, the particular composition selected, the dosing regimen to be followed, the timing of administration, the mode of administration, etc., all of which can be readily determined by one of ordinary skill in the art.

[0196] As used herein, the term "therapeutic" encompasses the full spectrum of treatments for a disease, condition, or disorder. The "therapeutic" agents of the invention may act in a prophylactic or preventative manner, including those incorporating procedures designed to target subjects who may be identified as at risk, or may act to slow the rate or extent of progression of at least one symptom of the disease or disorder being treated.

[0197] In a further aspect, the invention features veterinary compositions in which an antibody of the invention is provided for therapeutic or prophylactic use. The invention features methods for treating a canine, feline, or human subject with a particular antigen, e.g., an antigen associated with a disease or condition. The method comprises administering a therapeutically effective amount of an antibody specific for one or more TGFβ proteins, including antibodies of the invention as described herein.

[0198] The antigen-binding proteins of the present invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. The compounds of the present invention can be administered alone, in single or multiple doses, or in combination with pharmaceutically acceptable carriers, diluents, and / or excipients. The composition for administration will be designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable diluents, carriers, and / or excipients, such as dispersants, buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers, etc., will be used as appropriate.

[0199] Compositions comprising the antigen-binding proteins of the present invention can be administered to subjects exhibiting conditions or disorders such as those described herein using standard administration techniques, including intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The route of administration of the antibodies of the present invention may be parenteral. Injections are typically given by the intravenous route. Preferably, the antibodies of the present invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. As used herein, the term parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Peripheral systemic delivery by intravenous or intraperitoneal or subcutaneous injection is preferred. Furthermore, the subject of the methods of the present invention is also referred to as a patient, and is described herein as a canine, feline, or human.

[0200] As used herein, a TGFβ-associated disorder is one in which the regulation or overall levels of one or more TGFβ proteins results in a connective tissue disorder, fibrosis / fibrotic disorder, bone disorder, or cell proliferation disorder. TGFβ regulates a variety of cellular functions, including proliferation, apoptosis, differentiation, and inflammation, and therefore, dysregulation of these proteins can result in several named disorders.

[0201] As used herein, connective tissue disorders refer to a group of disorders involving protein-rich tissues that support organs and other parts of the body. Examples of connective tissues are fat, bone, and cartilage. These disorders often involve joints, muscles, and skin, but they can also involve other organs and organ systems, including the eyes, heart, lungs, kidneys, digestive tract, and blood vessels.

[0202] As described herein, fibrosis-associated diseases relate to pathological processes involving scar formation and excessive production of extracellular matrix by connective tissue in response to tissue injury. The molecular processes are not different from the normal formation of connective tissue and extracellular matrix in normal organs. Physiologically, fibrosis acts to deposit connective tissue, which can interfere with or completely inhibit the normal architecture and function of the underlying organ or tissue. Fibrosis can be used to describe the pathological state of excessive deposition of fibrous tissue and the process of connective tissue deposition during healing. Defined by the pathological accumulation of extracellular matrix (ECM) proteins, fibrosis results in scarring and thickening of affected tissues, essentially an exaggerated wound-healing response that interferes with normal organ function. Fibrosis formation involves interactions between many cell types and cytokines; when the balance is toward fibrosis, fibrosis occurs. Fibrosis is similar to the scarring process in that both involve stimulated fibroblasts, which overturn connective tissue containing collagen and glycosaminoglycans. The process begins when immune cells, such as macrophages, release soluble factors that stimulate fibroblasts. The best-characterized profibrotic mediator is TGFβ, which is released by macrophages and any damaged tissue between surfaces, called the interstitium. As defined herein, fibrotic conditions are selected from the group consisting of pulmonary fibrosis, including both cystic pulmonary fibrosis and idiopathic pulmonary fibrosis; liver cirrhosis; glial scarring in the brain; arthrofibrosis in the knee, shoulder, and other joints; retroperitoneal fibrosis; systemic sclerosis (scleroderma); and renal fibrosis, particularly leading to chronic kidney disease (CKD). Fibrosis is a progressive degenerative disease of blood vessels, skin, lungs, kidneys, heart, and GI tract that, to date, is considered an irreversible process and is classically treated with anti-inflammatory and immunosuppressive drugs, often causing harm.

[0203] Chronic kidney disease (CKD), as described herein, involves the loss of functional kidney tissue due to a long-term, progressive fibrotic process. While renal structural and functional changes are only loosely correlated, dramatic changes in renal structure can be observed. Disease typically exists for months or years before it becomes clinically apparent and is invariably irreversible. Many causes of CKD are associated with progressive interstitial fibrosis. The severity of interstitial fibrosis is positively correlated with the magnitude of GFR decline and negatively correlated with prognosis. The glomerular, tubulointerstitial, and vascular lesions seen in animals with systemic CKD are often similar, regardless of the initiating cause. Transforming growth factor (TGF)-β (TGF-β) has been described as the most important profibrotic mediator responsible for myofibroblast activation. It drives convergent pathways that integrate the effects of many other fibrogenic factors. TGF-β1 is the most abundant isoform and is synthesized by all cell types in the kidney. TGFβ functions as a profibrotic cytokine as discussed, but is also an abundant bone matrix protein that controls bone remodeling and influences the formation, function, and cell-cell interactions of osteoblasts and osteoclasts to maintain adequate bone mass, which suggests that TGFβ inhibition is a potential mechanism for reducing bone demineralization during SRHP due to CKD.

[0204] "Treatment," "treating," and the like refer to both therapeutic treatment and prophylactic or preventative measures. Animals in need of treatment include those already suffering from the disorder and those in which the disorder is to be prevented from developing or progressing. Treatment may also be described as delaying the onset of the symptoms or condition, or delaying the severity of the onset. The term "treatment" or "treating" a disease or disorder includes preventing or protecting against the disease or disorder (i.e., not causing the cause of clinical symptoms), inhibiting the disease or disorder (i.e., arresting or suppressing the development of clinical symptoms), and / or alleviating the disease or disorder (i.e., causing regression of clinical symptoms). As will be appreciated, it is not always possible to distinguish between "preventing" and "suppressing" a disease or disorder, as the ultimate inciting event or events may be unknown or may be latent. Thus, the term "prophylaxis" will be understood to constitute a species of "treatment" that encompasses both "preventing" and "suppressing." Accordingly, the term "treatment" includes "prophylaxis."

[0205] Before the present methods are described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

[0207] The invention disclosed herein relates to antigen-binding proteins (used interchangeably with the terms "antibody," "antagonist antibody," "antibody fragment," etc., as described herein) that specifically bind to one or more TGFβ proteins, in particular antibodies that specifically bind to TGFβ1 and TGFβ2 or TGFβ3 and therefore function as antagonists in that they prevent TGFβ from binding to the TGFβRII receptor and therefore prevent a signaling pathway from being activated by one of the TGFβ proteins, whether they are canine, feline, murine, human, or any other species, caninized, feline, humanized, or any other species-specific antibodies produced by recombinant methods, hybridoma technology, or phage display technology, or fully species-specific monoclonal antibodies. In one embodiment, the invention provides antibodies that bind only to TGFβ1, with or without TGFβ2 or TGFβ3. In one embodiment, the invention provides antibodies that bind to TGFβ1 and TGFβ3. In one embodiment, the invention provides antibodies that bind to TGFβ1 and TGFβ2 and TGFβ3.

