Humanized and variant TGF-β3 specific antibodies, and methods and uses thereof

Abstract

The present invention provides antibodies that bind to transforming growth factor β3 (TGF-β3), particularly humanized antibodies and variant antibodies and fragments thereof, which recognize human and murine TGF-β3, particularly antibodies and fragments that do not recognize or bind TGF-β1 or TGF-β2. Humanized antibodies and variant antibodies are provided, which specifically recognize and neutralize TGF-β3 and can be used for diagnosing and treating conditions associated with activated or elevated TGF-β3, including cancer, and for modulating immune cells and immune responses, including immune responses to cancer or cancer antigens, and for fibrotic conditions. The anti-TGF-β3 antibodies, their variable region or CDR domain sequences, and fragments thereof can also be used in lymphocyte-mediated therapies, including T cell-mediated therapies, and in therapies combined with chemotherapy, immunomodulators, or anti-cancer agents and / or with other antibodies or fragments thereof. Antibodies are exemplified as the antibodies herein, including antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D, the sequences of which are provided herein.

Description

TECHNICAL FIELD

[0001] The present invention relates to specific binding members, particularly antibodies, particularly humanized antibodies and variant antibodies and fragments thereof, which bind to transforming growth factor β3 (TGF-β3), particularly recognize human and murine TGF-β3 without recognizing or binding to TGF-β1 or TGF-β2. The humanized antibodies and variant antibodies can be used for diagnosing and treating conditions associated with activated or elevated TGF-β3, including cancer, and for modulating immune cells and immune responses, including immune responses to cancer or cancer antigens. The antibodies, their variable region or CDR domain sequences, and their fragments can also be used in lymphoid cell-mediated therapies, including T cell-mediated therapies, and in therapies combined with chemotherapy, radiotherapy, immunomodulators, cancer vaccines, cancer antigens or anticancer agents and / or with other antibodies or fragments. BACKGROUND OF THE INVENTION

[0002] The transforming growth factor β (TGF-β) family forms a group of three isotypes: TGF-β1, TGF-β2, and TGF-β3, whose structures are formed by interrelated dimeric polypeptide chains. The pleiotropic and redundant functions of the TGF-β family involve controlling many aspects and actions of cell functions in all tissues of the human body, including aspects of proliferation, differentiation, and migration (Poniatowski LA et al., 2015, Mediators Inflamm, 2015; 137823). Although the isotypes are similar in sequence (the active domain of TGF-β3 shares 86% similarity with TGF-β1 and 91% similarity with TGF-β2), protein crystal structure and NMR studies have shown that the active domain of TGF-β3 is different from TGF-β1. Comparison of the structures of TGF-β3 and TGF-β2 (Schlunegger MP, Grütter MG, 1992, Nature 358:430-434; Daopin S, Piez KA, Ogawa Y, Davies DR, 1992, Science 257:369-373) has revealed an almost identical central core. There are differences in the conformation of the N-terminal α-helix and the β-sheet loop (Mittl PR1, Priestle JP, Cox DA, McMaster G, Cerletti N, Grütter MG, 1996, Protein Science July 5 (7):1261-1271).

[0003] In most cells, three types of cell surface proteins mediate TGF-β signaling: TGF-β receptor I (TβRI), II (TβRII), and III (TβRIII) (Cheifetz S, Like B, Massagué J, J Biol Chem. July 25, 1986; 261(21):9972-8). The bioactive form of TGF-β is a dimer that is held together by hydrophobic interactions and, in most cases, also by inter-subunit disulfide bonds. The dimeric structure of these ligands suggests that they function by bringing together pairs of type I and type II receptors to form a heterotetrameric receptor complex (Sun PD, Davies DR, Annu Rev Biophys Biomol Struct. 1995;24:269-91). The binding of TGF-β to the extracellular domains of both receptors also induces the proper conformation of the intracellular kinase domains. These receptors undergo reversible post-translational modifications (phosphorylation, ubiquitination, and sumoylation), which regulate receptor stability and availability as well as SMAD and non-SMAD pathway activation.

[0004] Phosphorylation of the receptor activates the TGF-β signaling pathway - the ligand first binds to TβRII, followed by subsequent phosphorylation of the Gly-Ser regulatory region (GS domain) within TβRI. This results in the incorporation of TβRI and the formation of a large ligand-receptor complex consisting of a dimeric TGF-β ligand and two pairs of TβRI and TβRII (Shi Y, Massagué J, Cell. June 13, 2003;113(6):685-700). TGF-β1 and TGF-β3 bind to TβRII without the involvement of the type I receptor, while TGF-β2 interacts only with the combination of both receptors (Derynck R, Feng XH, Biochim Biophys Acta. October 24, 1997;1333(2):F105-50). It has been observed that different ligand / receptor engagements within the TGF-β family may contribute to qualitative and quantitative differences in signaling events and biological outcomes (Hart PJ et al Nat Struct Biol 2002 9(3):203-208). In addition, there are large spatio-temporal expression differences for some TGF-β isoforms during embryogenesis, indicating non-overlapping functions without compensation throughout development (Akhurst RJ et al Development 1990 110(2):445-460).

