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 of related applications This application asserts the benefit under § 119(e) of U.S. Patent Act pursuant to U.S. Provisional Patent Application No. 63 / 036,092, filed on 8 June 2020, which is incorporated herein by reference in its entirety.

[0002] This application relates to monoclonal antibodies, methods for producing them, and the therapeutic use of these antibodies. In certain embodiments, the monoclonal antibody targets transforming growth factor beta (TGFβ, TGFB, or TGFβ). In other embodiments, the antibody is a chimeric or bio-specified antibody. In other embodiments, therapeutic methods comprising the antibodies of the present invention are disclosed. [Background technology]

[0003] Transforming growth factor beta (TGFB, TGFβ, or TGFbeta as used interchangeably herein) is a cytokine that regulates many major cellular functions, including proliferation, differentiation, survival, migration, and epithelial-mesenchymal metastasis. It is a member of a superfamily of 38 cytokines, including TGFβ, bone morphogenesis proteins (BMPs), growth and differentiation factors, inhibitors, and activins. The TGFβ protein regulates a variety of biological processes, including extracellular matrix formation, wound healing, embryogenesis, osteogenesis, hematopoiesis, immune and inflammatory responses, and malignant transformation. Distortion of TGFβ leads to pathological conditions, including birth defects, cancer, chronic inflammation, autoimmune and fibrous diseases.

[0004] There are three known isoforms of TGFβ: 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, which are then cleaved near the carboxyl terminus to yield mature 112-amino acid polypeptides that share 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 latent-associated peptide (LAP) that forms a small latent complex (SLC) is biologically inactive and cannot bind to its receptor, TGFβRII. Through the formation of a disulfide bond, this complex loosely binds to latent TGFβ-binding protein (LTBP) to form a large latent complex (LLC). TGFβ is then secreted in a latent state and conserved in the extracellular matrix (ECM). TGFβ activation involves the release from its latent complex after exposure to numerous different factors, including integrins, proteases, metalloproteinases, reactive oxygen species (ROS), plasmins, and acids, which enable its binding to its cell surface receptor for the initiation of TGFβ signaling.

[0005] TGFβ1, 2, and 3 are multifaceted in their function and are expressed in different patterns across cell and tissue types. While they possess similar in vitro activity, individual knockouts in specific cell types suggest non-identical 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). When TGFβ binds to TGFβRII, the receptor's constitutive kinase activity phosphorylates and activates TGFβRII, thereby phosphorylating SMAD2 / 3, enabling association with SMAD4. This complex localizes to the nucleus and functions as a transcription factor for TGFβ-responsive genes. In addition to this classical signaling cascade, non-classical pathways transmit signals via other factors including p38, MAPK, PI3K, AKT, JUN, JNK, and NK-KB. The ultimate result is crosstalk of all these signaling pathways integrating the cellular state and environment.

[0006] Many severe diseases are associated with dysfunction of the TGFβ-induced signaling pathway. This invention addresses potential treatments for chronic kidney disease (CKD) in both canids and felines. CKD involves the loss of functional kidney tissue due to a long-term progressive process. Structural and functional changes in the kidneys are only loosely correlated, but dramatic changes in kidney structure can occur. The disease is usually present for months or years before it becomes clinically apparent, and it is always irreversible. Congenital disease results in a transient increase in prevalence in animals over 3 years of age, but prevalence increases with age progression beyond 5-6 years. In older populations, CKD affects 10% of dogs and 40-80% of cats. In the field of veterinary medicine, there is a clear and unmet need for treatment of CKD in both dogs and cats, a condition suggested to be affected by the overproduction of TGFβ protein. [Overview of the project]

[0007] The present invention provides novel anti-transforming growth factor beta (TGFB, TGF beta, or TGFβ as defined herein and used interchangeably) antigen-binding proteins (antibodies, antibody fragments, and 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 them from binding to their receptors, thereby preventing activation of the binding-related pathways. 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 the production of vectors and host cells. The present invention further provides a method for producing and using the antibody / antigen-binding protein, and a method for treating TGFβ disorders in canids, felines, and humans by administering the antibody of the present invention.

[0008] In one embodiment, the present invention provides an antibody / antigen-binding protein (as used interchangeably herein) that binds to TGFβ1. In one or more embodiments, the antigen-binding protein also specifically binds to TGFβ3. In one or more embodiments, the antigen-binding protein also specifically binds to TGFβ2 and TGFβ3. In one or more embodiments, the antigen-binding protein of the present invention binds to TGFβ1 in canids. In one or more embodiments, the antigen-binding protein of the present invention binds to TGFβ1 in felines. In one or more embodiments, the antigen-binding protein of the present invention is administered as a pharmaceutical composition further comprising excipients to treat TGFβ-related disorders. In one or more embodiments, TGFβ-related disorders include renal fibrosis / chronic kidney disease.

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

[0010] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but does not bind to TGFβ2 or TGFβ3, wherein the antigen-binding protein comprises a complementary determination region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41, and a region having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42. Complementarity-determining region 2 (CDR2) containing the amino acid sequence and sequence number 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)], where X1 contains lysine(K) or arginine(R), X2 contains threonine(T) or alanine(A), and X3 contains glutamic acid. X4 comprises a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least approximately 95% sequence identity to an amino acid sequence containing serine (S), glutamic acid (E), glutamine (Q), or aspartic acid (D), and a light chain variable region (VL) containing a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least approximately 95% sequence identity to an amino acid sequence containing SEQ ID NO: 44, a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least approximately 95% sequence identity to an amino acid sequence containing SEQ ID NO: 45, and a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least approximately 95% sequence identity to an amino acid sequence containing SEQ ID NO: 46, as well as any variant thereof having one or more conservative amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises a variant that is not a conservative amino acid substitution.

[0011] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to TGFβ2 or TGFβ3, wherein the antigen-binding protein comprises a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41, a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42, and sequences 43, 141, 142, 149, 150, 153, 154, 155, 156, 162, 166, 167, 169, 170, 171, and sequence A heavy chain variable region comprising: a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with an amino acid sequence selected from the group consisting of sequence number 172 and sequence number 178; and a light chain variable region (VL) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with an amino acid sequence containing sequence number 44; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with an amino acid sequence containing sequence number 45; and a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with an amino acid sequence containing sequence number 46; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

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

[0013] In one or more embodiments, the antigen-binding protein of the present invention that specifically binds to TGFβ1 of Canidae or Felidae animals but does not bind to TGFβ2 or TGFβ3 includes a complementarity-determining region 1 (CDR1) comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 4[], a complementarity-determining region 2 (CDR2) comprising an amino acid sequence having at least about 95% sequence identity to the 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 the amino acid sequence comprising SEQ ID NO: 141, and a heavy-chain variable region (VH) comprising any variants thereof having one or more conservative amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that are not conservative amino acid substitutions.

[0014] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 142; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0015] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 149; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0016] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 150; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0017] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 153; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0018] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 154; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0019] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 155; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0020] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41, a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42, and a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 156, and any variant thereof having one or more conserved amino acid substitutions, and in one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0021] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 162; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0022] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 166; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0023] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 167; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0024] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 169; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0025] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 170; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0026] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 171; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0027] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 172; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0028] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but not to TGFβ2 or TGFβ3, includes a heavy chain variable region (VH) comprising: a complementarity-determining region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 41; a complementarity-determining region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 42; a complementarity-determining region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 178; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0029] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 of canids or felines but not to TGFβ2 or TGFβ3, includes canine, feline, humanized, or chimeric antigen-binding proteins. In one or more embodiments, the antigen-binding protein is a canine antigen-binding protein. In one or more embodiments, the antigen-binding protein is a feline 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 present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to TGFβ2 or TGFβ3, and the antigen-binding protein includes a heavy chain variable region (VH) having at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 47, 55, 57, 231, 232, 239, 240, 243, 244, 245, 246, 252, 256, 257, 259, 260, 261, 262, and 268; a light chain variable region (VL) having at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 51, and 53; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0031] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 38, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 49, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0032] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 38, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0033] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 38, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 53, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0034] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 47, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 53, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0035] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 56, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0036] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 231, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0037] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 232, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0038] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines but not to 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 containing SEQ ID NO: 239, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions. In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines but not to 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 containing SEQ ID NO: 240, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises a variant that is not a conservative amino acid substitution.

[0039] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 243, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0040] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 244, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0041] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 245, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0042] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 246, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0043] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 252, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0044] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 256, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0045] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 257, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0046] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 259, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0047] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 260, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0048] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 261, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0049] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 262, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0050] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines, but not to 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 containing SEQ ID NO: 268, a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 51, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0051] In one or more embodiments, the present invention provides canine antigen-binding proteins that specifically bind to TGFβ1 of canids or felines but not to TGFβ2 or TGFβ3, the canine antigen-binding proteins further comprising a canine heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 127, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding proteins of the present invention include variants that do not have conserved amino acid substitutions.

[0052] In one or more embodiments, the present invention provides that the canine antigen-binding protein of the present invention, which specifically binds to TGFβ1 of canids or felines but not to TGFβ2 or TGFβ3, further comprises a canine light chain constant region comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 129. In one embodiment, the antigen-binding protein of the present invention comprises a heavy chain variable region (VH) having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 55, and a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 49; a constant region of the heavy chain (SEQ ID NO: 127); and a constant region of the light chain (SEQ ID NO: 129); and any variants thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0053] In one embodiment, the present invention provides an antigen-binding protein, including a feline antigen-binding protein, that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3. 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, 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 having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0054] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, wherein the antigen-binding protein comprises a heavy chain variable region (VH) having at least about 95% sequence identity with the amino acid sequence number 59, a light chain variable region (VL) having at least about 95% sequence identity with the amino acids including sequence number 61, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention comprises variants that do not have conserved amino acid substitutions.

[0055] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, wherein the antigen-binding protein further comprises a feline heavy chain constant region having an amino acid sequence having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 131, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0056] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, wherein the antigen-binding protein further comprises a feline light chain constant region having an amino acid sequence having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 133, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

[0057] In one embodiment, the present invention provides an antigen-binding protein, including a humanized antigen-binding protein, that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3. In one embodiment, the present invention provides an antigen-binding protein, including a chimeric antigen-binding protein and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding protein of the present invention includes variants that do not have conserved amino acid substitutions.

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

[0059] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, the protein 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-deletion 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 module immunotherapy (SMIP) nanobodies, and IgNAR molecules. In one embodiment, the antigen-binding protein is a monoclonal antigen-binding protein.

[0060] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, is intended for use in the treatment of canids for TGFβ-related disorders. In one or more embodiments, the antigen-binding protein of the present invention is intended for use in the treatment of felines for TGFβ-related disorders. In one or more embodiments, the antigen-binding protein of the present invention is intended for use in the treatment of humans for TGFβ-related disorders.

[0061] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, and the binding protein is used to reduce or eliminate TGFβ-related disorders. In one embodiment, the TGFβ-related disorder is selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders. In one embodiment, the TGFβ-related disorder includes a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, 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 TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, the pharmaceutical composition comprising a therapeutically effective amount of the antigen-binding protein of the present invention and a pharmaceutically acceptable carrier.

[0063] In one or more embodiments, the present invention provides a method for treating a subject for TGFβ-related disorders, wherein the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, is administered to the subject by a therapeutic dose of the pharmaceutical composition of the present invention. In one or more embodiments, the present invention provides a method for treating canids for TGFβ-related disorders. In one embodiment, the present invention provides a method for treating felines for TGFβ-related disorders. In one embodiment, the present invention provides a method for treating humans for TGFβ-related disorders. In one embodiment, the method provides administering a therapeutically effective dose of the pharmaceutical composition comprising the antigen-binding protein of the present invention. In one or more embodiments, the present invention provides that the TGFβ-related disorder is selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders. In one embodiment, the TGFβ-related disorder includes fibrotic disorders. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, 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 present invention provides a method for inhibiting TGFβ activity in a subject by administering a pharmaceutical composition containing the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3. In one embodiment, the subject includes canids, felines, or humans. In one embodiment, the subject includes canids. In one embodiment, the subject includes felines. In one embodiment, the subject includes humans.

[0065] In one or more embodiments, the present invention provides isolated nucleic acid sequences having at least about 95% sequence identity to the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, and any variant thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution. In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen-binding protein of the present invention, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 54, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 293, and any variant thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution.

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

[0067] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 58, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 60, and any variants thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution.

[0068] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 58, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 39, and any variants thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution.

[0069] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 37, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 60, and any variants thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution.

[0070] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen-binding protein of the present invention that specifically binds to TGFβ1 of canids or felines but does not bind to TGFβ2 or TGFβ3, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 58, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 60, a nucleotide sequence encoding the feline heavy chain constant region having 95% sequence identity to SEQ ID NO: 132, and a nucleotide sequence encoding the 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 a conserved amino acid substitution.

[0071] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen-binding protein of the present invention that specifically binds to TGFβ1 of canids or felines but does not bind to TGFβ2 or TGFβ3, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 58, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 39, a nucleotide sequence encoding the feline heavy chain constant region having 95% sequence identity to SEQ ID NO: 132, and a nucleotide sequence encoding the 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 a conserved amino acid substitution.

[0072] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding the antigen-binding protein of the present invention that specifically binds to TGFβ1 of canids or felines but does not bind to TGFβ2 or TGFβ3, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 37, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 60, a nucleotide sequence encoding the feline heavy chain constant region having 95% sequence identity to SEQ ID NO: 132, and a nucleotide sequence encoding the 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 a conserved amino acid substitution.

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

[0074] In one embodiment, the present invention provides a host cell comprising a nucleic acid sequence encoding the antigen-binding protein of the present invention, which specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3. In one embodiment, the present invention provides a host cell comprising a vector comprising a nucleic acid encoding the antigen-binding protein of the present invention. In one embodiment, the present invention provides a host cell that produces the antigen-binding protein of the present invention.

[0075] In one or more embodiments, the present invention provides a method for producing an antigen-binding protein of the present invention that specifically binds to TGFβ1 in canids or felines but does not bind to TGFβ2 or TGFβ3, the method comprising culturing host cells of the present invention under conditions that result in the production of the antigen-binding protein, and isolating the antigen-binding protein from the host cells or the culture medium of the host cells.

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

[0077] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein comprises a complementarity determination region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 5, a complementarity determination region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 6, and a complementarity determination region containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 7 The variants include a heavy chain variable region (VH) containing a determination region 3 (CDR3); a complementarity determination region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 8; a complementarity determination region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 9; a complementarity determination region 3 (CDR3) containing an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 10; and any variants thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variants of the antigen-binding protein of the present invention do not contain conserved amino acid substitutions.

[0078] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein includes a heavy chain variable region (VH) comprising a complementarity-determining region 1 (CDR1) containing amino acid sequence number 5, a complementarity-determining region 2 (CDR2) containing amino acid sequence number 6, and a complementarity-determining region 3 (CDR3) containing amino acid sequence number 7; and a light chain variable region (VL) comprising a complementarity-determining region 1 (CDR1) containing amino acid sequence number 8, a complementarity-determining region 2 (CDR2) containing amino acid sequence number 9, and a complementarity-determining region 3 (CDR3) containing amino acid sequence number 10; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variant of the antigen-binding protein of the present invention does not have a conserved amino acid substitution.