[0208] While the properties of antibodies make them very attractive therapeutic agents, they also have numerous limitations. As mentioned above, the majority of monoclonal antibodies (mAbs) are of rodent origin. When such antibodies are administered in a different species, the patient / subject may mount their own antibody response against the xenoantibody. Such a response may lead to the eventual neutralization and elimination of the antibody. As noted above, although antibody production is not limited to mice, mice are widely used for monoclonal antibody production. Species such as canines can be immunized with an antigen and the antibodies recovered and characterized. One problem with using antibodies produced by a particular species is that, for example, if originally produced in mice, non-mouse subjects being treated with the antibody may react to the mouse antibodies as if they were foreign and therefore generate a new set of antibodies against the mouse antibodies. The mouse antibodies are "seen" by the non-mouse, e.g., canine (or any other non-mouse species) immune system, which "sees" the xenoantibody as foreign and may then mount an immune response against the molecule. Those skilled in the art recognize that while antigen-specific antibodies can be used to treat a subject, it is necessary to have a specific antibody species for use. The portion of the reaction generated from interspecies antibody administration, e.g., a murine monoclonal antibody administered to a canine, can range from mild forms such as a rash to more extreme and life-threatening reactions such as renal failure. This immune response can also reduce the effectiveness of the treatment or lead to future reactions if the subject is given a subsequent treatment containing a murine antibody. Therefore, as described in the present invention, "caninization" or "dogification" of the antibodies of the present invention overcomes this drawback. In particular, this process focuses on the framework regions of immunoglobulin variable domains, but may also include the complementarity determinant regions (CDRs) of the variable domains. Enabling steps and reductions for carrying out this process are described in the present disclosure, and the process of affinity maturation centered on CDR sequences is well known in the art.

[0209] The process of modifying monoclonal antibodies (antigen-binding proteins, antagonist antibodies, etc., as described herein, and terms used interchangeably) from animals to make them less immunogenic for therapeutic administration to different species has been actively pursued and described in numerous publications (e.g., *Antibody Engineering: A Practical Guide*, Carl A.K. Borrebaeck ed. W.H. Freeman and Company, 1992). However, this process has not been routinely applied to non-human therapeutic or diagnostic development until recently. Indeed, little has been published regarding canine, feline, or other species-specific variable domains. Wasserman and Capra, Biochem. 6, 3160 (1977), determined the amino acid sequence of the variable regions of both canine heavy chains. Wasserman and Capra, Immunochem. 15, 303 (1978), determined the amino acid sequence of the K light chain from canine IgA. McCumber and Capra, Mol. Immunol. 16, 565 (1979) disclose the complete amino acid sequence of the canine mu chain. Tang et al., Vet. Immunology Immunopathology 80, 259 (2001) disclose a single canine IgG-Ay chain cDNA and four canine IgG-Ay chain protein sequences. Bergeron et al. describe the functional properties of four canine heavy chains. Until now, the lack of available information about canine antibodies has hindered their development as therapeutic agents for the treatment of canine diseases.

[0210] These notable limitations have prompted the development of an engineering technique known as "speciation," well known to those skilled in the art for the "humanization" of therapeutic antibodies. Antibody "caninization," "felineization," and "humanization" are some examples of "speciation" techniques, in which these molecules are generated as antibodies or fragments containing minimal sequence derived from non-target immunoglobulins. One example is a caninized antibody ("target species antibody"), in which residues from the recipient / target's complementarity-determining regions (CDRs) are replaced with residues from the CDRs of a non-target species, such as mouse (i.e., the "donor antibody" or "origin species antibody"), that possesses desired properties, such as specificity, affinity, and potency. This strategy is based on identifying the most suitable target (germline antibody sequence for CDR grafting). After extensive analysis of all available germline sequences for both the variable heavy and variable light chains, germline candidates were selected based on their homology to the mouse / donor mAb, and the CDRs from the mouse / donor precursor mAb were used to replace the native canine CDRs. The goal is always to maintain high affinity and ultimate in vivo efficacy when used as a therapeutic. Using canine antibody frameworks generally minimizes the potential for in vivo immunogenicity when administered to dogs. However, in some cases, framework region (FR) residues of canine immunoglobulins are replaced with corresponding non-canine residues if reduced affinity or function is observed. Backmutation of selected target framework residues to the corresponding donor residues may be required to restore and / or improve affinity as noted. Structure-based methods may also be used for caninization and affinity maturation, as described in U.S. Pat. No. 7,261,890. The above description uses canines as the target species and mice as the donor species. Speciation of antibodies is not limited to these targets and donors. Target species, such as felines, can also be used.

[0211] Another challenge in developing therapeutic antibodies that target proteins is that the epitopes on homologous proteins in different species are often different, and the potential for cross-reactivity with other proteins is also different. As a result, antibodies must be generated, tested, and developed for specific targets in the particular species being treated. Antibody binding between homologous targets in different species is unpredictable and requires testing and evaluation of efficacy.

[0212] Antibodies target antigens through their binding to specific epitopes on the antigen through interactions with the variable region of the antibody molecule. Furthermore, antibodies have the ability to mediate, inhibit (as in the case of the antagonist anti-TGFB antigen-binding proteins of the present invention), and / or initiate various biological activities. There are a wide range of functions for therapeutic antibodies; for example, antibodies can modulate receptor-ligand interactions as agonists or antagonists. Antibody binding can initiate intracellular signaling to stimulate cell proliferation, cytokine production, or apoptosis. Antibodies can also deliver drugs bound to their Fc region to specific sites. Antibodies also induce antibody-mediated cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and phagocytosis through binding of the antibody's Fc region to the respective intracellular molecules that trigger ADCC, CDC, and other functions. Some antibodies have been altered to eliminate ADCC, CDC, C1q binding, and phagocytic functions. In one embodiment, the present invention provides antigen-binding proteins containing alterations in the Fc region of an antibody that alter the antibody's effector function. The present invention further provides cells and cell lines expressing the antibodies of the present invention. Representative host cells include bacteria, yeast, mammalian, and human cells, such as CHO cells, HEK-293 cells, HeLa cells, CV-1 cells, and COS cells. Methods for generating stable cell lines after transformation of heterologous constructs into host cells are well known in the art. Representative non-mammalian host cells include insect cells (Potter et al. (1993) Int. Rev. Immunol. 10(2-3):103-112). Antibodies may also be produced in transgenic animals (Houdebine (2002) Curr. Opin. Biotechnol. 13(6):625-629) and transgenic plants (Schillberg et al. (2003) Cell Mol. Life Sci. 60(3):433-45).