[0005] In cancer animal models and cancer patients, the expression of transforming growth factor-β (TGF-β) is generally associated with tumor metastasis and poor prognosis (Donkor MK et al., 2012, OncoImmunology, 1(2):162-171). Members of the TGF-β family are potent regulatory cytokines that affect multiple cell types of the immune system mediating pro-inflammatory or anti-inflammatory responses. The actions of TGF-β on T cells are very multifaceted. In collaboration with other soluble factors, it controls the maturation, differentiation, and activity of various T cell subsets that prevent or initiate infections, graft-versus-host reactions, immune diseases, and cancer formation (Schon HT et al., 2014, Hepatobiliary Surg Nutr, 2014, Dec 3(6):386-406). Several studies indicate that TGF-β can promote cancer growth and metastasis by promoting tumor cell invasion and by inhibiting the function of immune cells via its actions on the tumor microenvironment (Flavell et al., 2010, Nat Rev Imm Aug;10(8):554-67).

[0006] Studies have demonstrated that blockade of TGF-β using the murine TGF-β pan-antibody 1D11, which recognizes TGF-β1, TGF-β2, and TGF-β3, will via CD8 +T cells synergistically enhance tumor vaccines in animal models (Terabe M et al. (2009) Clin Cancer Res 15:6560 - 6569; Takaku S et al. (2010) Int J Cancer 126(7):1666). Additionally, TGFβ production by tumor cells, myeloid - derived suppressor cells (MDSC), and stromal cells (such as cancer - associated fibroblasts (CAF)) present in the tumor site, as well as TGFβ immunosuppressive activity in the tumor site, imply that blocking TGFβ enhances antigen uptake, presentation, and activation of the anti - tumor immune response mediated by therapeutic vaccines. It has also been indicated that TGF - β drives the emergence of immunosuppressive plasma cells in various tumor types (e.g., hepatocellular carcinoma) (Shalapour et al. Nature. November 16, 2017; 551(7680):340 - 345; Shalapour et al. Nature. May 7, 2015; 521(7550):94 - 8). Combining anti - TGF - β blockade with programmed cell death protein 1 (PD1) blockade has been shown to induce anti - tumor immunity and tumor regression in different tumor models (Mariathasan et al., Nature. February 22, 2018; 554(7693):544 - 548. Tauriello et al. Nature. February 22, 2018; 554(7693):538 - 543).

[0007] The TGF-β ligands have been shown to be upregulated in many fibrotic disorders, and many TGF-β ligands are potent drivers of extracellular matrix formation, which is a hallmark of fibrotic disorders (Biernacka et al. Growth Factors. October 2011; 29(5):196-202). This also includes many types of cancer, in which the presence of fibrotic disorders has been indicated in tumor growth and metastatic spread (Principe et al. Cancer Res. May 1, 2016; 76(9):2525-39.; Caja et al. Int J Mol Sci. April 26, 2018; 19(5)). Thus, anti-TGF-β targeting has been proposed and is currently being tested as a treatment for various fibrotic disorders, including kidney, lung, heart, and skin (Walton et al. Front Pharmacol. July 14, 2017; 8:461). Other diseases in which TGF-β is involved are especially allergic diseases (Tirado-Rodriguez et al. J Immunol Res. 2014; 2014:318481) and Fanconi Anemia (Tummala and Dokal, Cell Stem Cell. May 5, 2016; 18(5):567-8).

[0008] Several publications have shown differences in the expression of TGF-β isoforms in various tissues, diseases, tumors, and tumor microenvironments. For example, Van Belle et al. showed that TGF-β1 is expressed by some melanocytes and almost uniformly by nevi and melanomas, while TGF-β2 and TGF-β3 were not detected in normal melanocytes but were found in nevi and all forms of melanoma (early and late primary and metastatic melanomas) in a tumor progression-related manner (P. Van Belle 1996 American J. of Pathology 148(6):1887-1894). There are many other examples of where different expression of isoforms has been shown, especially in glioblastoma, breast cancer, wound healing, and fibrosis (Roy et al. Int J Mol Sci. April 8, 2018; 19(4); Hachim et al., Tumour Biol. January 2018; 40(1); Lichtman et al., Wound Repair Regen. March 2016; 24(2):215-22).