[0079] In one or more embodiments, the present invention provides antigen-binding proteins that specifically bind to TGFβ1 and TGFβ3 in canids or felines, but not to TGFβ2, and the antigen-binding proteins include canine, feline, humanized, mouse, or chimeric antigen-binding proteins. In one embodiment, the antigen-binding protein includes a canine antigen-binding protein. In one embodiment, the antigen-binding protein includes 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 NOs: 12, SEQ ID NOs: 14, and SEQ ID NOs: 16; 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 NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, and SEQ ID NOs: 24; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the antigen-binding proteins of the present invention include variants that do not have conserved amino acid substitutions.

[0080] In one or more embodiments, the present invention provides antigen-binding proteins that specifically bind to TGFβ1 and TGFβ3 in canids or felines, but not to TGFβ2, and the antigen-binding proteins include canine, feline, humanized, mouse, or chimeric antigen-binding proteins. In one embodiment, the antigen-binding protein includes a canine antigen-binding protein. In one embodiment, the antigen-binding protein includes a heavy chain variable region (VH) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, SEQ ID NOs: 14, and SEQ ID NOs: 16; a light chain variable region (VL) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, and SEQ ID NOs: 24; and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variants of the antigen-binding proteins of the present invention do not have conserved amino acid substitutions.

[0081] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity with the amino acid sequence containing SEQ ID NO: 12, a light chain variable region (VL) having at least 95% sequence identity with the amino acid sequence containing SEQ ID NO: 24, and any variant thereof having one or more conserved amino acid substitutions, wherein the variant of the antigen-binding protein of the present invention does not have a conserved amino acid substitution.

[0082] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to the amino acid sequence containing SEQ ID NO: 14, a light chain variable region (VL) having at least 95% sequence identity to the amino acid sequence containing SEQ ID NO: 24, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variant of the antigen-binding protein of the present invention does not have a conserved amino acid substitution.

[0083] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to the amino acid sequence containing SEQ ID NO: 13, a light chain variable region (VL) having at least 95% sequence identity to the amino acid sequence containing SEQ ID NO: 23, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variant of the antigen-binding protein of the present invention does not have a conserved amino acid substitution.

[0084] In one or more embodiments, the present invention further provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 of canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein includes a canid heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 127. In one or more embodiments, the canid heavy chain constant region includes a conservative amino acid substitution. In one or more embodiments, the amino acid substitution is non-conservative.

[0085] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein includes a canid heavy chain constant region containing the amino acid sequence SEQ ID NO: 127. In one or more embodiments, the canid heavy chain constant region includes a conservative amino acid substitution. In one or more embodiments, the amino acid substitution is non-conservative.

[0086] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 of canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein includes a canid light chain constant region comprising an amino acid sequence having at least about 95% sequence identity with the amino acid sequence comprising SEQ ID NO: 129. In one or more embodiments, the present invention provides an antigen-binding protein comprising a canid light chain constant region comprising the amino acid sequence SEQ ID NO: 129. In one or more embodiments, the canid light chain constant region includes a conservative amino acid substitution. In one or more embodiments, the amino acid substitution is non-conservative.

[0087] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein comprises a heavy chain variable region (VH) having at least 95% sequence identity to the amino acid sequence containing SEQ ID NO: 12, a light chain variable region (VL) having at least 95% sequence identity to the amino acid sequence containing SEQ ID NO: 24, a canid heavy chain constant region containing an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 127, and a canid light chain constant region containing an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 129, and any variant thereof having one or more conserved amino acid substitutions, wherein the variant of the antigen-binding protein of the present invention is not a conserved amino acid substitution.

[0088] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, wherein the antigen-binding protein comprises a heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 12, a light chain variable region (VL) containing the amino acid sequence SEQ ID NO: 24, a canid heavy chain constant region containing the amino acid sequence SEQ ID NO: 127, and a canid light chain constant region containing the amino acid sequence SEQ ID NO: 129, and any variant thereof having one or more conserved amino acid substitutions, wherein the variant of the antigen-binding protein of the present invention is not a conserved amino acid substitution.

[0089] In one embodiment, the present invention provides an antigen-binding protein, including a feline antigen-binding protein, that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2. 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 NOs. 26, SEQ ID NOs. 28, and SEQ ID NOs. 30, and a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs. 32 or SEQ ID NOs. 34, and SEQ ID NOs. 36, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variant of the antigen-binding protein of the present invention does not have a conserved amino acid substitution.

[0090] In one embodiment, the present invention provides that an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines but does not bind to TGFβ2 comprises a heavy chain variable region (VH) having at least 95% sequence identity with the amino acid sequence comprising SEQ ID NO: 26, a light chain variable region (VL) having at least 95% sequence identity with the amino acid sequence comprising SEQ ID NO: 32, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variant of the antigen-binding protein of the present invention is not a conserved amino acid substitution.

[0091] In one or more embodiments, the present invention provides antigen-binding proteins that specifically bind to TGFβ1 and TGFβ3 in canids or felines, but not to 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 the amino acid sequence comprising SEQ ID NO: 131, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variants of the antigen-binding proteins of the present invention are not conserved amino acid substitutions.

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

[0093] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, the antigen-binding protein comprising a heavy chain variable region (VH) having at least 95% sequence identity to the amino acid sequence comprising SEQ ID NO: 26, a light chain variable region (VL) having at least 95% sequence identity to the 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 the 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 the amino acid sequence comprising SEQ ID NO: 133, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variant of the antigen-binding protein of the present invention is not a conserved amino acid substitution.

[0094] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, including a humanized antigen-binding protein. In another embodiment, the present invention provides an antigen-binding protein including a chimeric antigen-binding protein.

[0095] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 and TGFβ3 in canids or felines but does not bind to TGFβ2, is intended for use in the treatment of canids for TGFβ-related disorders. In one or more embodiments, the antigen-binding protein of the present invention is intended for use in the treatment of felines for TGFβ-related disorders. In one or more embodiments, the antigen-binding protein of the present invention is intended for use in the treatment of humans for TGFβ-related disorders.

[0096] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2, and the protein is 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-deletion 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 module immunotherapy (SMIP) nanobodies, and IgNAR molecules. In one embodiment, the antigen-binding protein is a monoclonal antigen-binding protein.

[0097] In one or more embodiments, the present invention provides antigen-binding proteins for use in treating TGFβ-related disorders that specifically bind to TGFβ1 and TGFβ3 in canids or felines, but not to TGFβ2, and that reduce or eliminate TGFβ-related disorders. In one embodiment, the TGFβ-related disorder is selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders. In one embodiment, the TGFβ-related disorder includes a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, 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 the antigen-binding protein of the present invention, which specifically binds to TGFβ1 and TGFβ3 in canids or felines but does not bind to TGFβ2, and a pharmaceutically acceptable carrier.

[0099] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1 and TGFβ3 in canids or felines but does not bind to TGFβ2, provides a method for treating a subject for TGFβ-related disorders, wherein the treatment involves administering a therapeutic dose of the pharmaceutical composition of the present invention to the subject. In one or more embodiments, the present invention provides a method for treating canids for TGFβ-related disorders. In one embodiment, the present invention provides a method for treating felines for TGFβ-related disorders. In one embodiment, the present invention provides a method for treating humans for TGFβ-related disorders. In one embodiment, the present invention provides a method for administering a therapeutically effective dose of the pharmaceutical composition comprising the antigen-binding protein of the present invention. In one or more embodiments, the present invention provides that the TGFβ-related disorder is selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders. In one embodiment, the TGFβ-related disorder includes fibrotic disorders. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, 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 a method for inhibiting TGFβ activity in a subject by administering a pharmaceutical composition containing the antigen-binding protein of the present invention, which specifically binds to TGFβ1 and TGFβ3 in canids or felines but does not bind to TGFβ2. In one embodiment, the subject includes canids, felines, or humans. In one embodiment, the subject includes canids. In one embodiment, the subject includes felines. In one embodiment, the subject includes humans.

[0101] In one or more embodiments, the present invention provides an antigen-binding protein of the present invention that specifically binds to TGFβ1 and TGFβ3 in canids or felines but does not bind to TGFβ2, and an isolated nucleic acid sequence having at least about 95% sequence identity to a nucleic acid sequence encoding any variant thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution.

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

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

[0104] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen-binding proteins of the present invention that specifically bind to TGFβ1 and TGFβ3 in canids or felines but not to TGFβ2, the sequences comprising a nucleotide sequence encoding VH having 95% sequence identity to SEQ ID NO: 13, a nucleotide sequence encoding VL having 95% sequence identity to SEQ ID NO: 23, and any variants thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution.

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

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

[0107] In one or more embodiments, the present invention provides isolated nucleic acid sequences encoding antigen-binding proteins of the present invention that specifically bind to TGFβ1 and TGFβ3 in canids or felines but not to TGFβ2, the nucleic acid sequences further comprising nucleotide sequences encoding 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 a conserved amino acid substitution.

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

[0109] In one embodiment, the present invention provides a host cell comprising a nucleic acid sequence encoding the antigen-binding protein of the present invention, which specifically binds to TGFβ1 and TGFβ3 in canids or felines, but does not bind to TGFβ2. In one embodiment, the present invention provides a host cell comprising a vector comprising a nucleic acid encoding the antigen-binding protein of the present invention. In one embodiment, the present invention provides a host cell that produces the antigen-binding protein of the present invention.

[0110] In one or more embodiments, the present invention provides a method for producing an antigen-binding protein of the present invention that specifically binds to TGFβ1 and TGFβ3 in canids or felines but does not bind to TGFβ2, the method comprising culturing host cells of the present invention under conditions that result in the production of the antigen-binding protein, and isolating the antigen-binding protein from the host cells or the culture medium of the host cells.

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

[0112] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1, TGFβ2, and TGFβ3 of canids or felines, wherein the antigen-binding protein comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 270 [Glycine(G)-Tyrosine(Y)-(X1)-Phenylalanine(F)-(X2)-(X3)-Tyrosine(Y)], wherein (X1) comprises threonine(T) or isoleucine(I), (X2) comprises isoleucine(I) or methionine(M), and (X3) comprises threonine(T) or lysine(K), and a complementation determining region 1 (CDR1) and an amino acid sequence containing SEQ ID NO: 271 [Phenylalanine(F)-Proline(P)-(X4)-(X5)-Glycine(G)-(X6)] Complementarity-determining region 2 (CDR2) includes an amino acid sequence having at least approximately 95% sequence identity, wherein (X4) contains alanine (A) or glycine (G), (X5) contains serine (S) or tryptophan (W), and (X6) contains serine (S), methionine (M), or valine (V); and heavy chain variable region (VH) includes an amino acid sequence having at least approximately 95% sequence identity to the amino acid sequence containing 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) contains aspartic acid (D) or tyrosine (Y);Complementarity-determining region 1 (CDR1) includes an amino acid sequence having at least about 95% sequence identity with the amino acid sequence including SEQ ID NO: 69; Complementarity-determining region 2 (CDR2) includes an amino acid sequence having at least about 95% sequence identity with the amino acid sequence including SEQ ID NO: 70; and Complementarity-determining region 3 (CDR3) includes an amino acid sequence having at least about 95% sequence identity with the amino acid sequence including SEQ ID NO: 273 [Glutamine(Q)-Glutamine(Q)-Asparagine(N)-(X8)-Glutamic acid(E)-Aspartic acid(D)-Proline(P)-Leucine(L)-(X9)], wherein (X8) is asparagine(N) or aspartic acid(D), and (X9) is threonine(T) or serine(S); and any variant 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 an antigen-binding protein that specifically binds to TGFβ1, TGFβ2, and TGFβ3 in canids or felines, wherein the antigen-binding protein comprises: Complementarity Determination Region 1 (CDR1) containing 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; Complementarity Determination Region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity to an amino acid sequence including SEQ ID NO: 67, SEQ ID NO: 278, SEQ ID NO: 279, and SEQ ID NO: 280; and at least about A heavy chain variable region (VH) comprising a complementarity determination region 3 (CDR3) containing an amino acid sequence having 95% sequence identity; a light chain variable region (VL) comprising a complementarity determination region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 69; a complementarity determination region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity to the amino acid sequence containing SEQ ID NO: 70; and a light chain variable region (VL) comprising a complementarity determination region 3 (CDR3) containing 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 present invention provides an antigen-binding protein that specifically binds to TGFβ1, TGFβ2, and TGFβ3 in canids or felines, wherein the antigen-binding protein comprises a complementation determination region 1 (CDR1) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: 66, a complementation determination region 2 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 67, and a complementation determination region 3 (CDR2) containing an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 68. A light chain variable region (VL) comprising: a heavy chain variable region (VH) including DR3); a complementarity determination region 1 (CDR1) including an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 69; a complementarity determination region 2 (CDR2) including an amino acid sequence having at least about 95% sequence identity with the amino acid sequence containing SEQ ID NO: 70; a complementarity determination region 3 (CDR3) including an amino acid sequence having at least about 95% sequence identity with the amino acid sequence SEQ ID NO: 71; 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.

[0115] In one or more embodiments, the present invention provides antigen-binding proteins that specifically bind to TGFβ1, TGFβ2, and TGFβ3 of canids or felines, wherein the antigen-binding proteins include canine, feline, humanized, or chimeric antigen-binding proteins. In one embodiment, the antigen-binding protein includes a canine antigen-binding protein.

[0116] In one embodiment, the present invention provides antigen-binding proteins that specifically bind to TGFβ1, TGFβ2, and TGFβ3 in canids or felines, and the antigen-binding proteins are sequence numbers 73, 75, 77, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, and 211. The variants include a heavy chain variable region (VH) having at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 213 and 215; a light chain variable region (VL) having at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 216, and 218; and any variants thereof having one or more conservative amino acid substitutions. In one or more embodiments, the variant is not a conservative amino substitution.