[0213] As discussed above, monoclonal, chimeric, species-specific, and speciation antibodies that have been modified, for example, by deleting, adding, or substituting other portions of the antibody, such as the constant region, are also within the scope of the present invention. For example, an antibody can be modified as follows: (i) by deleting the constant region; (ii) by replacing the constant region with another constant region, e.g., one intended to increase the half-life, stability, or affinity of the antibody, or a constant region from another species or antibody class; or (iii) by modifying one or more amino acids in the constant region to, for example, change the number of glycosylation sites, effector cell function, Fc receptor (FcR) binding, complement fixation, among others. In one embodiment of the present invention, an antibody of the present invention comprises an altered Fc region that alters the effector function of the antibody. In some embodiments of the present invention, the Fc region of the antigen-binding protein of the present invention has been replaced, modified, or removed.

[0214] Methods for altering antibody constant regions are known in the art. Antibodies with altered function, e.g., altered affinity for an effector ligand such as a cellular FcR or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388151 A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the contents of all of which are incorporated herein by reference).

[0215] For example, it is possible to alter the affinity of the Fc region of an antibody for FcR (e.g., Fc.gammaR1) or C1q binding by replacing a specified residue with a residue bearing an appropriate functional group on its side chain, or by introducing a charged functional group, e.g., glutamate or aspartate, or possibly an aromatic nonpolar residue, e.g., phenylalanine, tyrosine, tryptophan, or alanine (see, e.g., U.S. Pat. No. 5,624,821). Antibodies or binding fragments thereof can be conjugated with a cytotoxin, a therapeutic agent, or a radioactive metal ion. In one embodiment, the conjugated protein is an antibody or a fragment thereof. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Non-limiting examples include calicheamicin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin), and antimitotic agents (e.g., vincristine and vinblastine). Techniques for conjugating such moieties to proteins are well known in the art.

[0216] Compositions, derived compositions, and methods of making compositions The present invention encompasses compositions, including pharmaceutical compositions, comprising antigen binding proteins (such as "antibodies," "antibody fragments," and "antagonist antibodies," as used interchangeably herein), polypeptides, and polynucleotides comprising sequences encoding the antigen binding proteins or polypeptides of the invention.

[0217] As used herein, a composition comprises one or more antibodies, antigen-binding proteins, or polypeptides (which may or may not be antibodies) that bind to one or more of the TGFB proteins, and / or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind to one or more of the TGFB proteins. These compositions may further comprise suitable excipients, such as pharmaceutically / veterinarily acceptable excipients including buffers; excipients are well known in the art. The present invention also encompasses isolated antibody, polypeptide, and polynucleotide embodiments. The present invention also encompasses substantially pure antibody, polypeptide, and polynucleotide embodiments.

[0218] In one or more embodiments, the present invention provides novel antigen binding proteins that specifically bind to one or more TGFβ proteins. In one or more embodiments, the antigen binding protein is defined as an antibody or antibody fragment. In one or more embodiments, the antigen binding protein is fully canine, fully feline, feline, fully human, caninized, feline, or humanized. In one or more embodiments, the antigen binding protein of the invention binds to one or more canine, feline, or human TGFβ proteins. In one embodiment, the antigen binding protein is a monoclonal antibody. In one embodiment, the monoclonal antibody of the invention binds to one or more TGFβs and prevents them from binding to and activating their receptors, thus preventing the signaling cascade as described herein.

[0219] In one or more embodiments, the present invention provides an isolated recombinant antigen binding protein that binds to one or more TGFβ proteins, wherein the variable heavy chain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of an antigen binding protein of the invention as described herein, and the variable light chain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence comprising an antigen binding protein of the invention as described herein, and any variant thereof with one or more conservative amino acid substitutions in at least one of CDR1, CDR2 or CDR3 in either the variable light chain or variable heavy chain of the antigen binding protein.

[0220] The present invention provides recombinant antigen-binding proteins, in some embodiments described herein, monoclonal antibodies and antibody fragments, and their use in scientific procedures, including clinical administration and diagnostic procedures. Using molecular biology methods and recombinant technology, it is possible to produce antibodies and antibody-like molecules by recombinant means, thereby generating gene sequences that encode specific amino acid sequences found in the polypeptide structure of an antibody. Such antibodies can be produced either by cloning the gene sequences encoding the antibody polypeptide chains or by directly synthesizing the polypeptide chains and assembling the synthesized chains to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants. This allows for the rapid generation of antibodies with sequences characterized by neutralizing antibodies from different species and sources.

[0221] Regardless of the source of antibodies, how they are constructed, or how they are synthesized, whether in vitro or in vivo, using transgenic animals, large-scale laboratory cell cultures or commercial scale, using transgenic plants, or by direct chemical synthesis without using living organisms at any stage of the process, all antibodies have a similar overall three-dimensional structure. This structure is often provided as H2L2, referring to the fact that antibodies generally contain two light (L) chain amino acids and two heavy (H) chain amino acids. Both chains have regions capable of interacting with structurally complementary antigen targets. The target-interacting regions are referred to as "variable" or "V" regions and are characterized by differences in amino acid sequence from antibodies of different antigen specificities. The variable region of either the H or L chain contains amino acid sequences capable of specifically binding to antigen targets.

[0222] As used herein, the term "antigen-binding region" refers to a portion of an antibody molecule containing amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. The antibody-binding region includes "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the heavy or light chains that provide the antigen-binding region, there are smaller sequences called "hypervariable" regions, which are responsible for the extreme variability between antibodies of different specificities. Such hypervariable regions are also called "complementarity-determining regions" or "CDR regions." These CDR regions are responsible for the basic specificity of an antibody for a particular antigenic determinant structure.

[0223] Although CDRs represent non-contiguous stretches of amino acids within the variable region, the locations of these important amino acid sequences within the variable heavy and light chain regions have been found to have similar positions within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all antibodies each have three CDR regions, each non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) regions and variable regions, within the latter of which are CDRs and so-called "framework regions" composed of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

[0224] The present invention also provides vectors containing at least one of the above-described nucleic acids. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences may be identified, each of which may be capable of encoding that amino acid. The actual probability that a particular oligonucleotide constitutes an actual coding sequence can be estimated by considering unusual base pair relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an anti-TGFβ antibody or portion. Such "codon usage rules" are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Lathe's "codon usage rules" can be used to identify a single nucleotide sequence, or a set of nucleotide sequences, that contains the theoretically "most likely" nucleotide sequence capable of encoding an anti-TGFβ sequence. It is also contemplated that antibody coding regions for use in the present invention may be provided by altering existing antibody genes using standard molecular biology techniques to yield variants (agonists) of the antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions, and substitutions in the amino acid sequence of the anti-TGFβ antibody or peptide.