[0009] In addition, it has been reported that TGF-β3, rather than TGF-β1, immunostaining is associated with poor survival prognosis in breast cancer, and when combined with lymph node involvement, TGF-β3 is a very important prognostic factor for survival (Ghellal A1 2000 Anticancer Res 20:4413). Furthermore, compared with breast cancer patients without lymph node metastasis, the plasma levels of TGF-β3 and the complex of TGF-β3 and its receptor CD105 (TGF-β3-CD105) are significantly elevated in breast cancer patients with positive lymph nodes, and their levels are associated with the lymph node status (Li C1 1998 Int.J.Cancer 79:455). In glioma models, TGF-β3 has been proposed as a gatekeeper controlling downstream signaling and has thus been proposed as a target in gliomas (Mol Cancer Ther. June 2017;16(6):1177-1186).

[0010] In particular, studies have demonstrated that TGF-β3 is involved in the following: promoting epithelial-mesenchymal transition (EMT); elevated TGF-β3 levels in breast and prostate metastasis; and elevated TGF-β3 levels detected in advanced and invasive tumors such as breast, prostate, and lung.

[0011] Thus, it is evident that by targeting specific isoforms of TGF-β, one can avoid the detrimental inflammatory consequences of blocking all isoforms of TGF-β. In addition, the differential expression patterns of TGF-β isoforms in different cancer types give researchers a unique opportunity to target cancer cells with greater specificity and with greater efficacy. There is an unmet need in the art for therapeutic TGF-β antibodies that are generated against its isoforms, including particularly against TGF-β3. Additionally, tools developed to identify different TGF-β isoforms are a powerful source of diagnosis and prognosis. Furthermore, TGF-β-targeted therapies require available, effective, and neutralizing humanized antibodies against specific TGF-β isoforms, particularly TGF-β3, to provide clinically applicable therapies that reduce immunogenicity and the patient's immune response, while having stability and long lifespan in the human body. The present invention addresses this unmet need in the art and particularly with respect to TGF-β3.

[0012] The citations to references herein should not be construed as an admission that such references are prior art to the present invention. Summary of the Invention

[0013] In general aspects, the present invention provides novel transforming growth factor β (TGF-β) antibodies against human TGF-β3, particularly humanized antibodies. In one aspect, the TGF-β3 antibodies of the present invention are more specific for TGF-β3 binding than their binding to TGF-β1 or TGF-β2. In one aspect, the TGF-β3 antibodies of the present invention do not cross-react or bind with other members of the TGF-β family, and particularly do not cross-react or bind with TGF-β1 or TGF-β2. In one aspect, the present invention provides isolated specific binding members, particularly antibodies or fragments thereof, including Fab fragments and single-chain or domain antibodies, which specifically recognize TGF-β3. In a particular aspect, the antibody or its active fragment neutralizes TGF-β3 activity.

[0014] The present invention provides antibodies specific for TGFβ3 for diagnostic and therapeutic purposes. In particular, antibodies specific for TGFβ3 are provided, wherein the antibodies are humanized and recognize and are capable of binding and neutralizing human (and murine) TGFβ3 and do not recognize other forms of TGF-β (TGF-β1 or TGF-β2).

[0015] The antibodies of the present invention have diagnostic and therapeutic uses in cancer and immunomodulation, including modulating the immune response to cancer and cancer vaccines. In a further aspect, the antibodies of the present invention have diagnostic and therapeutic uses in fibrotic conditions and fibrotic diseases. The antibodies of the present invention can be applied to characterize and modulate the activity of TGF-β3, particularly to neutralize TGF-β3 activity.

[0016] In a further aspect, the present invention provides an antibody or a fragment thereof, particularly including a humanized antibody or a fragment thereof, which recognizes TGF-β3 and is selected from antibody 1901-1C comprising the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22); antibody 1901-1A comprising the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23); antibody 1901-1D comprising the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22); and antibody 1901-1B comprising the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23). In a particular aspect, the present invention provides an antibody or an active fragment thereof, which specifically recognizes and neutralizes TGF-β3 and is selected from antibody 1901-1C comprising the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22); antibody 1901-1A comprising the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23); antibody 1901-1D comprising the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22); and antibody 1901-1B comprising the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23).

[0017] In another aspect, the present invention provides an antibody or an active fragment thereof, which comprises the heavy chain 1901_VH_1G10m_02(J) (SEQ ID NO:36). In one such aspect, the antibody further comprises the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22).