[0117] In one embodiment, the present invention is a. Heavy chain variable region (VH) containing SEQ ID NO: 181, and light chain variable region (VL) containing SEQ ID NO: 85, b. Heavy chain variable region (VH) containing SEQ ID NO: 181, and light chain variable region (VL) containing SEQ ID NO: 216, c. Heavy chain variable region (VH) containing SEQ ID NO: 183, and light chain variable region (VL) containing SEQ ID NO: 85, d. Heavy chain variable region (VH) containing SEQ ID NO: 185, and light chain variable region (VL) containing SEQ ID NO: 85, e. Heavy chain variable region (VH) containing SEQ ID NO: 187, and light chain variable region (VL) containing SEQ ID NO: 85, f. Heavy chain variable region (VH) containing SEQ ID NO: 187, and light chain variable region (VL) containing SEQ ID NO: 218, g. Heavy chain variable region (VH) containing SEQ ID NO: 185, and light chain variable region (VL) containing SEQ ID NO: 218, h. Heavy chain variable region (VH) containing SEQ ID NO: 189, and light chain variable region (VL) containing SEQ ID NO: 218, i. Heavy chain variable region (VH) containing SEQ ID NO: 191, and light chain variable region (VL) containing SEQ ID NO: 218, j. Heavy chain variable region (VH) containing SEQ ID NO: 193, and light chain variable region (VL) containing SEQ ID NO: 218, k. Heavy chain variable region (VH) containing sequence number 195, and light chain variable region (VL) containing sequence number 218, l. Heavy chain variable region (VH) containing SEQ ID NO: 197, and light chain variable region (VL) containing SEQ ID NO: 218, m. Heavy chain variable region (VH) containing sequence number 199, and light chain variable region (VL) containing sequence number 218, n. Heavy chain variable region (VH) containing sequence number 199, and light chain variable region (VL) containing sequence number 85, o. Heavy chain variable region (VH) containing sequence number 193, and light chain variable region (VL) containing sequence number 85, p. Heavy chain variable region (VH) containing SEQ ID NO: 195, and light chain variable region (VL) containing SEQ ID NO: 85, q. Heavy chain variable region (VH) containing sequence number 191, and light chain variable region (VL) containing sequence number 85, r. Heavy chain variable region (VH) containing sequence number 197, and light chain variable region (VL) containing sequence number 85, s. Heavy chain variable region (VH) containing sequence number 189, and light chain variable region (VL) containing sequence number 85, t. Heavy chain variable region (VH) containing SEQ ID NO: 201, and light chain variable region (VL) containing SEQ ID NO: 218. u. Heavy chain variable region (VH) containing sequence number 203, and light chain variable region (VL) containing sequence number 218, v. Heavy chain variable region (VH) containing SEQ ID NO: 205, and light chain variable region (VL) containing SEQ ID NO: 218 w. Heavy chain variable region (VH) containing sequence number 201, and light chain variable region (VL) containing sequence number 85, x. Heavy chain variable region (VH) containing sequence number 205, and light chain variable region (VL) containing sequence number 85. y. Both the heavy chain variable region (VH) paired with the light chain variable region (VL) and the amino acid containing the heavy chain variable region (VH) including SEQ ID NO: 215, and the light chain variable region (VL) including SEQ ID NO: 218, and The present invention provides antigen-binding proteins comprising any variants thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variants are not conserved amino acid substitutions.

[0118] In one or more embodiments, the present invention provides an antigen-binding protein that specifically binds to TGFβ1, TGFβ2, and TGFβ3 of canids or felines, wherein the antigen-binding protein includes a canid heavy chain constant region comprising an amino acid sequence having at least about 95% sequence identity with the amino acid sequence comprising SEQ ID NO: 127. In one or more embodiments, the present invention provides an antigen-binding protein comprising a canid heavy chain constant region comprising the amino acid sequence SEQ ID NO: 127. In one or more embodiments, the canid heavy chain constant region includes a conservative amino acid substitution. In one or more embodiments, the variant does not include a conservative amino acid substitution.

[0119] In one or more embodiments, the present invention provides an antigen-binding protein comprising a canine light chain constant region comprising an amino acid sequence having at least about 95% sequence identity with respect to the amino acid sequence comprising SEQ ID NO: 129. In one or more embodiments, the present invention provides an antigen-binding protein 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 a conservative amino acid substitution. In one or more embodiments, the variant does not comprise a conservative amino acid substitution.

[0120] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1, TGFβ2, and TGFβ3 of canids or felines, comprising a feline 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 SEQ ID NO: 120 or SEQ ID NO: 122, a light chain variable region (VL) having at least 95% sequence identity to an amino acid sequence including SEQ ID NO: 99, and any variant thereof having one or more conserved amino acid substitutions. In one or more embodiments, the variant is not a conserved amino acid substitution.

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

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

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

[0124] In one embodiment, the present invention provides an antigen-binding protein that specifically binds to TGFβ1, TGFβ2, and TGFβ3 of canids or felines, including a humanized antigen-binding protein. In another embodiment, the present invention provides an antigen-binding protein that includes a chimeric antigen-binding protein.

[0125] In one or more embodiments, the present invention provides antigen-binding proteins that specifically bind to TGFβ1, TGFβ2, and TGFβ3 in canids or felines, 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-deletion 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 module immunotherapy (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 TGFβ1, TGFβ2, and TGFβ3 in canids or felines, and that these binding proteins are intended for use in reducing or eliminating TGFβ-related disorders. In one embodiment, the TGFβ-related disorder is selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders. In one embodiment, the TGFβ-related disorder includes a fibrotic disorder. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, 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 the antigen-binding protein of the present invention, which specifically binds to TGFβ1, TGFβ2, and TGFβ3 in canids or felines, and a pharmaceutically acceptable carrier.

[0128] In one or more embodiments, the antigen-binding protein of the present invention, which specifically binds to TGFβ1, TGFβ2, and TGFβ3 in canids or felines, provides a method for treating a subject for TGFβ-related disorders, wherein the treatment involves administering a therapeutic dose of the pharmaceutical composition of the present invention to the subject. In one or more embodiments, the present invention provides a method for treating canids for TGFβ-related disorders. In one embodiment, the present invention provides a method for treating felines for TGFβ-related disorders. In one embodiment, the present invention provides a method for treating humans for TGFβ-related disorders. In one embodiment, the method provides administering a therapeutically effective dose of the pharmaceutical composition comprising the antigen-binding protein of the present invention. In one or more embodiments, the present invention provides that the TGFβ-related disorder is selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders. In one embodiment, the TGFβ-related disorder includes fibrotic disorders. In one embodiment, the fibrotic disorder is selected from the group consisting of renal fibrosis / chronic kidney disease, pulmonary fibrosis, 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 a method for inhibiting TGFβ activity in a subject by administering a pharmaceutical composition comprising the antigen-binding protein of the present invention, which specifically binds to TGFβ1, TGFβ2, and TGFβ3 in canids or felines. In one embodiment, the subject includes canids, felines, or humans. In one embodiment, the subject includes canids. In one embodiment, the subject includes felines. In one embodiment, the subject includes humans.

[0130] In one or more embodiments, the present invention provides an antigen-binding protein of the present invention that specifically binds to TGFβ1, TGFβ2, and TGFβ3 of canids or felines, and an isolated nucleic acid sequence having at least about 95% sequence identity to a nucleic acid sequence encoding any variant thereof having one or more nucleic acid substitutions resulting in a conserved amino acid substitution.

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

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

[0133] In one or more embodiments, the present invention provides a vector comprising a nucleic acid sequence encoding the antigen-binding protein of the present invention, which specifically binds to TGFβ1, TGFβ2, and TGFβ3 in canids or felines.

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

[0135] In one or more embodiments, the present invention provides a method for producing the antigen-binding protein of the present invention, which specifically binds to TGFβ1, TGFβ2, and TGFβ3 in canids or felines, the method comprising culturing host cells of the present invention under conditions that result in the production of the antigen-binding protein, and isolating the antigen-binding protein from the host cells or the culture medium of the host cells. [Brief explanation of the drawing]

[0136] [Figure 1] Epitope mapping data of the anti-TGFβ antibody of the present invention that binds to TGFβ1 is presented. [Figure 2] This image shows the general structure of natural mouse immunoglobulin G (IgG), highlighting the antigen-binding site. [Figure 3] Mouse: This is a schematic diagram of the general structure of one embodiment of canine IgG. [Figure 4] This represents a heterochimeric molecule. [Figure 5] This represents the biological differentiation or canine differentiation of mouse IgG. [Figure 6] This is a graph showing the ZTS-426 SMAD3 inhibition curves for TGFβ1 and TGFβ3. [Figure 7] This represents a model of the secondary structure of a mature TGFβ1 dimer containing a Fab fragment bound to ZTS-426. [Figure 8] This represents a model of the superposition of the ZTS-426Fab complex with the TGFβ1-binding TGFβRI / TGFβRII complex. [Figure 9] This graph shows the regulation of disease progression in Alport dogs treated with ZTS-426 at different doses. [Figure 10] This represents a Phase 2 scenario plan for research related to the treatment of chronic kidney disease (CKD). [Figure 11A] This is a graph of survival curves related to the time to doubling of sCr after treatment with ZTS-426. [Figure 11B]This graph shows the survival curve after treatment with ZTS-426, displaying the time to stage 4 (sCr>5.0). [Figure 11C] This graph shows the survival curve after treatment with ZTS-426, specifically the time until Phos levels rise (>4.5 g / dl). [Figure 11D] This graph shows the survival curve after treatment with ZTS-426, death due to CKD, and the time until discontinuation of treatment. [Figure 11E] This graph shows the survival curve after treatment with ZTS-426 and the time until the end of the study. [Figure 11F] This graph shows the survival curve / time to uremic crisis after treatment with ZTS-426. [Figure 12] This is a graph showing the average ZTS-4155 concentration-time graph of canine serum. [Modes for carrying out the invention]

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

[0138] The present invention, as disclosed herein, provides anti-TGFβ antigen-binding proteins / antibodies / antibody fragments (terms are used interchangeably) that bind to TGFβ1 and / or TGFβ2 and / or TGFβ proteins with high affinity and specificity. The present invention further provides antigen-binding proteins and polypeptides that are variants of said antigen-binding proteins and bind to any one of the TGFβ proteins or polypeptides described herein, as well as methods for producing and using said proteins. In some embodiments, the present invention also provides polynucleotides encoding said antigen-binding proteins and / or polypeptides. The present invention, as disclosed herein, also provides methods for preventing and / or treating TGFβ-related disorders selected from the group of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders by administration of therapeutically effective amounts of the anti-TGFβ antigen-binding proteins of the present invention described herein and their respective variants.

[0139] General techniques and definitions It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and that these themselves may change. The terminology used herein is for the sole purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is defined solely by the claims. Unless otherwise defined, the scientific and technical terms used herein in connection with the present invention shall have meanings that are generally understood by those skilled in the art. Furthermore, unless otherwise required by the context, singular terms shall include plural forms and plural terms shall include singular forms. In general, the terminology and techniques used herein in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides 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 herein.

[0140] All specified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications that may be used in connection with the present invention. These publications are provided only for disclosures prior to the filing date of this 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 skilled in the art. Common techniques well known to those skilled in the art are performed according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures are generally performed according to conventional methods well known in the art and as described herein, but are not limited to the various general and more specific references cited and discussed throughout this specification. 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 & 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 See the Company (1993), and the different and current protocols used by those skilled in the art. Before describing the invention in detail, some terms used in the context of the invention are defined. In addition to these terms, other terms are defined elsewhere in this specification as necessary. Where 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 the meanings recognized in their respective art.

[0142] As used herein and in the claims, singular forms such as "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. For example, a reference to "antibody" refers to multiple such antibodies.

[0143] In this disclosure, terms such as “comprises,” “comprised,” “comprising,” “contains,” “containing,” “consisted of,” “essentially consisted of,” “includes,” and “included” are defined in accordance with standard U.S. patent law and international patent law practices.

[0144] The term “approximately” is used herein to indicate that a value includes the standard deviation of errors in the device or method used to determine that value. The term “at least approximately” is used herein to indicate a lower limit of a range. For example, in the term “having at least approximately 95% sequence identity,” it should be obvious to those skilled in the art that this includes 95% sequence identity through 100% sequence identity. The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated that it refers only to substitutes or that the substitutes are mutually exclusive, however, this disclosure supports the definitions of “substitutes only” and “and / or” respectively.

[0145] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner 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 are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes are chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0146] Amino acids may be referred herein by either their commonly known three-letter symbols or their single-letter symbols as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred to by their commonly accepted single-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 the locally regular three-dimensional structure within a polypeptide. These structures are commonly known as domains, e.g., enzyme domains, extracellular domains, transmembrane domains, pore domains, or cytoplasmic tail domains. Domains are parts of a polypeptide that form compact units of the polypeptide. Exemplary domains include those with enzymatic activity. Domains may consist of elongated β-sheets and smaller organized sections such as α-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 non-covalent 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 very similar to that of the given residue, and therefore does not result in a substantial decrease in polypeptide function (e.g., enzyme activity). Conservative amino acid substitutions are generally known in the art, and examples are described, for example, in U.S. Patents 6,790639, 6,774107, 6,194167, or 5350576. In preferred embodiments, a conservative amino acid substitution is any one occurring within one of the following six groups: ● Small aliphatic substantially nonpolar residues: Ala, Gly, Pro, Ser, and Thr, ●Large aliphatic nonpolar residues: Ile, Leu, and Val, Met, ● Negatively charged residues and their amides: Asp and Glu, ● Amides of negatively charged residues: Asn and Gln, His, ●Positively charged residues: Arg and Lys, His, and ●Large aromatic residues: Trp, Tyr, and Phe.

[0148] In preferred embodiments, a conserved amino acid substitution is one of the following listed as native residue (conserved 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. The polymers may be linear or branched, and may include modified amino acids, which may be interrupted by non-amino acids. These terms also encompass naturally modified or intervened amino acid polymers of any other operation or modification, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. Also included within the definition are polypeptides containing, for example, one or more analogues of amino acids (including, for example, non-natural amino acids), and other modifications known in the art. Since the polypeptides of the present invention are antibody-based, it is understood that the polypeptides may arise as single-chain or associated chains.

[0150] As used herein, “antibody,” “antigen-binding protein,” etc., refer to polypeptides containing a region encoded by an immunoglobulin gene or an antibody fragment thereof that specifically binds to and recognizes an antigen. Exemplary immunoglobulin (antibody) structural units may include tetramers, 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 involved in 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 by various peptidases. While various antibody fragments are defined in terms of the digestion of intact antibodies, those skilled in the art will understand that such fragments can either be chemically de novo synthesized or de novo synthesized by using recombinant DNA methodologies. Therefore, as used herein, the term “antibody” also includes any antibody fragment produced by modification of a whole antibody, or an antibody fragment de novo synthesized using recombinant DNA methodologies, or an antibody fragment identified using other methods known in the art.

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

[0152] The term "variable" region refers to the fact that certain parts of the variable domain, including framework and CDRs (also known as "hypervariable regions"), differ extensively in the sequence between antibodies and are used in the binding and specificity of each particular antibody to that particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called "complementarity-determining regions (CDRs)" or "hypervariable regions" in both the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The natural heavy and light chain variable domains each contain multiple FRs, primarily employing a β-sheet configuration connected by three hypervariable regions that form loops connecting β-sheet structures, and in some cases forming parts of them. The hypervariable regions within each chain are held together in close proximity by the fiber rearm (FR) and, together with the hypervariable regions from other chains, contribute to the formation of the antibody's antigen-binding site (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 domain does not directly participate in binding the antibody to the antigen, but exhibits various effector functions as described herein.