[0225] antibody derivative Antibody derivatives are included within the scope of the present invention. "Derivatives" of antibodies contain additional chemical moieties that are not normally part of the protein. Covalent modifications of proteins are included within the scope of the present invention. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art is useful for crosslinking antibodies or fragments to water-insoluble support matrices or other macromolecular carriers.

[0226] Derivatives also include radioactively labeled monoclonal antibodies, for example, radioactive iodine (251, 1311), carbon (C), sulfur (S), indium, tritium (H 3 ); conjugates of monoclonal antibodies containing biotin or avidin with enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose 6-phosphate dehydrogenase; and also conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine esters), or fluorescent agents (such as phycobyl proteins) are used.

[0227] Another derivative bifunctional antibody of the present invention is a bispecific antibody, generated by combining portions of two separate antibodies that recognize two different antigenic groups. This can be achieved by crosslinking or recombinant techniques. Additionally, moieties may be added to the antibody or portions thereof to increase its half-life in vivo (e.g., by increasing the time until clearance from the bloodstream). Such techniques include, for example, the addition of PEG moieties (also called pegylation), and are well known in the art. See U.S. Patent Application Publication No. 2003 / 0031671.

[0228] Recombinant expression of antibodies In some embodiments, nucleic acids encoding the antigen-binding proteins of the present invention are directly introduced into host cells, and the cells are incubated under conditions sufficient to induce expression of the encoded antibody. After the nucleic acid of interest is introduced into the cells, the cells are typically incubated, usually at 37°C, optionally under selection, for a period of about 1 to 24 hours to allow for antibody expression. In one embodiment, the antibody is secreted into the supernatant of the culture medium in which the cells are growing. Traditionally, monoclonal antibodies are produced as natural molecules in murine hybridoma lines. In addition to that technology, the present invention provides for recombinant DNA expression of monoclonal antibodies, allowing for the production of the antibody in a selected host species and the generation of a spectrum of antibody derivatives and fusion proteins.

[0229] A nucleic acid molecule, such as DNA, contains nucleotide sequences that contain transcriptional and translational regulatory information, and is said to be "capable of expressing" a polypeptide when such a sequence is "operably linked" to a nucleotide sequence that encodes the polypeptide. An operable linkage is one in which the regulatory DNA sequence and the DNA sequence to be expressed are joined in such a way as to permit gene expression as an anti-TGFβ antigen binding protein or antibody fragment in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the art.

[0230] Thus, the present invention encompasses expression of anti-TGFβ antigen binding proteins in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacterial, yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ, or in host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although any other mammalian cells may be used without limitation.

[0231] Expression vectors carrying chimeric, speciated antigen binding protein constructs or anti-TGFβ antigen binding proteins of the invention can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle bombardment (Johnston et al., 240 Science 1538 (1988)), or other techniques known to those skilled in the art, including, but not limited to,

[0232] For long-term, high-yield production of recombinant antibodies, stable expression can be used. For example, cell lines that stably express antibody molecules can be engineered. Rather than using expression vectors containing origins of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. Following introduction of the foreign DNA, engineered cells can be grown in an enriched medium and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow, forming foci that can then be cloned and expanded into cell lines. Such engineered cell lines can be particularly useful in screening and evaluating compounds / components that interact directly or indirectly with antibody molecules.

[0233] Once an antibody of the invention is produced, it can be purified by any method known in the art for the purification of immunoglobulin molecules, including, but not limited to, chromatography (e.g., ion exchange, affinity, particularly for a specific antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. In many embodiments, the antibody is secreted from the cells into the culture medium and harvested from the culture medium.

[0234] Pharmaceutical and veterinary uses The anti-TGFβ antigen-binding proteins or antibody fragments of the invention as described herein can be used, for example, in the treatment of TGFβ-related disorders in canines and felines. More specifically, the invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent and, as an active ingredient, an antibody or antibody fragment according to the invention. The antibody may be a chimeric, heterochimeric, caninized, felineized, humanized, or speciated antigen-binding protein for compatibility with different species. Intact immunoglobulins or binding fragments thereof are also contemplated. The antibodies of the invention and pharmaceutical compositions thereof are useful for parenteral administration, e.g., subcutaneous, intramuscular, or intravenous administration.

[0235] In some desirable embodiments, the antibodies of the present invention are administered by parenteral injection. For parenteral administration, the anti-TGFβ antibody or fragment can be formulated as a solution, suspension, emulsion, or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle can be a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, such as, but not limited to, an aqueous carrier, such as water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or serum albumin, glycine, or the like. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting agents, buffers, toxicity adjusters, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these formulations can vary widely, for example, less than about 0.5% by weight, usually from about 1% or more to up to 15% or 20% by weight, and will be selected primarily based on fluid volume, viscosity, etc., according to the particular mode of administration selected. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Actual methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described in more detail, for example, in REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

[0236] The antibodies of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilization and reconstitution technique can be used. Those skilled in the art will appreciate that lyophilization and reconstitution may result in varying degrees of antibody activity loss, which may require adjustment of use levels to compensate. The antibody compositions of the present invention may provide a cocktail that can be administered for the prevention of recurrence and / or therapeutic treatment of existing diseases. Suitable pharmaceutical carriers are described, inter alia, in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standard reference text in the art well known to those skilled in the art. In therapeutic applications, the composition is administered to a subject already suffering from a disease or condition in an amount sufficient to cure, or at least partially arrest, or alleviate, the disease or condition and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose" or "therapeutically effective amount."

[0237] The dose administered will, of course, vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health, and weight of the recipient; the nature and extent of the condition, type of concurrent treatment, frequency of treatment, and the desired effect.

[0238] As a non-limiting example, treatment of TGFβ-related conditions in dogs and cats can be provided in a range of doses as needed. Exemplary antibodies for canine or feline therapeutic use are high-affinity antibodies with potent in vivo anti-TGFB activity according to the present invention, as well as fragments, regions, and derivatives thereof. Single or multiple administrations of the compositions can be carried out, with dose levels and patterns selected by the treating veterinarian. In any event, the pharmaceutical formulation should provide a quantity of the antibody of the present invention sufficient to effectively treat the subject.

[0239] Diagnostic Use The present invention also provides the above-described anti-TGFB antibodies for use in diagnostic methods for detecting TGFβ in species known to have or suspected of having a TGFβ-related disorder, particularly canines, felines, or humans. The anti-TGFB antibodies of the present invention are useful for immunoassays to detect or quantify one or more TGFBs or anti-TGFB antibodies in a sample. Immunoassays for TGFβ typically involve incubating a clinical or biological sample in the presence of a detectably labeled, high-affinity (or high-avidity) anti-TGFB antibody of the present invention capable of selectively binding to TGFβ, and detecting labeled peptide or antibody binding in the sample. Various clinical assay procedures are well known in the art. Such samples include tissue biopsies, blood, serum, and fecal samples, or fluids collected from animal subjects and subjected to ELISA analysis, as known to those skilled in the art.