[0018] The binding of an antibody to its target antigen is mediated by the complementarity determining regions (CDRs) of its heavy and light chains. Thus, specific binding members based on the CDR regions of the heavy chain or light chain or both the heavy and light chains of the antibodies of the present invention (particularly any of antibodies 1901-1A, 1901-1B, 1901-1C or 1901-1D) will be useful specific binding members for therapy and / or diagnosis. In one aspect, the present invention provides a TGF-β3 antibody capable of binding and neutralizing TGF-β3, which comprises the light chain and heavy chain variable region CDR1, CDR2 and CDR3 sequences provided herein and Figure 7 and Figure 8 listed. In a specific aspect, the present invention provides a TGF-β3 specific antibody capable of specifically binding and neutralizing TGF-β3, wherein the antibody does not bind or neutralize TGF-β1 or TGF-β2, and the antibody comprises the sequences provided herein and Figure 7 and Figure 8The listed light and heavy chain variable region CDR1, CDR2, and CDR3 sequences. In one such aspect, antibodies are provided that comprise heavy chain variable region CDRs, the heavy chain variable region CDRs comprising a CDR1 sequence of SSWIH (SEQ ID NO:1), a CDR2 sequence of RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence of RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30). In another such aspect, antibodies are provided that comprise heavy chain variable region CDRs, the heavy chain variable region CDRs comprising a CDR1 sequence of SSWIH (SEQ ID NO:1) or GYTFSSSWIH (SEQ ID NO:28), a CDR2 sequence of RIYPGDGDTDYSEKFQ (SEQ ID NO:34) or WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and a CDR3 sequence of RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30). In a further aspect, antibodies are provided that further comprise light chain variable region CDRs, the light chain variable region CDRs comprising a CDR1 sequence of KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence of YASNRYT (SEQ ID NO:5), and a CDR3 sequence of QQDYSSPY (SEQ ID NO:33). In yet a further aspect, antibodies are provided that comprise light chain variable region CDRs, the light chain variable region CDRs comprising a CDR1 sequence of KASQSVINAVA (SEQ ID NO:4) or KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence of YASNRYT (SEQ ID NO:5) or LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence of QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0019] In one aspect, the present invention provides an antibody against TGF-β3, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising CDRs, the CDRs including a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence comprising the sequence RMITTQAAL (SEQ ID NO:37). In one such aspect, the antibody comprises a heavy chain variable region, the heavy chain variable region comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence comprising RMITTQAAL (SEQ ID NO:37). In one aspect, the antibody comprises a heavy chain variable region, the heavy chain variable region comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAAL (SEQ ID NO:37). In a further aspect, an antibody is provided, the antibody further comprising light chain variable region CDRs, the light chain variable region CDRs including a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33). In yet a further aspect, an antibody is provided, the antibody comprising light chain variable region CDRs, the light chain variable region CDRs including a CDR1 sequence KASQSVINAVA (SEQ ID NO:4) or KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence YASNRYT (SEQ ID NO:5) or LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6). In one aspect, the antibody specifically binds and neutralizes TGF-β3 without binding and / or without neutralizing TGF-β1 or TGF-β2.The present invention provides antibodies against TGF-β3, said antibodies comprising a heavy chain variable region sequence, said heavy chain variable region sequence comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35); or a CDR1 sequence GYTFSSSWIH (SEQ ID NO:28), a CDR2 sequence WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and a CDR3 sequence ARRMITTQAAL (SEQ ID NO:30). In one aspect, the present invention provides antibodies, said antibodies further comprising a light chain variable region sequence, said light chain variable region sequence comprising a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6); or a CDR1 sequence KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6). In one aspect, said antibodies comprise a variable region sequence that is humanized or altered or modified to increase its similarity to an antibody naturally produced by a human. In one aspect, said antibodies comprise a variable region sequence that is humanized or altered or modified in the framework region to increase its similarity to an antibody naturally produced by a human.

[0020] In a particular aspect, the isolated antibodies or fragments of the present invention neutralize TGF-β3. In a particular aspect, the isolated antibodies or fragments of the present invention do not react with TGF-β1 or TGF-β2. In one aspect, the isolated antibodies or fragments of the present invention bind and neutralize TGF-β3 and do not react with or bind to TGF-β1 or TGF-β2.

[0021] In one aspect, the present invention provides antibodies that specifically target and neutralize TGF-β3, wherein the antibodies do not bind or neutralize TGF-β1 or TGF-β2, and the antibodies comprise a heavy chain variable region sequence that comprises a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30). In a further aspect, the present invention provides antibodies that specifically target and neutralize TGF-β3, wherein the antibodies do not bind or neutralize TGF-β1 or TGF-β2, and the antibodies comprise a heavy chain variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30), and the light chain variable region sequence comprising a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33).