[0153] The characteristics of the four human IgG subclasses are well-established, each possessing distinct features and significantly different involvement in the immune system. Human IgG subclasses are differentiated by their binding affinity to immune effector proteins, including the neonatal Fc receptor (FcRn), the Fc gamma receptor (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 to these receptors is often used to characterize the functional properties of antibodies (Bruggemann et al., 1987). Higher affinity for FcγR1 and FcγRIII indicates that the antibody possesses ADCC activity, while binding of 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, exhibits complement activity that helps activate phagocytic cells and 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 functions of IgG subclasses assist 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 research focused on IgG, and Mazza et al. (1993) isolated four fractions from IgG-rich canine serum, separating each by gel filtration, protein A / G binding, and electrophoretic mobility. These fractions were used to obtain antibody reagents specific to canine IgG (Mazza et al., 1994). This study 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 solving these questions. Similar to human IgG, canine IgG consists of four subclasses. Based on accession numbers [AF354264, AF354265, AF354266, and AF354267], Bergeron et al (Veterinary Immunology and Immunopathology 157(2014)31-41) designated these canine IgG subclasses as 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 associated with two previously isolated known sequences from a feline spleen cDNA library. 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, tentatively called IgG2, and described the interactions of the three feline IgGs with identified feline FcγRI, FcγRIII, FcRn, and C1q. Additionally, feline kappa and lambda light chain regions were 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, and downregulation of cell surface receptors (e.g., B cell receptors, BCRs). Such effector functions generally require an Fc region that is combined with a binding domain (e.g., an antibody-variable domain), and can be evaluated using various assays known in the art for assessing such antibody effector functions.

[0157] A “natural sequence Fc region” includes an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. Non-limiting examples of sequences for a natural Fc region include amino acid sequences having about 80-99% sequence identity to SEQ ID NO: 292. In one embodiment, the antibody of the present invention includes a natural Fc region containing SEQ ID NO: 292. The natural Fc region as used herein preferably has at least about 80% sequence identity with the natural sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity, and more preferably at least about 95% sequence identity. A “variant Fc region,” or “mutated” or “mutant” Fc region, contains an amino acid sequence different from that of the natural sequence Fc region due to at least one amino acid modification, and may or may not retain at least one effector function of the natural sequence Fc region compared to the natural Fc region. Preferably, the variant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, compared to the natural sequence Fc region or the parent polypeptide Fc region, and preferably about 1 to about 5 amino acid substitutions in the natural sequence Fc region or the parent polypeptide Fc region. The variant Fc region described herein preferably has at least about 80% sequence identity with the natural sequence Fc region and / or the parent polypeptide Fc region, most preferably at least about 90% sequence identity, and more preferably at least about 95% sequence identity. The variant or mutated Fc region may also essentially eliminate the function of the antibody's Fc region. The variant or mutated Fc region may also add to or enhance the function of the antibody's Fc region. For example, an Fc region mutation may eliminate the effector function of the antibody. In another example, a mutated Fc region may enhance the effector function of the antibody. In yet another example, the mutated Fc region may alter the half-life of other intracellular factors that can determine the antibody's properties, or affect their binding. In one embodiment, the antibody of the present invention includes a mutated Fc region. In one embodiment, the antibody of the present invention includes a variant or mutated Fc region containing an amino acid sequence having approximately 80-99% sequence identity with SEQ ID NO: 288.In one embodiment, the antibody of the invention comprises a variant or mutated Fc region comprising the amino acid sequence comprising SEQ ID NO: 288.

[0158] As used herein, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are natural sequence FcRs. Furthermore, preferred FcRs are those that bind to 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 ("activating receptor") and FcyRIIB ("inhibitory receptor"), which mainly have similar amino acid sequences that differ 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 non-specific cytotoxic cells expressing Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and subsequently cause lysis of the target cell. The ADCC activity of a molecule of interest can be evaluated using, for example, an in vitro ADCC assay as described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, 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 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 evaluate complement activation, for example, a CDC assay can be performed as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996).

[0161] Many techniques known in the art can be used for the preparation of antibodies, such as recombinant, monoclonal, or polyclonal antibodies. Genes encoding the heavy and light chains of the 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 combinations of heavy and light chain gene products generate a large pool of antibodies with different antigen specificities. Techniques for producing single-chain or recombinant antibodies have been found in the art and can be adapted to produce antibodies against polypeptides according to the present invention. Phage display techniques can also be used to identify antibodies and heteromer fragments that specifically bind to selected antigens. Antibodies can also be bispecific, i.e., capable of recognizing two different antigens, or heteroconjugates, such as two covalently bound antibodies, or immunotoxins, can be produced.

[0162] "Natural antibodies" and "natural immunoglobulins" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (l) 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 links varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly separated intrachain disulfide crosslinks. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the 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. Certain amino acid residues are thought to form the interface between the light chain variable domain and the heavy chain variable domain. Figure 2 shows an example of the general structure of natural mouse immunoglobulin G (IgG) highlighting the antigen-binding site.

[0163] As used herein, terms such as “antigen-binding protein,” “antibody,” “antagonist antibody,” and “antigen-binding fragment,” which may be used interchangeably herein, refer to a polypeptide or fragment thereof containing an antigen-binding site. Therefore, isolated antibodies or fragments may be polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinant antibodies, chimeric antibodies, heterochimeric antibodies, canine antibodies, feline antibodies, humanized antibodies, fully canine antibodies, fully feline antibodies, or fully human antibodies.

[0164] In some embodiments, terms such as “antigen-binding protein,” “antibody,” and “antagonist antibody” preferably refer to monoclonal antibodies and their fragments that can bind to TGFβ protein and its fragments, as well as their immunological conjugation equivalents. Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies, their antigen-recognizing fragments, small module immunotherapy (SMIP) 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 aforementioned fragments and their chemically or genetically engineered counterparts, as well as other antibody fragments and their variants, fusion proteins containing antibody moieties, and any other modified configurations of immunoglobulin molecules containing antigen-recognizing sites. Antibodies and antigen-binding proteins can be produced, for example, by conventional hybridoma techniques (Kohler et al., Nature 256:495-499 (1975)), recombinant DNA methods (U.S. Patent 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 by other techniques used and well known by those skilled in the art.

[0165] As defined herein, a "monoclonal antibody" is an antibody of a single pure homogeneous type. All monoclonal antibodies produced are identical and have the same antigen specificity. A monoclonal antibody is a homogeneous population of antibodies composed of (naturally occurring and non-naturally occurring) amino acids that are involved in the selective binding of the antibody to an antigen. The population of monoclonal antibodies is highly specific and is directed against a single antigenic site. The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (Fab, Fab’, F(ab’)2, Fv, scFv, Fd, dAb, diabody, their antigen recognition fragments, small modular immunopharmaceutical (SMIP) nanobodies, IgNAR molecules, etc.), their variants, fusion proteins containing antibody portions, and any other modified constructs of immunoglobulin molecules that contain the antigen recognition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited to the source of the antibody or the manner in which the antibody is made (e.g., hybridoma, phage selection, recombinant expression, transgenic animal, etc.).

[0166] Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and a remaining "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-binding sites and can still cross-link antigens.

[0167] "Fv" is the smallest antibody fragment that contains the complete antigen recognition and antigen-binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in a close non-covalent association. The three hypervariable regions of each variable domain interact to form the V H -V LIt is in this configuration that the 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 Fv containing only the three antigen-specific hypervariable regions) has the ability to recognize and bind to the antigen, although it has a lower affinity than the entire binding site. The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is a notation used herein for Fab' in which the cysteine ​​residue of the constant domain has a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments having hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0168] The monoclonal antibodies described herein include, specifically, “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody from a particular species, while the remaining chain is identical or homologous to a corresponding sequence in an antibody from another species, and also fragments of such antibodies, insofar as they exhibit the desired biological activity. Typically, a chimeric antibody is an antibody in which the light chain and heavy chain genes are constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, a variable segment of a gene from a mouse monoclonal antibody (or an antibody from any other species, including humans and felines) can be ligated to a canine constant segment, 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, including SEQ ID NO: 290, of the feline heavy chain constant region is ligated to a variable segment of an antibody from another species (mouse, canine, human, etc.). Figure 3 is a schematic diagram of the general structure of one embodiment of mouse:canine IgG. In this embodiment, the antigen-binding site is derived from mouse, while the Fc portion is from a canine animal. This example does not limit the claimed invention to mouse / canine chimeras, but may also apply to combinations of antibodies from any of the canine, feline, mouse, and human species to enumerate some of them as described herein.

[0169] As defined herein, the term "heterochimeric" refers to an antibody in which one of its antibody chains (heavy or light) is speciesized (i.e., canine or felineized), while the other is chimeric. Figure 4 shows one embodiment of a heterochimeric molecule. In this embodiment, a canine variable heavy chain (all of the CDRs are mouse and all of the FRs are canids) is paired with a chimeric variable light chain (all of the CDRs are mouse and all of the FRs are mouse). In this embodiment, both the variable heavy chain and the variable light chain are fused to a canine constant region. As with chimeric antibodies, there are no restrictions on the combination of antibody species and parts.

[0170] The terms "canine antibody," "feline antibody," and "human antibody," as used herein, refer to antibodies (antigen-binding proteins) produced against a target and antibodies isolated from lymphocytes within the target species. These antibodies are recombinantly modified in vitro to include specific constant regions of the target species, as described herein. Additionally, antibodies as described herein were identified, isolated, modified to alter their constant regions, and subsequently expressed and isolated from in vitro cell culture systems known to those skilled in the art and routinely used.

[0171] The terms "recombinant canine antibody," "recombinant feline antibody," and "recombinant human antibody" all include, but are not limited to, antibodies prepared, expressed, created, or isolated by recombinant means such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combination canine (or feline, human, etc.) antibody library, antibodies isolated from animals transgenic for the canine immunoglobulin gene (e.g., mouse) (see, for example, Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving the recombination of the canine (or feline, human, etc.) immunoglobulin gene sequence with another DNA sequence.

[0172] For the sake of brevity, the following describes "canine" antibodies, however, this may also apply to feline, humanized, or any other "biologically modified" antigen-binding proteins. As an example, "canine" is defined as a method for transferring a non-canine antigen-binding region from a donor antibody to a less immunogenic canine antibody acceptor, resulting in a treatment useful as a therapeutic agent in dogs. A canine antibody is a canine antibody sequence in which the 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, cattle, horse, llama, camel, dromedary, shark, non-human primate, human, humanized, recombinant, or engineered sequences having desired properties, specificity, affinity, and capabilities. Furthermore, canine antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance. As described herein, modifications to the hypervariable region and / or framework region are not predictable prior to the experiment but are determined for each separately manipulated biospeciesized (canine) antibody based on experiments known to those skilled in the art. Canine antibodies may optionally include the entirety or at least a portion of the constant region (Fc) of an immunoglobulin, typically the constant region (Fc) of a canine immunoglobulin. Figure 5 illustrates one embodiment showing the biospeciesization or canineization of mouse IgG. In this embodiment, the mouse CDR is implanted onto a canine framework. In some cases, the mouse framework or its residues outside the hypervariable region are maintained. All descriptions of canineization of antigen-binding proteins, and all descriptions of canine antigen-binding proteins, may conceptually be applicable to any “biosexified” antibody, whether it is canine, feline, humanized, etc.

[0173] A “parent” antibody as described herein is an antibody encoded by an amino acid sequence used for variant preparation. Preferably, in the case of canine or canine antibodies, the parent antibody has a canine framework region and, if present, a canine antibody constant region. For example, the parent antibody may be a canine or canine antibody. The same applies to feline, humanized, equine, and bovine antibodies.

[0174] The term "reverse mutation" refers to the process in which some or all of the somatically mutated amino acids in a canine antibody are replaced by corresponding germline residues from a homologous germline antibody sequence. The heavy and light chain sequences of the canine antibody of the present invention are aligned separately with the germline sequence to identify the sequence with the highest homology. Differences in the canine antibody of the present invention are reversed by mutating defined nucleotide positions encoding such different amino acids. The role of each amino acid thus identified as a candidate for reverse mutation should be investigated for its direct or indirect role in antigen binding, and any amino acid found after a mutation affecting any desirable feature of the canine antibody should not be included in the final canine antibody; for example, activity-enhancing amino acids identified by a selective mutagenesis approach are not subjected to reverse mutation. To minimize the number of amino acids subjected to reverse mutations, amino acid positions found to be different from the nearest germline sequence but identical to the corresponding amino acid in the second germline sequence may be retained, as long as the second germline sequence is identical and collinear to the sequence of the canine antibody of the present invention. Reverse mutations 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 part of a molecule that can be bound by an antibody. Generally, an epitope consists of a classification of the chemically active surface of a molecule, such as an amino acid or sugar side chain, and possesses specific three-dimensional structural and specific charge characteristics. Epitopes are antigenic determinants on proteins recognized by the immune system. Components of the immune system that recognize epitopes 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 plus (MHC class II) amino acid lengths. Recognition of the displayed MHC-peptide complex by T cells is crucial for their activation. These mechanisms enable the proper recognition of "self" versus "non-self" proteins, such as bacteria and viruses. Independent amino acid residues, not necessarily adjacent, 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 (New York edition: 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, even linear epitopes or epitopes found in continuous stretches of protein sequences often have discontinuous amino acids that represent key points of contact with antibody paratopes or B cell receptors. Epitopes recognized by antibodies and B cells can be amino acids and conformations containing common contact regions on the protein in three-dimensional space, and depend 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” refer to transformed growth factor beta protein 1 (TGFβ1), transformed growth factor beta protein 2 (TGFβ2), and transformed growth factor beta protein 3 (TGFβ3), respectively, where used interchangeably herein. TGFβ proteins are part of a superfamily of related growth factors that exert multifaceted effects on wound healing by modulating cell proliferation and migration, cell differentiation, apoptosis, ECM (extracellular matrix) production, and immunomodulation. As used herein, inhibition of TGFβ proteins by the use of the antigen-binding proteins of the present invention is used to treat TGFβ-related disorders such as fibrotic disorders, bone disorders, and cell proliferation disorders.

[0177] As used herein, “anti-TGFβ antigen-binding protein” may be interchangeably referred to as “anti-TGFβ antibody,” “anti-TGFβ antagonist antibody,” “anti-TGFβ antigen-binding fragment,” “anti-TGFβ antigen-binding moiety,” etc., and 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 the respective TGFβ signaling pathways associated with them. In some embodiments, the present invention provides that the anti-TGFβ antigen-binding protein binds to the TGFβ1 protein. In some embodiments, the antigen-binding protein of the present invention may have stronger binding and functional inhibition to TGFβ1, but still weaker binding and functional inhibition to TGFβ2 and / or TGFβ3. In some embodiments, the present invention provides that the anti-TGFβ antigen-binding protein binds to the TGFβ2 protein. In some embodiments, the present invention provides that the anti-TGFβ antigen-binding protein binds to the TGFβ3 protein. In some embodiments, the anti-TGFβ antigen-binding protein binds to the TGFβ1, 2, and 3 proteins. In some embodiments, the anti-TGFβ antigen-binding protein binds to TGFβ1 and TGFβ2. In some embodiments, the TGFβ antigen-binding protein of the present invention binds to TGFβ1 and TGFβ3 proteins. The anti-TGFβ antigen-binding protein of the present invention comprises binding proteins and antibodies that block, antagonistize, 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 by inducing receptor binding and / or cellular responses to TGFβ1 and / or TGFβ2 and / or TGFβ3 proteins.For the purposes of the present invention, the terms “anti-TGFβ antigen-binding protein,” “anti-TGFβ antagonist antibody,” or “TGFβB antibody” are expressly understood to encompass all previously identified terms, titles, and functional states and characteristics, including but not limited to their ability to mediate any aspect of the development or treatment of TGFβ-related disorders such as fibrotic disorders, bone disorders, and / or cell proliferation disorders, in which the biological activity of TGFβ itself, or the consequences of its biological activity, is substantially neutralized, reduced, or neutralized to any degree of its significance. Examples of anti-TGFβ antigen-binding proteins are provided herein.