[0240] A "solid phase support" or "carrier" refers to any support capable of binding a peptide, antigen, or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride (PVDF), dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the support may be soluble to some extent or insoluble for purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding one or more TGFβ proteins or anti-TGFβ antibodies. Thus, the configuration of the support may be spherical, as in beads, or cylindrical, as in the interior surface of a test tube, or a rod-like external surface. Alternatively, the surface may be flat, such as a sheet, culture dish, test strip, etc. For example, the support may comprise polystyrene beads. Those of skill in the art will know many other suitable carriers for binding antibodies, peptides, or antigens, or will be able to ascertain such by routine experimentation. Well-known method steps can determine the binding activity of a given lot of anti-TGFB peptides and / or antibodies or antigen-binding proteins. Those skilled in the art can determine operational and optimal assay conditions by routine experimentation.

[0241] Detectably labeling TGFβ-specific peptides and / or antibodies can be achieved by linking them to an enzyme for use in enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA). The linked enzyme reacts with an exposed substrate to produce a chemical moiety that can be detected, for example, but not limited to, by spectrophotometric, fluorimetric, or visual means. Enzymes that can be used to detectably label TGFβ-specific antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Radiolabeling TGFβ-specific antibodies allows for the detection of TGFβ through the use of radioimmunoassays (RIA). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes that are particularly useful for the purpose of the present invention include: 3 H, 125 I, 131 I, 35 S, and 14 C is one example.

[0242] It is also possible to label TGFβ-specific antibodies with fluorescent compounds. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can then be detected due to fluorescence. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine, among others known to those skilled in the art. TGFβ-specific antibodies or antigen-binding proteins can also be labeled with 125Fluorescence-emitting metals such as Eu or others of the lanthanide series can be used to delectably label TGFβ-specific antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0243] A TGFβ-specific antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescently-labeled antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0244] Similarly, a bioluminescent compound can be used to label the TGFβ-specific antibodies, portions, fragments, polypeptides, or derivatives of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase, and aequorin.

[0245] Detection of TGFβ-specific antibodies, portions, fragments, polypeptides, or derivatives can be accomplished, for example, by a scintillation counter if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material. In the case of an enzyme label, detection can be achieved by colorimetric methods which employ a substrate for the enzyme. Detection can also be achieved by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

[0246] For the purposes of the present invention, the TGFβ detected by the above assay can be present in a biological sample. Any sample containing TGFβ can be used. For example, the sample can be a biological fluid such as blood, serum, lymph, urine, feces, inflammatory exudate, cerebrospinal fluid, amniotic fluid, tissue extract, or homogenate, and any biopsy-related material. The present invention is not limited to assays using only these samples; however, in light of this specification, those skilled in the art can determine suitable conditions that allow the use of other samples.

[0247] In situ detection can be achieved by removing a histological specimen from an animal subject and providing a labeled antibody combination of the present invention to such specimen. The antibody (or a portion thereof) can be provided by applying or overlaying the labeled antibody (or a portion thereof) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of TGFβ, but also the distribution of TGFβ in the examined tissue. Using the present invention, those skilled in the art will readily recognize that any of a variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0248] The antibodies, fragments, or derivatives of the invention can be adapted for use in immunometric assays, also known as "two-site" or "sandwich" assays. In a typical immunometric assay, an amount of unlabeled antibody (or antibody fragment) is bound to a solid support that is insoluble in the liquid being tested, and an amount of detectably labeled soluble antibody is added, allowing for detection and / or quantitation of the ternary complex formed between the solid-phase antibody, antigen, and labeled antibody.

[0249] Antibodies can be used to quantitatively or qualitatively detect one or more TGFβ proteins in a sample, or to detect the presence of cells expressing one or more TGFβ proteins. This can be achieved by immunofluorescence techniques (see below) using fluorescently labeled antibodies coupled with fluorescence microscopy, flow cytometry, or fluorimetric detection. For diagnostic purposes, antibodies can be either labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) reactive with the antibody, such as antibodies specific for canine immunoglobulin constant regions. Alternatively, antibodies can be directly labeled. A variety of labels may be used, including radionuclides, fluoresceins, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (particularly haptens). Numerous types of immunoassays, such as those discussed above, are available and well known to those skilled in the art. Importantly, the antibodies of the present invention can be useful in the diagnosis of TGFβ-related disorders in canines, felines, humans, and the like. More specifically, the antibodies / antigen-binding proteins of the present invention can identify overexpression of TGFβ in companion animals. Thus, the antibodies of the present invention can provide an important immunohistochemistry tool. The antibodies of the present invention may be used in antibody arrays, which are highly suitable for measuring gene expression profiles and other diagnostic tools well known to those skilled in the art.

[0250] kit Kits for carrying out the subject methods are also included within the scope of the present invention. The kits include at least one or more of the antibodies of the present invention, nucleic acids encoding them, or cells containing them. The antibodies of the present invention may be provided in a container, usually in lyophilized form. The antibodies, which may or may not be conjugated to a label or toxin, are typically included in the kit along with buffers such as Tris, phosphate, or carbonate, stabilizers, biocides, and inert proteins such as serum albumin. Generally, these materials are present in an amount of less than 5% by weight based on the amount of active antibody, and usually in a total amount of at least about 0.001% by weight, again based on the antibody concentration. It is often desirable to include an inert bulking agent or excipient to dilute the active ingredient; the excipient may be present in an amount of about 1% to 99% by weight of the total composition. If a second antibody capable of binding to the primary antibody is used in the assay, it is usually present in a separate vial. The second antibody is typically conjugated to a label and formulated in a manner similar to the antibody formulations described above. The kit also typically includes a set of instructions for use.

[0251] Before describing the compositions and methods of the present invention, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The present invention will now be further described by the following non-limiting examples. [Example]

[0252] The present invention is further illustrated and supported by the following examples. However, these examples should in no way be construed to further limit the scope of the present invention. On the contrary, those skilled in the art will readily appreciate that there are other embodiments, modifications, and equivalents of the present invention without departing from the spirit of the present invention and / or the scope of the appended claims.

[0253] Example 1: Anti-TGFβ1,3 antibody Identification of a murine monoclonal antibody that recognizes canine TGF-β1 AJ, CF-1, and BalbC mice were immunized with a combination of amphibian and trout TGFβ1 formulated with Freund's complete adjuvant and then incomplete adjuvant according to standard RIMMS protocols. After vaccination, serum antibody titers from immunized animals were determined using enzyme-linked immunosorbent assays (ELISAs). To determine antibody titers against individual immunogens and probe for cross-reactive antibody responses to canine TGFβ1, individual ELISAs were performed using canine TGFβ1, SEQ ID NO: 220 (50 ng / well), or a mixture of amphibian and trout TGFβ1 (100 ng / well) immobilized on polystyrene microplates and used as capture antigens. Serum from immunized animals was diluted in phosphate-buffered saline with 0.05% Tween-20 (PBST). The presence of anti-TGFβ1 antibodies was detected with a goat anti-mouse secondary antibody (KPL, Inc., Gaithersburg, MD) labeled with horseradish peroxidase (HRP). A chromogenic substrate (SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate, KPL, Inc., Gaithersburg, MD) was added, and after 10 minutes of incubation at room temperature (RT), the reaction was stopped by adding 100 μL of 0.1 N HCl. The absorbance of each well was determined at an optical density (OD) of 450 nm. Under these conditions, animals in all groups demonstrated robust responses to their respective immunogens; however, they were unable to generate an immune response directed against the canine form of TGFβ1. The highest response was exemplified by group CF-1, where the titer for all three animals was 1:31250 (data not shown).