[0022] In one aspect, the present invention provides antibodies that specifically target and neutralize TGF-β3, wherein the antibodies do not bind or neutralize TGF-β1 or TGF-β2, and the antibodies comprise a heavy chain variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30), and the light chain variable region sequence comprising a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6); or a CDR1 sequence KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0023] In one aspect, the present invention provides an antibody that specifically targets and neutralizes TGF-β3, wherein the antibody does not bind or neutralize TGF-β1 or TGF-β2. The antibody comprises a heavy chain variable region sequence and a light chain variable region sequence. The heavy chain variable region sequence comprises a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35); or a CDR1 sequence GYTFSSSWIH (SEQ ID NO:28), a CDR2 sequence WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and a CDR3 sequence ARRMITTQAAL (SEQ ID NO:30). The light chain variable region sequence comprises a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6); or a CDR1 sequence KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0024] In one aspect, the present invention provides antibodies that specifically target and neutralize TGF-β3, wherein the antibodies do not bind or neutralize TGF-β1 or TGF-β2. The antibodies comprise a heavy chain variable region sequence and a light chain variable region sequence. The heavy chain variable region sequence comprises a CDR1 sequence of SSWIH (SEQ ID NO:1) or GYTFSSSWIH (SEQ ID NO:28), a CDR2 sequence of RIYPGDGDTDYSEKFQ (SEQ ID NO:34) or WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and a CDR3 sequence of RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30). The light chain variable region sequence comprises a CDR1 sequence of KASQSVINAVA (SEQ ID NO:4) or KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence of YASNRYT (SEQ ID NO:5) or LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence of QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0025] In a further aspect, the present invention provides antibodies, particularly humanized antibodies, that comprise a heavy chain variable region sequence of LCR1901_VH_1G10m (SEQ ID NO:18) or LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) or a variant thereof, the variant having at least 90% amino acid identity with the heavy chain variable region sequence of LCR1901_VH_1G10m (SEQ ID NO:18) or LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) or comprising one or more amino acid substitutions in 1 to 3 of the heavy chain CDR regions in Figure 7 (SEQ ID NO:1, 34, and 35 or 30, SEQ ID NO:28, 29, and 30, or SEQ ID NO:1, 34, and 35), wherein the variant retains TGF-β3 reactivity and neutralizing activity and lacks TGF-β1 and TGF-β2 reactivity. In a specific aspect, the present invention provides a humanized antibody that comprises a heavy chain variable region sequence of LCR1901_VH_1G10m (SEQ ID NO:18) or LCR1901_VH_1G10m_03(K) (SEQ ID NO:19).

[0026] In a further aspect, the invention provides an antibody, particularly a humanized antibody, comprising a heavy chain variable region sequence LCR1901_VH_1G10m_02(J) (SEQ ID NO: 36) or a variant thereof, said variant having at least 90% amino acid identity with the heavy chain variable region sequence LCR1901_VH_1G10m_02(J) (SEQ ID NO: 36) or comprising Figure 7 from 1 to 3 amino acid substitutions in one or more of the heavy chain CDR regions in (SEQ ID NO: 1, 34 and 35 or 30, SEQ ID NO: 28, 29 and 30, or SEQ ID NO: 1, 34 and 35), wherein said variant retains TGF-β3 reactivity and neutralization and lacks TGF-β1 and TGF-β2 reactivity. In a particular aspect, the invention provides a humanized antibody comprising the heavy chain variable region sequence LCR1901_VH_1G10m_02(J) (SEQ ID NO: 36).

[0027] The antibody of the present invention may comprise heavy chain variable region CDR domain region sequences: CDR1 sequence SSWIH (SEQ ID NO: 1), CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO: 34), and CDR3 sequence RMITTQAALDY (SEQ ID NO: 35) or ARRMITTQAAL (SEQ ID NO: 30); CDR1 sequence SSWIH (SEQ ID NO: 1), CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO: 34), and CDR3 sequence RMITTQAALDY (SEQ ID NO: 35); or CDR1 sequence GYTFSSSWIH (SEQ ID NO: 28), CDR2 sequence WIGRIYPGDGDTDYSEKFQ (SEQ ID NO: 29), and CDR3 sequence ARRMITTQAAL (SEQ ID NO: 30), and a light chain variable region. The antibody of the present invention may comprise heavy chain variable region CDR domain region sequences: CDR1 sequence SSWIH (SEQ ID NO: 1) or GYTFSSSWIH (SEQ ID NO: 28), CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO: 34) or WIGRIYPGDGDTDYSEKFQ (SEQ ID NO: 29), and CDR3 sequence RMITTQAALDY (SEQ ID NO: 35) or ARRMITTQAAL (SEQ ID NO: 30), and a light chain variable region. In one aspect, the TGF-β3 antibody further comprises light chain variable region CDR sequences: CDR1 sequence KASQSVINAVA (SEQ ID NO: 4), CDR2 sequence YASNRYT (SEQ ID NO: 5), and CDR3 sequence QQDYSSPY (SEQ ID NO: 33) or QQDYSSPYT (SEQ ID NO: 6); or CDR1 sequence KASQSVINAVAWY (SEQ ID NO: 31), CDR2 sequence LLIYYASNRYT (SEQ ID NO: 32), and CDR3 sequence QQDYSSPY (SEQ ID NO: 33) or QQDYSSPYT (SEQ ID NO: 6). In one aspect of the present invention, TGF-β3 specific neutralizing antibodies with alternative heavy and light chain CDR sequences compete with each other for TGF-β3 binding.