[0178] A "variant" anti-TGFβ antibody, as used herein, refers to a molecule whose amino acid sequence differs from that of the "parent" anti-TGFβ antibody due to the addition, deletion, and / or substitution of one or more amino acid residues in the parent antibody sequence, and which 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 animals, and the ability to inhibit TGFβ-mediated SMAD signaling in cell-based assays. In one embodiment, the variant comprises one or more amino acid substitutions in one or more hypervariable and / or framework regions of the parent antibody. For example, the variant may comprise at least one, or about 1 to about 10, or about 2 to about 5 substitutions in one or more hypervariable and / or framework regions of the parent antibody. Typically, a variant has an amino acid sequence having at least 50% amino acid sequence identity with the heavy or light chain variable domain sequence of the parent antibody, or sequence identity between at least approximately 65%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the parent antibody. This sequence identity or homology is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to parent antibody residues after aligning the sequences and, if necessary, introducing gaps to achieve the maximum sequence identity percentage. N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence should not be interpreted as affecting sequence identity or homology. The variant may retain the ability to bind to the TGFβ variant and may possess stronger binding affinity, enhanced ability to reduce, inhibit, or neutralize TGFβ activity in animals, and / or enhanced ability to inhibit TGFβ-mediated SMAD signaling in cell-based assays.

[0179] A "TGFβ receptor" refers to a polypeptide that is bound to or activated by the TGFβ protein. TGFβ receptors are single-pass serine / threonine kinase receptors belonging to the TGFβ receptor family. They exist in several different isoforms, which can be homodimers or heterodimers. Three TGFβ receptors specific to the TGFβ protein 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 homodimer TGFβ1 and TGFβ2, and in addition to heterodimer TGFβ1 and 2. The TGFβ receptor also binds to TGFβ3. Mechanistically, the TGFβ protein first binds to the TGFβR2 receptor, which recruits and phosphorylates TGFβR1. TGFβR1 then phosphorylates the receptor regulatory SMAD (R-SMAD), which can then bind to co-SMAD SMAD4. The R-SMAD / co-SMAD complex functions as a transcription factor and accumulates in the nucleus, participating in the regulation of target gene expression.

[0180] As used herein with respect to the activity of the monoclonal antibodies of the present invention, the term “neutralizing” means the ability to substantially antagonistize, prohibit, prevent, limit, delay, disrupt, eliminate, stop, reduce, or reverse the progression or severity of what is inhibited, including but not limited to biological activity or properties, disease or condition. Inhibition or neutralization is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more. An antigen-binding protein is said to “neutralize” an antigen if the antibody that binds to the antigen results in partial or complete inhibition or reduction of the biological function of the antigen. Neutralization of the biological activity of a 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 herein in the in vitro assays described herein. In vivo neutralization of TGFβ can lead to a shift in TGFβ-induced cellular phenotypes, inhibition of cell proliferation, and inhibition of cell survival in disease states.

[0181] As used herein, "immunologically specific" binding of an antibody refers to an antigen-specific binding interaction that occurs between the antigen-combining site of the antibody and a particular antigen recognized by that antibody (i.e., the antibody reacts with the protein in an ELISA or other immunoassay and does not react detectably with unrelated proteins, and additionally means that the antibodies of the invention will also bind to the target antigen at the epitope in vivo). An epitope that "specifically binds to" or "preferentially binds to" (used interchangeably herein) an antibody or polypeptide 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 more frequently, more rapidly, for a longer duration, and / or with a higher affinity with a particular cell or substance containing the antigen as compared to alternative cells or substances. An antibody is said to "specifically bind to" or "preferentially bind to" a target if it binds with a higher affinity, avidity, more readily, and / or for a longer 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 a higher affinity, avidity, more readily, and / or for a longer duration than it binds to other epitopes or non-TGFB epitopes.

[0182] The term "specifically" in the context of antibody binding refers to the high avidity and / or high affinity binding of an antibody to a particular antigen, i.e., a polypeptide, or epitope. An antibody that specifically binds to an antigen binds more strongly than the binding of the same antibody to other antigens. An antibody that specifically binds to a polypeptide may be able to bind to other polypeptides at a weak but detectable level (e.g., less than 10% of the binding shown to the polypeptide of interest). Such weak binding, or background binding, can be readily distinguished from the binding of the specific antibody to the target polypeptide, for example, by the use of appropriate controls. Generally, a specific antibody is 10 -7 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 below M D It binds to the antigen due to its binding affinity.

[0183] As used herein, the term "affinity" refers to the strength of binding of a single antigen combination site to an antigenic determinant. Affinity depends on the tightness of the stereochemical fit between the antibody or antigen-binding protein combination site and the antigenic determinant, the size of the contact area between them, the charge and hydrophobic group distribution, 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) that 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 there, and SPR causes a reduction in reflected light intensity at specific combinations of angle and wavelength. A bimolecule binding event causes a change in refractive index in the surface layer, which is detected as a change in the SPR signal.

[0184] "K D The term "dissociation constant" as used herein is intended to refer to the dissociation constant of antibody-antigen interactions. D , and the association constant K a k is a quantitative measure of affinity. In equilibrium, free antigen (Ag) and free antibody (Ab) are in equilibrium with the antigen-antibody complex (Ag-Ab), and the rate constant is k. a and k d This quantifies the rate of individual reactions. At equilibrium, ka[Ab][Ag]=kd[Ag-Ab]. Dissociation constant, K d is, K D =kd / ka=[Ag][Ab] / [Ag-Ab] is given by K. D K is a unit of concentration, most typically M, mM, μM, nM, pM, etc. DWhen comparing antibody affinities expressed as such, a lower value indicates higher affinity for TGFB. (Association constant, K) a This is given by Ka = ka / kd = [Ag - Ab] / [Ag][Ab]. a M is the unit of the reciprocal of concentration, most typically M -1 , mM -1 μM -1 nM -1 pM -1 These include, etc. As used herein, the term "avidity" refers to the strength of antigen-antibody binding after the formation of a reversible complex. Anti-TGFB antibodies are "approximately (lower K)." D Value) ~ approximately (higher K D Dissociation constant (K) within the range of values D ) has a bond, and K for their binding to TGFB protein D It can be characterized in respect to this.

[0185] The terms “nucleic acid,” “polynucleotide,” and “nucleic acid molecule” may be used interchangeably herein and refer to a set of nucleotide bases (also called “nucleotides”) in DNA and RNA. Nucleic acids may include deoxyribonucleotides, ribonucleotides, and / or analogs thereof. The term “nucleic acid” includes, for example, single-stranded and double-stranded molecules. Nucleic acids may be, for example, genes or gene fragments, exons, introns, DNA molecules (e.g., cDNA), RNA molecules (e.g., mRNA), recombinant nucleic acids, plasmids, and other vectors, primers, and probes. Both 5'→3' (sense) and 3'→5' (antisense) polynucleotides are included. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides may include modified nucleotides such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if present, may be conferred before or after the 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 labeling components. Other types of modifications include, for example, "capping," substitution of one or more naturally occurring nucleotides by analogs, modifications between nucleotides, such as those having uncharged links (e.g., methylphosphonates, triesters of phosphate, phosphoamidates, cabamates, etc.) and charged links (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant portions, 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 having modified links (e.g., alpha-anomeric nucleic acids, etc.), and the unmodified form of polynucleotides.Furthermore, any of the hydroxyl groups normally present in the sugar may be replaced, for example, with a phosphonate group, a phosphate group, protected with a standard protecting group, activated to prepare additional linkage to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH groups may be phosphorylated or replaced with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized with standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azid-ribose, carbocyclic sugar analogs, anomeric sugars, epimeric sugars, e.g., arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and debasic nucleoside analogs, e.g., methylriboside. One or more phosphodiester linkages may be replaced by alternative linking groups. Examples of these alternative linking groups, but not limited to, include embodiments in which the 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 optionally a substituted or unsubstituted alkyl (1 to 20 C) containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in the polynucleotide are to be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0186] As used herein, “vector” means 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, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells such as producer cells. A vector has an expression regulatory sequence, meaning a nucleic acid sequence that directs the transcription of the nucleic acid, as described herein. The expression regulatory sequence may be a promoter, such as a constitutive or inductive promoter, or an enhancer. The expression regulatory sequence is “operably ligated” to the nucleic acid sequence to be transcribed. A nucleic acid is “operably ligated” when it is positioned in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably ligated to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably ligated to a coding sequence if it is positioned to facilitate translation. Generally, "operably ligated" means that the DNA sequences to be ligated are adjacent, and in the case of a secretion reader, they are adjacent and within the reading frame. However, enhancers do not need to be adjacent.

[0187] Just as polypeptides can contain conserved amino acid substitutions, their polynucleotides can contain conserved codon substitutions. A codon substitution is considered conserved if, when expressed, it produces the aforementioned conserved amino acid substitutions. Degenerate codon substitutions that do not result in amino acid substitutions can also be useful in the polynucleotides of the present invention. Therefore, for example, a polynucleotide encoding a selected polypeptide useful in one embodiment of the present invention can be mutated with degenerate codon substitutions to approximate the codon usage frequency shown by the expressing host cell transformed with it, or to improve its expression in other ways.

[0188] In this specification, a “variant” nucleic acid refers to a molecule whose sequence differs from that of the “parent” nucleic acid. Deviations from a polynucleotide sequence can result from mutational changes such as the deletion, substitution, or addition of one or more nucleotides. Each of these changes may occur one or more times in a given sequence, either individually or in combination.

[0189] The term “isolated” means that the material (e.g., an antigen-binding protein or nucleic acid as described herein) is separated and / or recovered from components of its natural environment. Components of the natural environment are those that would interfere with the diagnostic or therapeutic use of the material and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. With respect to nucleic acids, isolated nucleic acids may include those separated from the 5'→3' sequence that it normally associates in chromosomes. In preferred embodiments, the material is purified to more than 95% by mass, most preferably more than 99% by mass. Since at least one component of the material’s natural environment is absent, the isolated material contains the material in situ within recombinant cells. However, typically, isolated material is prepared by at least one purification step as used herein.

[0190] The terms “cell,” “cell line,” and “cell culture” may be used interchangeably. These terms also include all subsequent generations, their offspring. It is understood that all offspring may not be identical due to planned or accidental mutations. In the context of heterologous nucleic acid sequence expression, “host cell” refers to a prokaryotic or eukaryotic cell (e.g., bacterial cells, yeast cells, mammalian cells, and insect cells), whether located in vitro or in vivo. For example, a host cell may be located in a transgenic animal. A host cell may include any transformable organism that can be used as a recipient for a vector and is capable of replicating the vector and / or expressing the 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 detectable itself (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition.

[0192] "Subject" or "patient" refers to an animal requiring treatment that may be affected by the molecules of the present invention. Examples of animals that can be treated according to the present invention include vertebrates, and specifically, mammals such as canids, felines, or humans are particularly preferred examples.

[0193] "Composition" is intended to mean a combination of an active agent, whether a chemical composition, a biological composition, or a biological treatment (in particular, an antigen-binding protein as described herein), and another compound or composition that may be inactive (e.g., labeled) 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, physiological saline, buffered physiological saline, phosphate buffer, alcohol / aqueous solutions, emulsions, or suspensions. Other conventionally used diluents, adjuvants, and excipients may be added according to conventional techniques. Such carriers include, but are not limited to, 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. Typical 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, trimmethamine hydrochloride, or phosphate buffers. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixative oil. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents (e.g., EDTA), and inert gases, 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 components having appropriate pH isotonicity, stability, and other conventional characteristics is within the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, publ., 2000, and The Handbook of Pharmaceutical Excipients, 4th sup.th edit, eds. RCRowe et al, APhA Publications, 2003.

[0195] The “therapeutic effective dose” (or “effective dose”) refers to the amount of an active ingredient sufficient to produce a beneficial or desired outcome when administered to a subject or patient, such as the drug according to the present invention. The effective dose may be administered in one or more doses. The therapeutic effective dose of a composition according to the present invention can be readily determined by those skilled in the art. In the context of the present invention, the “therapeutic effective dose” is the amount that produces a sufficiently objectively measurable change in one or more parameters associated with a TGFB-related condition to produce a beneficial or desired outcome, including clinical outcomes such as relief or reduction of pain sensation. The effective dose may be administered in one or more doses. For the purposes of the present invention, the effective dose of a composition is sufficient to prevent, treat, reduce, or eliminate a TGFβ-related disorder as defined herein as a fibrotic disorder, a bone disorder, or a cell proliferation disorder. The therapeutic effective dose varies depending on the specific subject and condition being treated, the subject’s weight and age, the severity of the condition, the specific composition selected, the dosing regimen to be followed, the timing of administration, the mode of administration, and so on, all of which can be readily determined by those skilled in the art.

[0196] As used herein, the term “therapeutic” encompasses the entire spectrum of treatment for a disease, condition, or disorder. The “therapeutic” agents of the present invention may act in a preventive or anticipatory manner, including incorporating procedures designed to target subjects that may be identified as being at risk, or they may act to slow the rate or degree of progression of at least one symptom of the disease or disorder being treated.

[0197] In a further embodiment, the present invention features a veterinary composition in which the antibody of the present invention is provided for therapeutic or prophylactic use. The present invention features a method for treating a canid, feline, or human subject having a specific antigen, for example, an antigen associated with a disease or condition. The method comprises administering a therapeutically effective amount of one or more antibodies specific to TGFβ protein, accompanied by the antibody of the present invention as described herein.

[0198] The antigen-binding protein of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a target. The compounds of the present invention may be administered alone or in combination with pharmaceutically acceptable carriers, diluents, and / or excipients in single or multiple dosing regimens. The administration composition is designed to be suitable for the selected administration method, and may use pharmaceutically acceptable diluents, carriers, and / or excipients, such as dispersants, buffers, surfactants, preservatives, solubilizers, isotonic agents, stabilizers, etc., as appropriate.

[0199] Compositions comprising the antigen-binding protein of the present invention may be administered to subjects exhibiting conditions or disorders as described herein using standard administration techniques, including intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, intramuscular, intranasal, buccal, sublingual, or suppository administration. The route of administration of the antibody of the present invention may be parenteral. Infusion is typically given via an intravenous route. Preferably, the antibody 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, intraperitoneal, or subcutaneous injection is preferred. Furthermore, subjects of the method of the present invention are also referred to as patients and are described herein as canids, felines, or humans.