[0254] To overcome self-tolerance that may result from the conserved nature of TGFβ across species, canine TGFβ1 (SEQ ID NO: 220) was conjugated to a carrier protein, an inactive mutant (non-toxic) form of diphtheria toxin (CRM197), using standard cross-linking chemistry to create the immunogen referred to herein as TGFβ1-CRM. Two additional immunizations with TGFβ1-CRM were performed using mice from groups AJ and CF-1, two weeks apart. These efforts did not result in any of the mice mounting an immune response to the canine TGFβ1 protein, as assessed by ELISA, as described above.

[0255] A final strategy involved giving two booster doses of alum-adjuvanted TGFβ1-CRM, administered intraperitoneally (IP), two weeks apart. These efforts yielded a single mouse with sufficient titers to perform fusions. A pool of donor splenocytes from this mouse was used for fusions, and based on the results of the primary screen for anti-TGFβ1 antibodies, eight wells were selected for expansion and secondary screening. The secondary screen confirmed that five fusions retained the ability to produce anti-TGFβ1 antibodies. Spent supernatant from 15 ml of culture was purified using protein A / G resin to obtain an enriched population of IgG for further evaluation.

[0256] DNA sequence encoding mouse antibody 04H09 Ribonucleic acid (RNA) was isolated from 04H09 hybridoma cells using an RNeasy Mini Kit (Qiagen, Inc., Germantown, MD) and the manufacturer's protocol. Briefly, one million frozen cells were harvested by centrifugation, and RNA was purified from the cell lysate using RNeasy spin columns. RNA was eluted from each column and immediately used for quantification and cDNA preparation. RNA yield and purity were analyzed by measuring its absorbance at 260 nm and 280 nm using a GeneQuant Pro spectrophotometer (GE Healthcare, Uppsala, Sweden). After isolation, the remaining RNA was stored at -80°C for further use.

[0257] Oligonucleotide primers designed for amplification of mouse immunoglobulin (Ig) variable domains were used according to the manufacturer's instructions (EMD Chemicals, Inc., Gibbstown, NJ). cDNA was prepared from total hybridoma RNA by reverse transcription (RT) using a ThermoScript RT kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. Briefly, 200–400 ng of RNA was added to individual reaction tubes containing a 3′ Ig constant region primer that hybridizes to the Ig gene at a position proximal to the variable Ig region, thus transcribing first-strand cDNA representing the variable region of the mouse antibody. Individual RT reactions were performed using a 3′ constant heavy chain primer and a 3′ constant kappa light chain primer.

[0258] These cDNAs from the 04H09 hybridoma were used as templates in polymerase chain reactions (PCR) to amplify variable IgG heavy and kappa light chain cDNAs for sequencing purposes. Multiple reactions were performed for each PCR using degenerate 5' primers or primer pools designed to anneal to the signal sequence-coding regions of the mouse Ig variable domains. Separate PCR reactions were performed using degenerate primers or primer pools for amplification of the mouse variable heavy and variable light chain regions. PCR was performed on 1 μl of cDNA reaction using the Expand High Fidelity DNA Polymerase Kit (Roche Diagnostics Corp., Indianapolis, IN) according to the manufacturer's protocol. Thermal cycling parameters for PCR were as follows: 94°C for 2 minutes, 35 cycles (94°C for 15 seconds, 55°C for 30 seconds, 72°C for 1 minute), 72°C for 7 minutes. The PCR-amplified fragments were separated by gel electrophoresis on a 1% agarose gel and purified using a Qiagen gel extraction kit (Qiagen, Inc., Germantown, MD). The forward primers for the heavy and light chain variable regions incorporate EcoRI or SalI sites, and the reverse primers incorporate a HindIII site to facilitate cloning into the pUC19 plasmid. The purified PCR fragments and pUC19 plasmid were digested with the above restriction endonucleases (New England Biolabs (NEB), Inc., Ipswich, MA) at 37°C for 1-2 hours. After digestion, the PCR fragments were purified using a Qiaquick PCR cleanup kit (Qiagen, Inc., Germantown, MD). The digested plasmids were separated by gel electrophoresis on a 1% agarose gel and purified using a Qiagen gel extraction kit. Purified PCR fragments representing the variable IgG heavy chain and kappa light chain DNA were ligated into the pUC19 plasmid overnight at 4°C using T4 DNA ligase and ligation buffer (NEB, Inc., Ipswich, Mass.).3 ul of each ligation reaction was used to transform E. coli TOP10 cells (Invitrogen Corp., Carlsbad, Calif.).

[0259] Plasmids were isolated from positive clones representing the variable regions of each hybridoma using a Qiagen miniprep kit (Qiagen 27106) according to the manufacturer's protocol. DNA sequences for each cloned insert were amplified using BigDye sequencing reactions (Applied Biosystems by Life Technologies Corp., Carlsbad, CA) according to the manufacturer's protocol using M13 forward and reverse primers. Sequencing reactions were purified using a 96-well purification kit (Zymo Research, Irvine, CA) according to the manufacturer's protocol. Samples were loaded onto an ABI-3730 capillary sequencer, and the resulting sequence traces were analyzed for the presence of complete open reading frames using Sequencher (GeneCodes v.4.2). The mouse anti-TGFβ variable sequences determined for antibody 04H09 are as follows: SEQ ID NO: 1 04H09 VH nucleotide sequence, SEQ ID NO: 2 04H09 VH amino acid sequence, SEQ ID NO: 3 04H09 VL nucleotide sequence, SEQ ID NO: 4 04H09 VL amino acid sequence. Additionally, the six CDRs for the 04H09 monoclonal antibody are as follows:

[0260] Table 1 TIFF0007798805000001.tif36121

[0261] Construction of the recombinant mouse:canine chimera 04H09 Antibody variable domains are involved in antigen binding; therefore, grafting the complete variable domains of the 04H09 antibody onto a different constant region, e.g., a constant region from a different species, should have little or no effect on the antibody's ability to bind to the canine TGFβ1 immunogen. As such, expression vectors have been designed to produce recombinant chimeric or fully canine antibodies in mammalian expression systems. The chimeric antibodies described herein consist of variable sequences (both CDRs and frameworks) from a host species antibody grafted onto the respective heavy and light constant regions of an IgG molecule from a different species. For example, the variable regions can be from a mouse host species, e.g., SEQ ID NOS:2 and 4 of 04H09, and a heavy chain constant region from a canine species (SEQ ID NO:127), which will be referred to herein as a mouse:canine chimera. To produce the desired chimeric antibody, synthetic DNA sequences for the variable heavy (VH) and variable light (VL) sequences of a selected antibody were constructed containing unique restriction endonuclease sites, a Kozak consensus sequence, and an N-terminal secretory leader to facilitate expression and secretion of the recombinant antibody from mammalian cell lines.