[0028] In one aspect, the TGF-β3 specific antibody of the present invention comprises a heavy chain variable amino acid SEQ ID NO:18 or SEQ ID NO:19. In one aspect, the TGF-β3 specific antibody of the present invention comprises a heavy chain variable amino acid SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:36. In a specific aspect, the TGF-β3 antibody of the present invention comprises a heavy chain and a light chain variable region amino acid sequence listed in any one of Figure 10 (SEQ ID NO:18 and 22), Figure 11 (SEQ ID NO:18 and 23), Figure 12 (SEQ ID NO:19 and 22) or Figure 13 (SEQ ID NO:19 and 23). The TGF-β3 antibody of the present invention may comprise an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence listed in Figure 10 、 11 、12 or 13 (SEQ ID NO:18 and 22, 18 and 23, 19 and 22, 19 and 23). In one aspect, the TGF-β3 antibody of the present invention comprises a heavy chain variable region SEQ ID NO:36. In a further aspect, the TGF-β3 antibody of the present invention comprises a heavy chain variable region SEQ ID NO:36 and a light chain variable region SEQ ID NO:22. In a further aspect, the TGF-β3 antibody of the present invention comprises a heavy chain variable region SEQ ID NO:36 and a light chain variable region SEQ ID NO:22 or SEQ ID NO:23. The TGF-β3 specific antibody of the present invention that can specifically bind to TGF-β3 and does not bind to TGF-β1 or TGF-β2 may comprise an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the heavy chain variable region amino acid sequence SEQ ID NO:18 or SEQ ID NO:19. The TGF-β3 specific antibody of the present invention that can specifically bind to TGF-β3 and does not bind to TGF-β1 or TGF-β2 may comprise an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the heavy chain variable region amino acid sequence SEQ ID NO:36. In a further aspect, the TGF-β3 specific antibody of the present invention that can specifically bind to TGF-β3 and does not bind to TGF-β1 or TGF-β2 may comprise an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the light chain variable region amino acid sequence SEQ ID NO:22 or SEQ ID NO:23.

[0029] The TGF-β3 specific antibodies of the present invention that can specifically bind to TGF-β3 and do not bind to TGF-β1 or TGF-β2 may comprise amino acid sequences having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the heavy chain variable region amino acid sequence SEQ ID NO:18 or SEQ ID NO:19 and further having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the light chain variable region amino acid sequence SEQ ID NO:22 or SEQ ID NO:23. The TGF-β3 specific antibodies of the present invention that can specifically bind to TGF-β3 and do not bind to TGF-β1 or TGF-β2 may comprise amino acid sequences having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the heavy chain variable region amino acid sequence SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:36 and further having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity with the light chain variable region amino acid sequence SEQ ID NO:22 or SEQ ID NO:23.

[0030] In a particular aspect, the antibodies of the present invention or their active fragments neutralize human and murine TGF-β3. In one aspect, the antibodies of the present invention neutralize and block TGF-β3-mediated signal transduction in mammals, particularly in humans or mice, in vivo. In one aspect, the antibodies of the present invention or their active fragments neutralize and block TGF-β3-mediated signal transduction in mammals in vivo, without neutralizing or blocking TGF-β1 or TGF-β2 signal transduction in mammals in vivo.

[0031] Accordingly, specific binding proteins (such as antibodies) based on the CDRs of one or more antibodies (particularly including the heavy chain CDRs identified herein) will be useful for targeting TGF-β3 in diseases or cancers, particularly cells expressing TGF-β3, or TGF-β3 activity in immune responses. Since the target of the antibodies of the present invention is specifically TGF-β3 rather than TGF-β1 and / or TGF-β2, in one aspect of the present invention, the antibodies of the present invention do not significantly bind to TGF-β forms or family members other than TGF-β3, and it is expected that the TGF-β3 specific antibodies of the present invention will have less toxicity and inflammatory or adverse immune responses or reactions in cell targets or in animals, particularly as compared to more non-specific TGF-β antibodies, such as pan-TGF-β antibodies that recognize more than one form of TGF-β or all forms of TGF-β.

[0032] In another aspect of the present invention, there is provided one or more antibodies or one or more antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3). In a specific embodiment, there is provided one or more antibodies or one or more antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3) to the extent that the antibodies or antigen-binding fragments thereof described herein compete with themselves for binding to TGF-β3 (e.g., human TGF-β3).