[0200] As used herein, TGFβ-related disorders are disorders in which the regulation or overall level of one or more TGFβ proteins results in connective tissue disorders, fibrosis / fibrous disorders, bone disorders, or cell proliferation disorders. 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 tissue include fat, bone, and cartilage. These disorders often involve joints, muscles, and skin, but they may also involve other organs and organ systems, including the eyes, heart, lungs, kidneys, digestive tract, and blood vessels.

[0202] Fibrosis-related disorders, as described herein, relate to pathological processes involving scar formation and overproduction of extracellular matrix by connective tissue in response to tissue damage. The molecular processes are no 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 may be used to describe the pathological condition of excessive deposition of fibrous tissue and the process of connective tissue deposition in healing. Defined by the pathological accumulation of extracellular matrix (ECM) proteins, fibrosis results in scarring and thickening of affected tissue, which is essentially an exaggerated wound healing response that interferes with normal organ function. Fibrosis formation involves interactions between many cell types and cytokines, and when the balance becomes fibrotic, fibrosis formation occurs. Fibrosis is analogous to the scarring process in that it involves stimulated fibroblasts that lay down connective tissue, both containing collagen and glycosaminoglycans. The process is initiated when immune cells, such as macrophages, release soluble factors that stimulate fibroblasts. The most well-characterized pre-fibrotic 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 and idiopathic pulmonary fibrosis; cirrhosis; glial scarring in the brain; articular fibrosis in the knees, shoulders 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 the blood vessels, skin, lungs, kidneys, heart, and GI ducts, and to date has been considered an irreversible process, classically treated with anti-inflammatory and immunosuppressive drugs, which have repeatedly caused harm.

[0203] Chronic kidney disease (CKD), as described herein, involves the loss of functional kidney tissue due to a long-term, progressive fibrous process. While structural and functional changes in the kidney are only loosely correlated, dramatic changes in kidney structure can occur. The disease is typically present for months or years before it becomes clinically apparent, and it is always irreversible. Many causes of CKD are associated with progressive interstitial fibrosis. The severity of interstitial fibrosis is positively correlated with the magnitude of the decrease in glomerular filtration rate (GFR) and negatively correlated with prognosis. Glomerular, tubulointerstitial, and vascular lesions observed in animals with systemic CKD are often similar, regardless of the cause of onset. TGFβ is described as the most important pre-fibrous mediator responsible for myofibroblast activation. It drives a convergent pathway that integrates the effects of many other fibrogenic factors. TGFβ1 is the most abundant isoform and is synthesized by all cell types of the kidney. As discussed, TGFβ functions as a prefibrotic cytokine and is also a rich bone matrix protein that influences the formation, function, and cell-cell interactions of osteoblasts and osteoclasts in order to regulate bone remodeling and maintain adequate bone mass. It has been shown that TGFβ inhibition is a potential mechanism for reducing bone mineralization in SRHP caused by CKD.

[0204] The terms "treatment" and "treating" refer to both therapeutic measures and preventive or protective measures. Animals requiring treatment include those already suffering from a disorder, and those for which the onset or progression of the disorder should be prevented. Treatment may also be described as delaying the onset of symptoms or a condition, or delaying the severity of the onset. The terms "treatment" or "treating" of a disease or disorder include prevention or protection against the disease or disorder (i.e., not causing the cause of clinical symptoms), inhibition of the disease or disorder (i.e., preventing or suppressing the onset of clinical symptoms, and / or mitigation of the disease or disorder (i.e., causing regression of clinical symptoms)). As can be understood, it is not always possible to distinguish between "preventing" and "suppressing" a disease or disorder, as the final inducing event or event may be unknown or latent. Therefore, the term "prophylaxis" will be understood to constitute a type of "treatment" that encompasses both "preventing" and "suppressing." Thus, the term "treatment" includes "prevention."

[0205] Before describing this method, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and such methods and conditions may vary. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the invention pertains. Any methods and materials similar to or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference in their entirety.

[0207] The present invention, as disclosed herein, relates to an antigen-binding protein (as used interchangeably with terms such as “antibody,” “antagonist antibody,” and “antibody fragment,” as described herein) that specifically binds to one or more TGFβ proteins, particularly an antibody that specifically binds to TGFβ1 and TGFβ2 or TGFβ3 and thus functions as an antagonist in that it prevents TGFβ from binding to the TGFβRII receptor and thus prevents the activation of a signaling pathway by one of the TGFβ proteins, regardless of whether it is a canid, feline, mouse, human or any other species-specific antibody produced by a recombinant method, hybridoma technology or phage display technology, or a fully species-specific monoclonal antibody. In one embodiment, the present invention provides an antibody that binds only to TGFβ1, with or without TGFβ2 or TGFβ3. In one embodiment, the present invention provides an antibody that binds to both TGFβ1 and TGFβ3. In one embodiment, the present invention provides antibodies that bind to TGFβ1, TGFβ2, and TGFβ3.

[0208] The properties of antibodies make them very attractive therapeutic agents, but they also have numerous limitations. The majority of monoclonal antibodies (mAbs), as mentioned earlier, are of rodent origin. When such antibodies are administered in a different species, the patient / subject may develop an increased antibody response of their own to such heterologous antibodies. Such a response can lead to the eventual neutralization and elimination of the antibody. As mentioned above, antibody production is not limited to mice, but mice are widely used for the production of monoclonal antibodies. Species such as canids can be immunized with antigens and recovered and characterized antibodies. One problem in the use of antibodies produced by specific species is that, for example, if originally produced in mice, non-mouse subjects treated with those antibodies may react to the mouse antibodies as if they were foreign substances, and thus create a new set of antibodies against the mouse antibodies. Mouse antibodies are "seen" by the immune system of a non-mouse, e.g., a canid (or any other non-mouse species), and the immune system may "recognize" the heterologous antibody as a foreign substance and then initiate an immune response against the molecule. Those skilled in the art will recognize that while a subject can be treated with antigen-specific antibodies, there is a need to have a specific antibody species for use. Interspecies antibody administration, for example, the portion of the reaction generated from mouse monoclonal antibodies administered to canids, can range from mild forms such as rashes to more extreme and life-threatening responses such as renal failure. This immune response may also reduce the effectiveness of the treatment or cause future reactions if the subject is subsequently given treatment containing mouse antibodies. Therefore, we describe overcoming this drawback by "canineization" or "canineization" of the antibody of the present invention, as described in the present invention. In particular, this process focuses on the framework region of the immunoglobulin variable domain, but may also include the complementarity determinant region (CDR) of the variable domain. Enabling steps and reductions for carrying out this process are described in this disclosure, and the affinity maturation process centered on the CDR sequence is well known in the art.

[0209] The process of modifying monoclonal antibodies from animals (such as antigen-binding proteins, antagonist antibodies, and other interchangeable terms as described herein) to reduce their immunogenicity for therapeutic administration to different species has been actively pursued and described in numerous publications (e.g., Antibody Engineering: A Practical Guide. Carl AKBorrebaeck ed. WH Freeman and Company, 1992). However, this process has not been routinely applied until recently for the development of therapeutic or diagnostic applications in non-humans. In fact, little has been published regarding canid, feline, or other species-specific variable domains. Wasserman and Capra, Biochem. 6, 3160 (1977) determined the amino acid sequences of the variable regions of the heavy chains of both canids. Wasserman and Capra, Immunochem. 15, 303 (1978) determined the amino acid sequences of the K light chain from canid IgA. McCumber and Capra, Mol. Immunol. 16, 565 (1979) disclose the complete amino acid sequence of a canid muon chain. Tang et al., Vet. Immunology Immunopathology 80, 259 (2001) disclose a single canid IgG-Ay chain cDNA and four canid IgG-Ay chain protein sequences. Bergeron et al. describe the functional properties of four canid heavy chains. To date, the lack of available information on canid antibodies has hindered their development as therapeutic agents for the treatment of canid diseases.

[0210] These notable limitations have spurred the development of manipulation techniques known to those skilled in the art as "bio-speciesization," which are the "humanization" of therapeutic antibodies. "Canine," "feline," and "humanized" antibodies are some examples of "bio-speciesization" techniques, in which these molecules are generated as antibodies or fragments containing minimal sequences derived from non-target immunoglobulins. As an example, a canine antibody ("target species antibody") is created in which residues from the recipient / target complementarity-determining region (CDR) are replaced by residues from the CDR of a non-target species (i.e., a "donor antibody" or "origin species antibody") such as mouse, possessing desired properties such as specificity, affinity, and potency. This strategy is based on identifying the most appropriate target (germline antibody sequence for CDR transplantation). After extensive analysis of all available germline sequences for both variable heavy and variable light chains, candidate germline sequences were selected based on their homology to mouse / donor mAbs, and natural canine CDRs were replaced using CDRs from mouse / donor precursor mAbs. The objective is to consistently maintain high affinity and ultimate in vivo efficacy when used as a therapeutic agent. Using a canine antibody framework 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. Revert mutations of selected target framework residues to corresponding donor residues may be necessary to restore and / or improve the affinity of interest. Structure-based methods may also be used for canineization and affinity maturation, as described in US7,261,890. The above description uses canines as the target species and mice as the donor species. Biospecies-based antibodies are not limited to these targets and donors. Felines, etc., can be used as target species.

[0211] Another challenge in developing therapeutic antibodies that target proteins is that epitopes on homologous proteins frequently differ across different species, and the potential for cross-reactivity with other proteins also varies. Consequently, antibodies must be fabricated, tested, and developed specifically for specific targets in the particular species being treated. Antibody binding between homologous targets in different species is unpredictable and requires efficacy testing and evaluation.

[0212] Antibodies target antigens through their binding to specific epitopes on the antigen via interaction 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 protein of the present invention), and / or initiate a variety of biological activities. There is 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 deliver drugs bound to their Fc region to specific sites. Antibodies also induce antibody-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and phagocytosis through the binding of the antibody's Fc region to each intracellular molecule that induces ADCC, CDC, etc. Some antibodies have been altered so that their ADCC, CDC, C1q binding, and phagocytic functions are eliminated. In one embodiment, the present invention provides an antigen-binding protein comprising alterations in the Fc region of the antibody that alter the effector function of the antibody. The present invention further provides cells and cell lines expressing the antibodies of the present invention. Typical 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 host cells with xenoconstitutes are well known in the art. Typical 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 biospecies-specific antibodies, for example, modified by deletion, addition, or substitution of other parts of the antibody, such as the constant region, are also within the scope of the present invention. For example, an antibody may be modified as follows: (i) by deleting the constant region; (ii) by replacing the constant region with another constant region, such as a constant region that increases 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 change, for example, the number of glycosylation sites, effector cell function, Fc receptor (FcR) binding, or complement fixation. In one embodiment of the present invention, the antibody of the present invention includes a modified 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 is replaced, modified, or removed.

[0214] Methods for altering the constant region of an antibody are known in the art. Antibodies with altered function, such as altered affinity for effector ligands like FcR on cells or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant region of the antibody with a different residue (see, for example, EP388151A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260, all of which are incorporated herein by reference).

[0215] For example, the affinity of the antibody's Fc region for FcR (e.g., Fc. gamma R1) or C1q binding can be altered by replacing a specified residue with a residue having an appropriate functional group on its side chain, or by introducing a charged functional group, such as a glutamate or aspartate, or possibly an aromatic nonpolar residue, such as phenylalanine, tyrosine, tryptophan, or alanine (see, for example, U.S. Patent No. 5,624,821). The antibody or its conjugated fragment can be conjugated with a cytotoxin, a therapeutic agent, or a radioactive metal ion. In one embodiment, the protein to be conjugated is an antibody or its fragment. Examples of cytotoxins or cytotoxic agents include any agent that is harmful to cells. Non-exclusive examples include calicheamycin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and their analogues or homologues. 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., mechloretamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mitramycin, and anthramycin), and antimitotic agents (e.g., vincristine and vinblastine). Techniques for conjugating such moieties into proteins are well known in the art.

[0216] Composition, derivative composition, and method for preparing the composition The present invention encompasses compositions comprising a pharmaceutical composition comprising an antigen-binding protein (such as "antibody," "antibody fragment," and "antagonist antibody," as used interchangeably herein), a polypeptide, and a polynucleotide comprising a sequence encoding the antigen-binding protein or polypeptide of the present invention.

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

[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 entirely canid, entirely feline, entirely human, canine, feline, or humanized. In one or more embodiments, the antigen-binding protein of the present invention binds to one or more canid, feline, or human TGFβ proteins. In one embodiment, the antigen-binding protein is a monoclonal antibody. In one embodiment, the monoclonal antibody of the present invention binds to one or more TGFβ proteins, preventing their binding to their receptors and their activation, and thus preventing the signal transduction cascade as described herein.

[0219] In one or more embodiments, the present invention provides isolated recombinant antigen-binding proteins that bind 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 the antigen-binding protein of the present 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 the amino acid sequence comprising the antigen-binding protein of the present invention as described herein, and any variant thereof having one or more conserved amino acid substitutions in at least one of CDR1, CDR2, or CDR3 in either the variable light chain or the variable heavy chain of the antigen-binding protein.

[0220] The present invention provides recombinant antigen-binding proteins, monoclonal antibodies in some embodiments described herein, and antibody fragments, as well as their use in scientific procedures, including clinical administration and diagnostic procedures. By using molecular biology methods and recombinant techniques, antibodies and antibody-like molecules can be produced by recombinant means, and thereby gene sequences encoding specific amino acid sequences found in the polypeptide structure of the antibody can be generated. Such antibodies can be produced by either cloning the gene sequence encoding the polypeptide chain of the antibody, or by directly synthesizing the polypeptide chain and assembling the synthesized chain to form an active tetramer (H2L2) structure having affinity for a specific epitope and antigenic determinant. This has enabled the immediate production of antibodies having sequences characterized by neutralizing antibodies from different species and sources.

[0221] Regardless of the source of antibodies, how they are constructed or synthesized—whether in vitro or in vivo, using transgenic animals, in large-scale laboratory cell cultures or commercial-scale, using transgenic plants, or through direct chemosynthesis without the use of 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-chain (L) amino acids and two heavy-chain (H) amino acids. Both chains have regions capable of interacting with structurally complementary antigen targets. The target-interacting region is referred to as the "variable" or "V" region and is characterized by differences in the amino acid sequence from antibodies of different antigen specificities. The variable region of either the H or L chain contains an amino acid sequence capable of specifically binding to the antigen target.

[0222] As used herein, the term “antigen-binding region” refers to a portion of an antibody molecule that contains amino acid residues that interact with an antigen and confer its specificity and affinity to the antigen to the antibody. The antibody-binding region contains “framework” amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable region of the H or L chain that provides the antigen-binding region, there is a smaller sequence called “hypervariable,” which is the reason 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 fundamental specificity of the antibody to a particular antigenic determinant structure.