[0262] For the mouse:canine 04H09 chimera, referred to herein as chi04H09, each mouse variable region (SEQ ID NOs: 1 and 3) was cloned into a mammalian expression plasmid containing either the canine IgG heavy chain (SEQ ID NO: 127) or light chain (SEQ ID NO: 129) constant region.

[0263] Plasmids encoding the heavy and light chains under the control of a CMV promoter were cotransfected into HEK293 cells using standard methods. After 6 days of expression, the chimeric mAb was purified from 50 ml of transiently transfected HEK293FS cell supernatant using MabSelect Sure Protein A resin (GE Healthcare, Uppsala, Sweden) according to standard methods for protein purification. The eluted fraction was neutralized and concentrated to approximately 0.5–1.0 mL using Amicon Ultra centrifugal devices (Millipore Sigma, Burlington, MA) with a nominal 10,000 MW cutoff. The eluted fraction was dialyzed overnight at 4°C against 20 mM sodium acetate, pH 5.0, 85 g / L sucrose, + / - 0.05 g / L EDTA, and stored at 4°C for further use.

[0264] The affinity and cell-based efficacy of 04H09 and chi04H09 were evaluated using surface plasmon resonance (SPR). To characterize the binding affinity of candidate monoclonal antibodies (mAbs) to TGFβ, surface plasmon resonance (SPR) was evaluated using a Biacore T200 system (Biocore Life Sciences (GE Healthcare), Uppsala, Sweden). To avoid affinity differences associated with different surface preparations that may arise when immobilizing antibodies to a surface, TGFβ1, TGFβ2, and TGFβ3 (R&D Systems) were directly conjugated to individual surfaces. Immobilization was achieved by amine coupling at 5 μg / mL using N-hydroxysuccinimide (NHS) / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. The chip was quenched with ethanolamine, and the affinity of all candidate mAbs bound to the immobilized TGFβ was evaluated. All curves were fitted to a 1:1 model. -11Affinities below this range are below the quantitation limit of detection for the instrument. This led to the identification of a single murine antibody, designated 04H09, with an affinity range for TGFβ1 > TGFβ3 > TGFβ2 (see KD data in Table 3 below). Murine anti-TGFβ 04H09 was further subcloned to generate hybridoma-produced homogenous antibodies and for sequencing of the variable heavy and light chains.

[0265] A primary cell-based assay measuring inhibition of TGFβ1-induced SMAD3 phosphorylation in canine mitral valve interstitial cells (CMVIC) to assay for potency. For this assay, TGFβ1, 2, or 3 was added to cells with or without antibody, and SMAD3 signaling was determined via the AlphaLISA detection kit. The affinity of both the murine (04H09) and chimeric (chi04H09) forms of anti-TGFβ antibodies for the TGFβ1, 2, and 3 surfaces, as well as the potency of each antibody, are shown in Table 3 (below) (KD data and CMVIC data, respectively).

[0266] These observations indicate that conversion to the chimeric form results in some loss of affinity for all three isoforms. However, they also demonstrate increased potency through blockade of TGFβ1 and 3-mediated pSmad signaling in CMVIC cells (compare CMVIC data for 04H09 and chi04H09 in Table 3). These results may be due to the higher level of homogeneity and purity for the recombinant mouse:canine chimera compared to purified material from mouse hybridoma subclones. Blockade of TGFβ1 and 3 was encouraging, leading to further studies pursuing a caninized form of this antibody.

[0267] Caninization of antibody 04H09 The generation of anti-drug antibodies (ADA) can be associated with a loss of efficacy for any biotherapeutic protein, including monoclonal antibodies. While examples of immunogenic fully human mAbs and non-immunogenic chimeric mAbs can be found, speciation of monoclonal antibodies can reduce the tendency of mAbs to become immunogenic. To help mitigate the risks associated with ADA formation in the 04H09 monoclonal antibody provided herein, a caninization strategy was used. This caninization strategy is based on identifying the most suitable canine germline antibody sequences for CDR grafting.

[0268] After extensive analysis of available canine germline sequences for both the variable heavy and variable light chains, germline candidates were selected based on their homology to the framework regions of the murine 04H09 antibody variable regions, and the CDRs from the murine precursor 04H09 antibody (SEQ ID NOS: 5-10) were used to replace the native canine CDRs. The goal was to retain high affinity and cell-based activity using the canine antibody framework to minimize the potential for immunogenicity in vivo.

[0269] Synthetic nucleotide constructs representing caninized variable heavy and light chains for the 04H09 antibody were generated. Caninization efforts using the murine antibody 04H09 focused on three canine VH frameworks and four canine VL frameworks as follows:

[0270] Table 2 TIFF0007798805000002.tif47125

[0271] Each variable chain was subcloned into a plasmid containing the respective canine heavy chain (SEQ ID NO: 127) or light chain (kappa) constant region (SEQ ID NO: 129), and the plasmids were then co-transfected in all 12 possible combinations (can04H09 VH1 / VL1, can04H09 VH1 / VL2, can04H09 VH1 / VL3, can04H09 VH1 / VL4, can04H09 VH2 / VL1, can04H09 VH2 / VL2, can04H09 VH2 / VL3, can04H09 VH2 / VL4, can04H09 VH3 / VL1, can04H09 VH3 / VL2, can04H09 VH3 / VL3, and can04H09 VH3 / VL4) for antibody expression in HEK293 cells. All combinations were able to transiently produce antibodies, except for any heavy chain paired with can04H09-VL2.

[0272] After expression, the caninized mAbs were characterized for their affinity for binding to canine TGFβ1, 2, and 3, in addition to assessing their potency in cell-based assays as described above (see data in Table 3 below). In general, all combinations of canine frameworks with grafted 04H09 CDRs resulted in comparable affinity for canine TGFβ2 and 3. The most dramatic loss of affinity was observed for TGFβ1 with antibodies can04H09 VH1 / VL1, can04H09 VH2 / VL1, and can04H09 VH3 / VL1. As observed by converting murine 04H09 to chi04H09, caninization had variable effects on the potency of each antibody with respect to cellular pSmad signaling in response to each TGFβ isoform. Similar to murine 04H09 and chi04H09, none of the framework combinations resulted in activity against TGFβ2-mediated cell signaling. With the exception of the canine VL2 framework, representative pairs from each heavy and light canine framework produced antibodies capable of inhibiting signaling induced by TGFβ1 and 3. Together, these data demonstrate that the CDRs from murine antibody 04H09 form robust combinations with candidate canine frameworks, and that speciesing can be achieved without any modifications to the canine germline sequence to retain binding and potency phenotypes.