[0033] In another specific embodiment, there is provided a first antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3), wherein the competition is exhibited as a reduction of more than 60% (e.g., 65%, 70%, 75%, 85%, 90%, 95%, or 98%, or between 60% and 65%, 65% and 70%, 70% and 75%, 75% and 80%, 80% and 85%, 85% and 95%, or 95% and 100%) in the binding of the first antibody or antigen-binding fragment thereof to TGF-β3 (e.g., human TGF-β3). In another specific embodiment, there is provided a first antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3), wherein the competition is exhibited as a reduction of more than 60% (e.g., 65%, 70%, 75%, 85%, 90%, 95%, or 98%, or between 60% and 65%, 65% and 70%, 70% and 75%, 75% and 80%, 80% and 85%, 85% and 95%, or 95% and 100%) in the binding of one or more of the antibodies or antigen-binding fragments of antibody 1901-1A, 1901-1B, 1901-1C, or 1901-1D in the presence of and / or following binding of the first antibody or antigen-binding fragment thereof.

[0034] In a specific aspect, there is provided an antibody that binds to a polypeptide comprising (i) a VL domain comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the VL CDRs of the antibodies listed in Figure 8 or Figures 10 - 13 ; and (ii) an VH domain comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of the VH CDRs of the antibodies listed in Figure 7 or Figures 10 - 13The VH domains of the VH CDR1, VH CDR2, and VH CDR3 of the amino acid sequences of the CDRs of the antibodies listed in compete (e.g., in a dose-dependent manner) for specific binding to TGF-β3 (e.g., human TGF-β3). In a specific aspect, provided herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to TGF-β3 (e.g., human TGF-β3) with an antibody comprising a VH domain, optionally further comprising or further comprising a VL domain, the VH domain comprising the CDR1 sequence SSWIH (SEQ ID NO:1), the CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and the CDR3 sequence RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30), and the VL domain comprising the CDR1 KASQSVINAVA (SEQ ID NO:4)), the CDR2 sequence YASNRYT (SEQ ID NO:5), and the CDR3 sequence QQDYSSPY (SEQID NO:33).

[0035] In a particular embodiment, provided herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to TGF-β3 (e.g., human TGF-β3) with an antibody comprising the VH CDRs of 1901-1A, 1901-1B, 1901-1C, or 1901-1D, particularly SEQ ID NOs: 1, 34, and 35 or 30, SEQ ID NOs: 28-30, or SEQ ID NOs: 1, 34, and 35. In a particular embodiment, provided herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to TGF-β3 (e.g., human TGF-β3) with an antibody comprising the VL CDRs of 1901-1A, 1901-1B, 1901-1C, or 1901-1D, particularly SEQ ID NOs: 4, 5, and 33, SEQ ID NOs: 31-33, or SEQ ID NOs: 4-6.

[0036] In a specific embodiment, the antibodies described herein are antibodies that are competitively blocked (e.g., in a dose-dependent manner) from specifically binding to TGF-β3 (e.g., human TGF-β3) by antibodies comprising a VH domain having the amino acid sequence SEQ ID NO:18 or SEQ ID NO:19. In a specific embodiment, the antibodies described herein are antibodies that are competitively blocked (e.g., in a dose-dependent manner) from specifically binding to TGF-β3 (e.g., human TGF-β3) by antibodies comprising a VH domain having the amino acid sequence SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:36. In a specific embodiment, the antibodies described herein are antibodies that are competitively blocked (e.g., in a dose-dependent manner) from specifically binding to TGF-β3 (e.g., human TGF-β3) by antibodies comprising a VH domain having the amino acid sequence SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:36 and a VL domain having the amino acid sequence SEQ ID NO:22 or SEQ ID NO:23.

[0037] In other aspects, the invention provides isolated nucleic acids that comprise a sequence encoding a specific binding member or antibody as defined above or herein, and methods of making the specific binding members or antibodies of the invention, the methods comprising expressing the nucleic acid under conditions that cause expression of the binding member or antibody and recovering the binding member or antibody. In one such aspect, provided is a nucleic acid encoding an antibody variable region sequence having a heavy chain amino acid sequence as set forth in Figure 10 , 11 , 12 or 13, or provided is an antibody having a heavy chain CDR domain sequence as set forth in Figure 7 and SEQ ID NO:1, 34, 35, 28, 29, 30 or Figure 10 , 11 , 12 or 13. In one aspect, provided is a nucleic acid encoding an antibody light chain variable region having a light chain amino acid sequence as set forth in Figure 10 , 11 , 12 or 13, or provided is an antibody having a light chain CDR domain sequence as set forth in Figure 8 and SEQ ID NO:4, 5, 6, 31, 32, 33 or Figure 10 , 11, an antibody having a light chain CDR domain sequence listed in 12 or 13. The invention also relates to a recombinant DNA molecule or a cloned gene encoding the antibody of the invention or a degenerate variant thereof; preferably a nucleic acid molecule encoding the VH of the antibody, particularly the CDR region sequence and optionally additionally encoding the VL, particularly the CFR region sequence, particularly a recombinant DNA molecule or a cloned gene, which is capable of encoding a heavy chain sequence SEQ ID NO:18 or 19 or 36 and a light chain sequence SEQ ID NO:22 or 23, or such a combination of heavy and light chain variable region sequences (including those listed as Figure 10 , 11 , 12 and 13).