[0223] CDRs represent discontinuous ranges of amino acids within the variable region, and it has been found that, regardless of species, the positions of these important amino acid sequences within the variable heavy and light chain regions are similar within the amino acid sequence of the variable chain. All antibody variable heavy and light chains each have three CDR regions, each discontinuous with the others. In all mammalian species, antibody peptides contain both constant (i.e., highly conserved) regions and variable regions, the latter of which include so-called "framework regions" consisting of CDRs and amino acid sequences that are within the variable region of the heavy or light chain but outside the CDRs.

[0224] The present invention further provides vectors comprising at least one of the nucleic acids described above. Because the genetic code is degenerate, more than one codon may be used to code for a particular amino acid. Using the genetic code, one or more different nucleotide sequences may be identified, each of which may be capable of coding for that amino acid. The factual probability that a particular oligonucleotide constitutes an actual coding sequence can be estimated by considering the unusual base pairing relationships and the frequency with which a particular codon is actually used (to code for a particular amino acid) in eukaryotic or prokaryotic cells expressing an anti-TGFB antibody or portion thereof. Such “codon usage rules” are disclosed by Lathe, et a. I., 183 J. Molec. Biol. 1-12 (1985). Using Lathe’s “codon usage rules,” a single nucleotide sequence, or set of nucleotide sequences, containing the theoretically “most likely” nucleotide sequence capable of coding an anti-TGFB sequence can be identified. Furthermore, antibody coding regions for use in the present invention may also be provided by modifying existing antibody genes using standard molecular biology techniques resulting in 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 anti-TGFβ antibodies or peptides.

[0225] antibody derivative The scope of this invention includes antibody derivatives. “Derivatives” of antibodies typically contain additional chemical portions that are not part of the protein. Covalent modifications of proteins are included within the scope of this invention. Such modifications can be introduced into a molecule by reacting target amino acid residues of an antibody with an organic derivatizing agent that can react with selected side-chain or terminal residues. For example, derivatization using bifunctional agents well known in the art is useful for crosslinking antibodies or fragments to a water-insoluble support matrix or other macromolecule carrier.

[0226] The derivatives also include radioactively labeled monoclonal antibodies, such as radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, and tritium (H). 3 Labeling with ) etc.; conjugate a monoclonal antibody containing biotin or avidin with an enzyme such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase; and also use conjugates of a monoclonal antibody with a bioluminescent agent (such as luciferase), a chemiluminescent agent (such as acridine ester), or a fluorescent agent (such as phycovir protein).

[0227] Another derivative bifunctional antibody of the present invention is a bispecific antibody produced by combining portions of two distinct antibodies that recognize two different antigenic groups. This can be achieved by crosslinking or recombinant techniques. Additionally, portions may be attached to the antibody or its portion to increase its in vivo half-life (e.g., by prolonging the time to clearance from the bloodstream). Such techniques include, for example, the attachment of a PEG moiety (also known as pegylation), which is well known in the art. See U.S. Patent Application Publication No. 2003 / 0031671.

[0228] Recombinant expression of antibodies In some embodiments, the nucleic acid encoding the antigen-binding protein of the present invention is directly introduced into host cells, and the cells are incubated under conditions sufficient to induce the expression of the encoded antibody. After the introduction of the target nucleic acid into the cells, the cells are incubated for a period of about 1 to 24 hours, typically at 37°C and optionally under selective conditions, 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. Conventionally, monoclonal antibodies have been produced as innate molecules in mouse hybridoma strains. In addition to that technique, the present invention provides recombinant DNA expression of monoclonal antibodies. This enables the production of said antibodies in selected host species, as well as the generation of spectra of antibody derivatives and fusion proteins.

[0229] Nucleic acid molecules such as DNA contain nucleotide sequences that provide transcriptional and translational regulatory information, and if such sequences are "operably linked" to nucleotide sequences that encode polypeptides, then the polypeptide is said to be "capable of expression." An operable linkage is a linkage in which a regulatory DNA sequence and the DNA sequence to be expressed are connected in such a manner that gene expression as an anti-TGFβ antigen-binding protein or antibody fragment is possible in a recoverable amount. The exact properties of the regulatory regions required for gene expression can vary from organism to organism, as is well known in similar technologies.

[0230] Therefore, the present invention encompasses the expression of anti-TGFβ antigen-binding proteins in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacteria, yeast, insects, fungi, birds, and mammalian cells, in vivo or in situ, or in host cells of mammalian, insect, bird, or yeast origin. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cells may be used without limitation.

[0231] The chimeric constructs of the present invention, or expression vectors carrying anti-TGFβ antigen-binding proteins, 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, as well as mechanical means such as electroporation, direct microinjection, and particle guns. (Johnston et al., 240 Science 1538 (1988), or other techniques known to those skilled in the art, but not limited to these.)

[0232] Stable expression can be used for the long-term high-yield production of recombinant antibodies. For example, cell lines that stably express antibody molecules can be engineered. Rather than using expression vectors containing the origin of the replicas, host cells can be transformed with immunoglobulin expression cassettes and selectable markers. Following the introduction of exogenous DNA, the engineered cells may be grown in concentrated medium and then switched to selective medium. Selectable markers in recombinant plasmids confer resistance to selection, allowing cells to stably incorporate the plasmid into their chromosomes and grow, thereby forming a focus that can 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 the antibodies of the present invention are produced, they can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, but not limited to, chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, solubility difference, or any other standard technique for protein purification. In many embodiments, the antibodies are secreted from cells into a culture medium and then collected from the culture medium.

[0234] Pharmaceutical and veterinary uses The anti-TGFβ antigen-binding proteins or antibody fragments of the present invention, as described herein, may be used, for example, in the treatment of TGFβ-related disorders in canids and felines. More specifically, the present invention further provides pharmaceutical compositions comprising the antibody or antibody fragment according to the present invention as a pharmaceutically acceptable carrier or diluent and as an active ingredient. The antibodies may be chimeric, heterochimeric, canine, feline, humanized, or biospeciesized antigen-binding proteins adapted to different species. Intact immunoglobulins or their binding fragments are also envisioned. The antibodies and pharmaceutical compositions thereof of the present invention are useful for parenteral administration, for example, subcutaneous, intramuscular, or intravenous administration.

[0235] In some preferred embodiments, the antibodies of the present invention are administered by parenteral injection. For parenteral administration, the anti-TGFβ antibody or fragment may be formulated as a solution, suspension, emulsion, or lyophilized powder associated with a pharmaceutically acceptable parenteral vehicle. For example, the vehicle may be a solution of the antibody or its cocktail dissolved in an acceptable carrier, such as an aqueous carrier, such as water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or serum albumin, glycine, etc. Non-aqueous vehicles such as liposomes and fixative oils may 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 may contain pH adjusters and pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as buffers and toxicity modifiers, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The antibody concentration in these formulations can vary widely, for example, from less than about 0.5% by mass, typically from about 1% by mass or more to a maximum of 15% or 20% by mass, and will be selected according to the specific administration mode chosen, mainly based on fluid volume, viscosity, etc. 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 formulations are sterilized by commonly used techniques. Practical methods for preparing parenterally administered compositions are known or obvious to those skilled in the art and are described in detail, for example, in REMINGTON'S PHARMA.SCI. (15th ed., Mack Pub.Co., Easton, Pa., 1980).

[0236] The antibodies of the present invention may be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional immunoglobulins. Any suitable lyophilization and reconstitution technique may be used. Those skilled in the art will understand that lyophilization and reconstitution may result in varying degrees of antibody activity loss, which may need to be compensated for by adjusting the usage level. The antibody compositions of the present invention may provide a cocktail of which can be administered for the prevention and / or therapeutic treatment of recurrence of pre-existing diseases. Suitable pharmaceutical carriers are, in particular, described 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 use, the composition is administered to a subject already suffering from a disease or condition in an amount sufficient to cure, at least partially prevent, or alleviate the disease or condition and its complications. An amount sufficient to achieve this is defined as a "therapeutic effective dose" or "therapeutic amount."

[0237] The dosage administered will naturally vary depending on known factors, such as the pharmacodynamic characteristics of a particular drug and its method and route of administration; the recipient's age, health, and weight; the nature and severity of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect.

[0238] As a non-limiting example, treatment of TGFβ-related conditions in dogs and cats may be provided in doses as needed. Exemplary antibodies for therapeutic use in canids or felines are high-affinity antibodies having 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 composition may be carried out at dose levels and patterns selected by the treating veterinarian. In any case, the pharmaceutical formulation should provide an amount of the antibody of the present invention sufficient to effectively treat the subject.

[0239] Diagnostic use The present invention also provides the above-mentioned anti-TGFB antibodies for use in diagnostic methods for detecting TGFβ in species known to have or suspected to have TGFβ-related disorders, particularly canids, 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. An immunoassay for TGFβ typically comprises incubating a clinical or biological sample in the presence of the detectably labeled high-affinity (or high-avidity) anti-TGFB antibody of the present invention, which can selectively bind to TGFβ, and detecting a labeled peptide or antibody bound in the sample. Various clinical assay procedures are well known in the art. Such samples include, as known to those skilled in the art, tissue biopsies, blood, serum, and fecal samples, or liquids collected from animal subjects and subjected to ELISA analysis.

[0240] "Solid support" or "carrier" refers to any support capable of binding to peptides, antigens, or antibodies. 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 properties of the carrier may be partially soluble or insoluble for the purposes of this invention. The support material may have substantially any possible structural configuration, as long as the coupled molecules can bind to one or more TGFβ proteins or anti-TGFβ antibodies. Therefore, the configuration of the support may be spherical, as in beads, or cylindrical, as in the inner surface of a test tube, or a rod-shaped outer surface. Alternatively, the surface may be flat, such as a sheet, culture dish, or test strip. For example, the support may include polystyrene beads. Those skilled in the art know of, or can confirm by routine experiments, many other suitable carriers for binding to antibodies, peptides, or antigens. A well-known method step can determine the binding activity of a given lot of anti-TGFB peptide and / or antibody or antigen-binding protein. Those skilled in the art can determine the operational and optimal assay conditions through routine experiments.

[0241] The detection of TGFβ-specific peptides and / or antibodies can be achieved by linking them to an enzyme for use in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts with the exposed substrate to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorescence quantification, or visual means. Enzymes that can be used to detectably label the TGFβ-specific antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Radiolabeling of TGFβ-specific antibodies makes it possible to detect TGFB through the use of a radioimmunoassay (RIA). Radioactive isotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. Isotopes particularly useful for the purposes 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 using fluorescent compounds. When a fluorescently labeled antibody is exposed to light of an appropriate wavelength, its presence can then be detected due to fluorescence. The most commonly used fluorescent labeling compounds, known to those skilled in the art, include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescein. TGFβ-specific antibodies or antigen-binding proteins can also be labeled. 125These can be delectably labeled using fluorescent metals such as Eu or other lanthanides. These metals can be attached to TGFβ-specific antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0243] TGFβ-specific antibodies can also be detected by coupling them with chemiluminescent compounds. The presence of a chemiluminescently labeled antibody is then determined by detecting the presence of light emitted during the course of the chemical reaction. Examples of useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium esters, imidazole, acridinium salts, and oxalate esters.

[0244] Similarly, bioluminescent compounds 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 catalytic proteins increase the efficiency of chemiluminescent reactions. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Key bioluminescent compounds for labeling purposes are luciferin, luciferase, and aequorin.

[0245] Detection of TGFβ-specific antibodies, moieties, fragments, polypeptides, or derivatives can be achieved, for example, by a scintillation counter if the detectable label is a radioactive gamma emitter, or by a fluorometer if the label is a fluorescent material. In the case of enzyme labeling, detection can be achieved by a colorometric method using the substrate against the enzyme. Detection can also be achieved by a visual comparison of the degree of enzymatic reaction of the substrate compared to a similarly prepared standard.

[0246] For the purposes of the present invention, TGFβ detected by the above assay may be present in a biological sample. Any sample containing TGFβ may be used. For example, the sample may 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 enable the use of other samples.

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

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

[0249] Antibodies may 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 fluorescence measurement detection. For diagnostic purposes, antibodies may be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the antibody, such as antibodies specific to the constant region of canine immunoglobulins. Alternatively, antibodies can be directly labeled. A variety of labels may be used, such as radionuclides, fluor, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (especially 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 may be useful in the diagnosis of TGFB-related disorders in canines, felines, humans, and other animals. More specifically, the antibody / antigen-binding protein of the present invention can identify TGFβ overexpression in companion animals. Therefore, the antibody of the present invention can provide an important immunohistochemical tool. The antibody 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 The scope of the present invention also includes kits for carrying out the methods in question. A kit comprises at least one of the following: the antibody of the present invention, the nucleic acid encoding it, or cells containing it. The antibody of the present invention may be supplied in a container, usually in a lyophilized form. Antibodies that can be conjugated with a label or toxin, or that can not be conjugated, are typically included in a kit with buffers such as Tris, phosphates, or carbonates, stabilizers, biocides, and inactive proteins, such as serum albumin. Generally, these materials are present in less than 5% by mass based on the amount of active antibody, and usually again in a total amount of at least about 0.001% by mass based on the antibody concentration. Frequently, it is desirable to include inactive extenders or excipients for diluting the active ingredient, which may be present in about 1% to 99% by mass 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 with a label and formulated in a manner similar to the antibody preparations described above. The kit also typically includes a set of user instructions.

[0251] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods, and experimental conditions described, and that such compositions, methods, and conditions may vary. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them. The present invention is further described here by the following non-limiting examples. [Examples]

[0252] The present invention is further illustrated and supported by the following embodiments. However, these embodiments should not be considered in any way to further limit the scope of the invention. Rather, those skilled in the art will readily understand that other embodiments, modifications, and equivalents of the invention exist without departing from the spirit and / or the accompanying claims of the invention.

[0253] Example 1: Anti-TGFβ1,3 antibody Identification of a mouse monoclonal antibody that recognizes canid TGFβ1. AJ, CF-1, and BalbC mice were immunized according to a standard RIMMS protocol using combinations of amphibian and trout TGFβ1 formulated with Freund's complete adjuvant followed by an incomplete adjuvant. After vaccination, serum antibody titers from the immunized animals were determined using enzyme-coupled immunosorbent assay (ELISA). Individual ELISAs were performed to determine antibody titers against individual immunogens and to probe for cross-reactive antibody responses to canine TGFβ1, 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 as capture antigens. Serum from the immunized animals was diluted in phosphate-buffered saline with 0.05% tween-20 (PBST). The presence of anti-TGFβ1 antibodies was detected using horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (KPL, Inc., Gaithersburg, MD). A chromogenic substrate (SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate, KPL, Inc., Gaithersburg, MD) was added, and the mixture was incubated at room temperature (RT) for 10 minutes. The reaction was then stopped by adding 100 μL of 0.1N HCl. The absorbance of each well was determined by optical density (OD) at 450 nm. Under these conditions, animals in all groups showed a robust response to their respective immunogens; however, they were unable to produce an immune response directed towards the canine form of TGFβ1. The best response was exemplified by group CF-1, with titers of 1:31250 in all three animals (data not shown).