[0273] Table 3 TIFF0007798805000003.tif161134

[0274] Epitope mapping of caninized ZTS-426 Co-crystallization of ZTS-426 Fab (VH SEQ ID NO: 12 and VL SEQ ID NO: 24) and TGFβ1, and subsequent analysis of the antigen-binding mAb Fab fragment structure were performed to a final resolution of 2.2 Å. Figure 7 shows a representation of the secondary ZTS-426:TGFβ1 dimer crystal structure. The complex structure provides structural insight into the key recognition elements that govern the interaction of ZTS-426 Fab with residues of mature TGFβ1.

[0275] Mammalian systems consist of at least three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3), therefore, subtype specificity was additionally probed. Based on the analysis of the interaction of ZTS-426 Fab with mature TGFβ1, approximately 16 unique antigenic residues proximal to the Fab were identified.

[0276] The overall architecture was determined from the crystal structure asymmetric unit, containing two ZTS-426 Fab molecules bound diagonally opposite a reference TGFβ1 homodimer (Figure 7), and showed approximately two-fold rotational symmetry from the overall complex center of mass. The mature TGFβ1 homodimer is held together by a series of covalent disulfide bonds formed by cysteine ​​side chain thio functionalities within and between TGFβ1 monomers. Most notably, each Fab fragment interacts exclusively with only one of the TGFβ1 monomers. X-ray factor B analysis suggests that the TGFβ1 and Fab interface residues can be adequately resolved to reveal the key interactions contributing to binding affinity.

[0277] The complex X-ray structure was prepared using the Protein Preparation module within the MAESTRO modeling application suite from Schrödinger to provide a complete description of the complex, lacking any missing atoms / residues, and with minimal energy to relax any excessive steric activity. This final structure, along with the Kabat numbering scheme for the Fab residues, was used for all subsequent analyses. One of the antigen-antibody binding hypotheses suggests that lipophilic antibody CDR residues prefer to bury their hydrophobic surface on the antigen surface. Therefore, the surface property probe was constructed at approximately 388.5 Å, including residues F8, Q19, Y21, H34, E35, H40, V89, R94, P96, and V98. 2 We identified a discontinuous but proximal patch covering the hydrophobic surface area of ​​. This region extends to approximately 343.1 Å. 2The ZTS-426 residues L_I2, L_Y30, L_Y49, L_A50, L_F91, L_W92, H_W33, H_Y52, H_H95, and H_Y96 from the Fab light (L) and heavy (H) chains span a hydrophobic surface region that serves as docking sites. Overall, the ZTS-426:(TGFβ1)2:ZTS-426 complex spans approximately 3514 Å. 2 of van der Waals surface area, approximately 1939 Å 2 is contributed by hydrophobic surface moieties. Qualitatively, light chain CDR-1 and -2 contribute minimally, while CDR-3 residues show a significant contribution.

[0278] Table 4 Hypervariable region residues of ZTS-426 Fab at the interface of the mature TGFβ1 dimer. Kabat numbering used for CDR definition. TIFF0007798805000004.tif218140

[0279] Table 4 lists some of the proximal residues and specific interactions that account for the binding strength of the interface between mature TGFβ1 and ZTS-426 Fab. Specifically, an ionic bond between the R25 side chain and the heavy chain D54 and D56 carboxylate functional groups anchors the antigen-Fab complex. This is further supported by additional ionic bonds between the antigen side chains of Fab heavy chain residues K31 and D54. A network of explicit H-bond interactions between the side chains of Q19...L_N53, E35...L_Y96, K37...H_Q50, and H40...H_Y96 pairs further contributes to binding strength.

[0280] Superimposition of the structures of the TGFβ2 and TGFβ3 complexes onto the current X-ray structure revealed that 10 of the antigenic residues are completely conserved among TGFβ subfamily members. However, four of the unique residues are of homologous polarity (compared to human sequences), and two residues (Q19P and P87T) are identified as polarity switches that may affect binding and subsequent potency. Any effect on the binding of ZTS-426 to other TGFβ subfamily members may be due to differences in these residues.

[0281] Additional structural superposition of the ZTS-426 Fab-bound TGFβ1 complex with the GC1008 scFv-bound TGFβ1 complex reported in the literature reveals that the current antibody does not occupy the same binding region as that of GC1008. Furthermore, structural superposition indicates non-competitive binding between the GC1008 and ZTS-426 antibodies. However, superposition of the current complex with the ternary complex of the TGFβ1-bound TGFβRI / TGFβRII extracellular domain receptor suggests competitive binding.

[0282] A field study investigating the effect of ZTS-426 on the clinical progression of advanced CKD in client-owned dogs A proof-of-concept (PoC) field study was performed to investigate whether the beneficial effects noted in Alport dogs treated with ZTS-501 (the study described in the Examples below) translated to canine veterinary patients with naturally occurring CKD. The PoC study described herein evaluated the safety and mechanism of action of ZTS-426 on the clinical progression of advanced-stage CKD in client-owned dogs with naturally occurring CKD.

[0283] The study was conducted as a randomized, double-blind, multicenter clinical study in the United States. Dogs diagnosed with advanced CKD (IRIS stages 2, 3, and 4) were enrolled from 11 general veterinary practices. IRIS staging was based on fasting blood creatinine or fasting blo...

Claims

1. A caninized antibody that specifically binds to canine transforming growth factor beta 1 (TGFβ1) and canine transforming growth factor beta 3 (TGFβ3), a. a heavy chain variable region (VH), i. a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:5; ii. a complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 6; iii. A heavy chain variable region (VH) comprising a complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 7; and b. A light chain variable region (VL), i. a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 8; ii. a complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 9; iii. A light chain variable region (VL) comprising a complementarity-determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO:

10. The caninized antibody comprising:

2. The caninized antibody of claim 1, comprising a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:

24.

3. A pharmaceutical composition comprising the caninized antibody of claim 1 or 2 for use in treating canines for chronic kidney disease.

4. A method for treating a canine for chronic kidney disease, comprising administering to the canine a therapeutic amount of the pharmaceutical composition of claim 3.

5. 10. A method of inhibiting TGFβ1 and TGFβ1 activity in a canine by administering the pharmaceutical composition of claim 3.

6. An isolated nucleic acid encoding the caninized antibody of claim 2, wherein the nucleic acid comprises a nucleotide sequence encoding a VH comprising the sequence of SEQ ID NO: 11, and a nucleotide sequence encoding a VL comprising the sequence of SEQ ID NO:

23.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6.

9. A host cell comprising the vector of claim 7.

10. A host cell that produces the caninized antibody of claim 1 or 2.

11. 10. A method for producing a caninized antibody according to claim 1 or 2, comprising culturing a host cell according to claim 8 or 9 under conditions that result in the production of said caninized antibody, and isolating said caninized antibody from said host cell or the culture medium of said host cell.

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