[0254] To overcome potential self-tolerance stemming from the conserved properties of TGFβ across species, canid 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 an immunogen referred to herein as TGFβ1-CRM. Two additional immunizations with TGFβ1-CRM were performed using mice from AJ and CF-1 groups, separated by two weeks. These efforts did not result in increased immune response to the canid TGFβ1 protein in any of the mice, as assessed by ELISA, as described above.

[0255] A final strategy was employed involving the administration of two additional doses of alum-adjuvanted TGFβ1-CRM via intraperitoneal (IP) injection, spaced two weeks apart. These efforts yielded a single mouse with sufficient titer to perform fusion. A pool of donor splenocytes from this mouse was used for fusion, and based on the results of primary screening to detect anti-TGFβ1 antibodies, eight wells were selected for expansion and secondary screening. Secondary screening confirmed that five fusions retained the ability to produce anti-TGFβ1 antibodies. Using protein A / G resin, the consumed supernatant from 15 ml of culture was purified to obtain an IgG-enriched population for further evaluation.

[0256] DNA sequence encoding mouse antibody 04H09 Ribonucleic acid (RNA) was isolated from 04H09 hybridoma cells using the RNeasy-Mini Kit (Qiagen, Inc., Germantown, MD) and the manufacturer's protocol. Briefly, 1 million frozen cells were collected by centrifugation, and RNA was purified from the cell lysates using an RNeasy spin column. 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 the mouse immunoglobulin (Ig) variable domain 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 thermal script 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 3' Ig constant region primers that hybridize to the Ig gene at a position proximal to the variable Ig region, thus transcribing the first strand cDNA representing the variable region of the mouse antibody. Individual RT reactions were performed using 3' constant heavy chain primers and 3' constant kappa light chain primers.

[0258] These cDNAs from 04H09 hybridomas were used as templates in polymerase chain reactions (PCR) to amplify variable IgG heavy chain and kappa light chain cDNA 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 region of the mouse Ig variable domain. Separate PCR reactions were performed using degenerate primers or primer pools for amplification of the mouse variable heavy chain and variable light chain regions. PCR was performed using an Expand High Fidelity DNA Polymerase Kit (Roche Diagnostics Corp., Indianapolis, IN) with a 1 μl cDNA reaction, according to the manufacturer's protocol. The 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 fragments amplified from PCR were separated by gel electrophoresis on a 1% agarose gel and purified using the Qiagen gel extraction kit (Qiagen, Inc., Germantown, MD). Forward primers for the heavy and light chain variable regions incorporated the EcoRI or SalI cleavage site, and reverse primers incorporated the HindIII cleavage site to facilitate cloning to the pUC19 plasmid. The purified PCR fragments and pUC19 plasmid were digested with the restriction endonuclease (New England Biolabs (NEB), Inc., Ipswich, MA) at 37°C for 1-2 hours. After digestion, the PCR fragments were purified using the Qiaquick PCR cleanup kit (Qiagen, Inc., Germantown, MD). The digested plasmids were separated by gel electrophoresis on a 1% agarose gel and purified using the Qiagen gel extraction kit. Purified PCR fragments representing variable IgG heavy chain and kappa light chain DNA were ligated to pUC19 plasmids overnight at 4°C using T4 DNA ligase and ligation buffer (NEB, Inc., Ipswich, MA).E. coli TOP10 cells (Invitrogen Corp., Carlsbad, CA) were transformed using 3 µl of each ligation reaction.

[0259] Plasmids were isolated from positive clones representing the variable region of each hybridoma using the Qiagen MiniPrep Kit (Qiagen 27106) according to the manufacturer's protocol. DNA sequences for each cloned insert were amplified using the BigDye sequencing reaction (Applied Biosystems by Life Technologies Corp., Carlsbad, CA) with M13 forward and reverse primers, according to the manufacturer's protocol. The sequencing reaction products were purified using a 96-well purification kit (Zymo Research, Irvine, CA) according to the manufacturer's protocol. Samples were loaded into an ABI-3730 capillary sequencing instrument, 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: Sequence ID 1 04H09 VH nucleotide sequence, Sequence ID 2 04H09 VH amino acid sequence, Sequence ID 3 04H09 VL nucleotide sequence, Sequence ID 4 04H09 VL amino acid sequence. Furthermore, the following are the six CDRs for the 04H09 monoclonal antibody:

[0260] Table 1 TIFF2023530663000002.tif36121

[0261] Recombinant mice: Construction of canid chimera 04H09 The antibody variable domain is involved in antigen binding, and therefore, transplantation of the complete variable domain of the 04H09 antibody to different constant regions, for example, constant regions from different species, should have little to no effect on the antibody's ability to bind to the canine TGFβ1 immunogen. For this reason, expression vectors were designed to produce recombinant chimeric or complete canine antibodies in mammalian expression systems. The chimeric antibodies described herein consist of variable sequences (both CDRs and frameworks) from host species antibodies transplanted onto the respective heavy and light constant regions of IgG molecules from different species. For example, the variable regions may be from the mouse host species, e.g., SEQ ID NOs. 2 and 4 of 04H09, and from the heavy chain constant region of a canine species (SEQ ID NO: 127), which would be referred to herein as a mouse:canine chimera. To produce desired chimeric antibodies, synthetic DNA sequences were constructed for the variable weight (VH) and variable light weight (VL) sequences of selected antibodies containing specific restriction endonuclease sites, Kozak consensus sequences, and N-terminal secretion leaders, thereby facilitating the expression and secretion of recombinant antibodies from mammalian cell lines.

[0262] For the mouse:canid 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 a canid IgG heavy chain (SEQ ID NO: 127) or a light chain (SEQ ID NO: 129) constant region.

[0263] Under the control of the CMV promoter, plasmids encoding each heavy and light chain were co-transfected into HEK293 cells using standard methods. After 6 days of expression, the chimeric mAbs were 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 fractions were neutralized and concentrated to approximately 0.5–1.0 mL using an Amicon Ultra centrifuge device (Millipore Sigma, Burlington, MA) with a nominal 10,000 MW cutoff. The fractions were dialyzed overnight at 4°C in 20 mM sodium acetate, pH 5.0, 85 g / L sucrose, and + / - 0.05 g / L EDTA, and stored at 4°C for further use.

[0264] The affinity and cell-based potency of 04H09 and chi04H09 were evaluated using surface plasmon resonance (SPR). SPR was assessed using the Biacore T200 system (Biocore Life Sciences (GE Healthcare), Uppsala, Sweden) to characterize the affinity of candidate monoclonal antibodies (mAbs) to TGFβ. To avoid affinity differences associated with different surface preparations that may occur when immobilizing antibodies to surfaces, 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 tips were quenched with ethanolamine, and the affinity with all candidate mAbs bound to immobilized TGFβ was evaluated. All curves were fitted to a 1:1 model. 10 -11Affinity values ​​below a certain level are below the lower limit of detection for the instrument. This led to the identification of a single mouse antibody, designated 04H09, with affinity ranges for TGFβ1 > TGFβ3 > TGFβ2 (see KD data in Table 3 below). Mouse anti-TGFβ 04H09 was further subcloned to generate hybridoma-producing homogeneous antibodies and for sequencing of variable heavy and light chains.

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

[0266] These observations indicate that conversion to chimeric morphology results in some loss of affinity for all three isoforms. However, they also show 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 levels of homogeneity and purity of recombinant mouse:canid chimeras compared to purified material from mouse hybridoma subclones. Blockade of TGFβ1 and 3 is encouraged and has led to further research into the canine morphology of this antibody.

[0267] Canine transformation of antibody 04H09 The formation of anti-drug antibodies (ADAs) can be associated with a loss of efficacy for any biological therapeutic protein, including monoclonal antibodies. While examples of immunogenic fully human mAbs and non-immunogenic chimeric mAbs can be found, biospeciesization of monoclonal antibodies may reduce the tendency for mAbs to become immunogenic. A canine-like strategy was employed to help mitigate the risks associated with ADA formation in the 04H09 monoclonal antibody provided herein. This canine-like strategy is based on identifying the most appropriate canine germline antibody sequences for CDR transplantation.

[0268] Following extensive analysis of available canine germline sequences for both variable heavy and variable light chains, candidate germline sequences were selected based on their homology to the framework region of the variable region of the mouse 04H09 antibody, and the native canine CDRs were replaced with CDRs from mouse precursor 04H09 antibodies (SEQ ID NOs. 5-10). The objective was to maintain high affinity and cell-based activity using the canine antibody framework to minimize potential immunogenicity in vivo.

[0269] Synthetic nucleotide constructs representing the canine variable heavy and light chains for the 04H09 antibody were constructed. The canine transformation efforts using the mouse antibody 04H09 focused on three canid VH frameworks and four canid VL frameworks, as described below.

[0270] Table 2 TIFF2023530663000003.tif47125

[0271] Each variable chain was subcloned into plasmids containing the respective canine heavy chain (SEQ ID NO: 127) or light chain (kappa) constant region (SEQ ID NO: 129). These plasmids were then simultaneously transfected in HEK293 cells using 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). All combinations, except for any heavy chain paired with can04H09-VL2, were able to transiently produce antibodies.

[0272] After expression, in addition to evaluating their potency in cell-based assays as described above, the canine mAbs were characterized for their affinity to canine TGFβ1, 2, and 3 (see data in Table 3 below). In general, all combinations of canine frameworks with transplanted 04H09 CDRs yielded comparable affinity to canine TGFβ2 and 3. The most dramatic loss of affinity was observed for TGFβ1 with the antibodies can04H09 VH1 / VL1, can04H09 VH2 / VL1, and can04H09 VH3 / VL1. As observed by the conversion of mouse 04H09 to chi04H09, canine modification had a variability effect on the potency of each antibody with respect to cellular pSmad signaling in response to each TGFβ isoform. As with mouse 04H09 and chi04H09, none of the framework combinations yielded activity against TGFβ2-mediated cellular signaling. With the exception of the Canid VL2 framework, representative pairs from each heavy and light Canid framework produced antibodies capable of inhibiting TGFβ1 and β3-induced signaling. Together, these data demonstrate that CDRs from mouse antibody 04H09 form robust combinations with candidate Canid frameworks, and that biospeciesization can be achieved without any modifications to the Canid germline sequence to preserve binding and potency phenotypes.

[0273] Table 3 TIFF2023530663000004.tif161134

[0274] Epitope mapping of canine-like ZTS-426 The cocrystallization of ZTS-426Fab (VH SEQ ID NO: 12 and VL SEQ ID NO: 24) and TGFβ1, followed by the structure of the antigen-binding mAb Fab fragment, was analyzed to a final resolution of 2.2 Å. Figure 7 shows the crystal structure of the secondary ZTS-426:TGFβ1 dimer. The complex structure provides structural insight into the key recognition elements governing the interaction of ZTS-426Fab with residues of mature TGFβ1.

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

[0276] The overall architecture was determined from a crystalline asymmetric unit containing two ZTS-426 Fab molecules bound diagonally opposite to a reference TGFβ1 homodimer (Figure 7), exhibiting nearly twofold rotational symmetry from the overall complex centroid. The mature TGFβ1 homodimer is held together by a series of covalent disulfide bonds formed by cysteine ​​side-chain thio functional groups within and between TGFβ1 monomers. Most notably, each Fab fragment interacts exclusively with only one of the TGFβ1 monomers. X-ray B factor suggests that TGFβ1 and Fab interface residues are appropriately degraded to reveal the key interactions contributing to binding affinity.

[0277] The complex X-ray structure was prepared using the protein preparation module within the complete MAESTRO modeling application suite from Schrödinger, ensuring a complete description of the complex lacking any missing atoms / residues and having the energy minimized to relax any excessive stereochemistry. This final structure, along with the Kabat numbering scheme for Fab residues, is used for all subsequent analyses. One of the antigen-antibody binding hypotheses suggests that lipophilic antibody CDR residues prefer to embed hydrophobic surfaces on the antigen surface. Therefore, the surface characterization probe contains residue...

Claims

1. A caninized antigen-binding protein that specifically binds to canine transforming growth factor beta-1 (TGFβ1) and canine transforming growth factor beta 3 (TGFβ3), comprising: a. a heavy chain variable region (VH), i. 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:5; ii. 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:6; iii. A heavy chain variable region (VH) comprising 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:7; and b. A light chain variable region (VL), i. 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:8; ii. 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:9; iii. A light chain variable region (VL) comprising 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: 10; and as well as any variant thereof having one or more conservative amino acid substitutions.

2. An antigen-binding protein comprising: a. 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:12, SEQ ID NO:14, SEQ ID NO:16; and b. a light chain variable region (VL) having at least about 95% sequence identity to the amino acid sequence SEQ ID NO:24; and 2. The antigen binding protein of claim 1, including any variant thereof having one or more conservative amino acid substitutions.

3. The antigen binding protein of claim 1, wherein the antigen binding protein comprises a heavy chain variable region (VH) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:12, and a light chain variable region (VL) having at least about 95% sequence identity to an amino acid sequence comprising SEQ ID NO:24, and any variant thereof having one or more conservative amino acid substitutions.

4. An antigen-binding protein according to any one of claims 1 to 3 for use in treating a canine for a TGFβ-related disorder.

5. The antigen-binding protein of claim 4, wherein 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.

6. The antigen-binding protein of claim 5, wherein the TGFβ-related disorder comprises a fibrotic disorder.

7. The antigen-binding protein of claim 6, wherein 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.

8. The antigen-binding protein of claim 7, wherein the TGFβ-related disorder is renal fibrosis / chronic kidney disease.

9. A pharmaceutical composition comprising a therapeutically effective amount of an antigen-binding protein according to any one of claims 1 to 7.

10. A method for treating a non-human subject for a TGFβ-related disorder, comprising administering to the non-human subject a therapeutic amount of the pharmaceutical composition described in claim 9.

11. The method of claim 10, wherein the non-human subject comprises a canine.

12. The method of claim 10, wherein the TGFβ-related disorder is selected from the group consisting of fibrotic disorders, connective tissue disorders, bone disorders, and cell proliferation disorders.

13. The method of claim 12, wherein the TGFβ-related disorder comprises a fibrotic disorder.

14. The method of claim 13, wherein 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.

15. The method described in claim 14, wherein the TGFβ disorder is renal fibrosis / chronic kidney disease.

16. A method for inhibiting TGFβ1 and TGFβ1 activity in a non-human subject by administering the pharmaceutical composition of claim 9.

17. The method of claim 16, wherein the subject comprises a canine.

18. An isolated nucleic acid encoding the antigen binding protein of claim 3, said nucleic acid comprising 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.

19. A vector comprising the nucleic acid described in claim 18.

20. A host cell comprising the nucleic acid described in claim 18.

21. A host cell comprising the vector described in claim 19.

22. A host cell producing an antigen-binding protein according to any one of claims 1 to 8.

23. A method of producing an antigen-binding protein according to any one of claims 1 to 8, comprising culturing a host cell according to claim 20 or 21 under conditions which result in the production of said antigen-binding protein, and isolating said antigen-binding protein from said host cell or from culture medium of said host cell.

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