[0038] The unique specificity and affinity of the antibodies and fragments of the invention provide diagnostic and therapeutic uses for identifying, characterizing and targeting disorders associated with TGF-β3 expression, activity or activation. Accordingly, methods and aspects thereof are provided according to the invention. In one aspect, the antibodies of the invention that target TGF-β3 can be used to modulate the immune response, including modulating the immune response against cancer, cancer cells or tumor cells and cancer antigens or tumor antigens. In another aspect, the antibodies of the invention that target TGF-β3 can be used for the therapeutic treatment or management of cancer, enhancing the anti-cancer immune response and enhancing cancer vaccines. The antibodies can be applied to enhance the therapeutic effect of one or more cancer therapies (including conventional anti-cancer agents and compounds and cell therapies, including cancer-targeted T cell therapies), including anti-cancer and / or anti-cell effects. The antibodies can be applied to enhance the therapeutic effect of one or more radiotherapy treatments, including anti-cancer and / or anti-cell effects. In a particular aspect, the antibodies of the invention can be applied to treat, manage and / or prevent cancer, including cancer recurrence and metastasis. In one aspect, the TGF-β3 antibodies of the invention can be applied to treat or modulate breast cancer, melanoma, prostate cancer or lung cancer.

[0039] According to the invention, methods for treating, alleviating or modulating cancer are provided herein, the methods comprising administering an antibody of the invention or a pharmaceutical composition thereof. In a further aspect, methods for stimulating or enhancing an immune response or an immunomodulatory agent or radiotherapy against a vaccine or antigen in a mammal are provided, the methods comprising administering an antibody of the invention or a pharmaceutical composition thereof.

[0040] In one aspect of the invention, one or more TGF-β3 antibodies, particularly one or more TGF-β3 neutralizing antibodies as provided herein, particularly humanized antibodies can be combined with or administered in a composition of one or more cancer antigens and one or more adjuvants, including administering to a patient to facilitate a more robust initiation and activation of an adaptive anti-tumor response to enhance immunotherapy against cancer. Additional inhibitors of TGFβ activity, such as small molecules, antisense or aptamers, can also be used to inhibit TGFβ activity, including or particularly TGF-β3.

[0041] Thus, in one aspect of the present invention, the one or more anti-TGF-β3 antibodies can be administered alone or in combination with other treatments, therapies or agents or cell therapies, either simultaneously or sequentially, depending on the condition to be treated. Immunomodulators can be included in a composition having one or more TGF-β3 antibodies or administered with and / or at different times from one or more TGF-β3 antibodies to enhance immunomodulation and / or cancer therapy, including immunotherapy or cell therapy against cancer. The immunomodulator can be an adjuvant. In a further aspect, the TGF-β3 antibodies of the present invention can also be used as one or more immunostimulants or one or more adjuvants, in combination with antigenic materials such as but not limited to proteins, peptides or nucleic acids, in order to generate a protective immune response, such as a T cell or CTL response against the administered antigen.

[0042] The present invention provides methods for improving, facilitating or enhancing chimeric antigen receptor (CAR) T cell therapy, the methods comprising administering one or more anti-TGF-β3 antibodies or fragments thereof either simultaneously or sequentially with one or more CAR T cells. In one aspect of the method, the one or more anti-TGF-β3 antibodies or fragments thereof are added to the CAR T cell culture prior to administration or infusion. In one aspect, the one or more anti-TGF-β3 antibodies or fragments thereof, such as their scfv, are expressed by or expressed on one or more CAR T cells. In another method of the present invention, one or more anti-TGF-β3 antibodies or fragments thereof are administered in combination with activated T cells or T cells directed against cancer antigens or cell cycle regulators.

[0043] According to the present invention, methods are provided for treating, alleviating or modulating fibrotic conditions or fibrotic diseases, the methods comprising administering the antibodies of the present invention or pharmaceutical compositions thereof. In one aspect, methods are provided for treating, alleviating or modulating conditions or diseases in which there is an alteration in extracellular matrix formation, the methods comprising administering the antibodies of the present invention or pharmaceutical compositions thereof. In one aspect of these methods, the antibodies of the present invention can be administered in combination or conjunction with one or more anti-inflammatory agents, immunosuppressive agents, immunoresponse modulators, antioxidants or antifibrotic drugs or agents. In one such aspect, one or more antibodies of the present invention are administered in combination or conjunction with a treatment for fibrosis. In one such aspect, methods for treating or modulating pulmonary fibrosis are provided. In one aspect, one or more antibodies of the present invention are administered in combination or conjunction with a fibrosis treatment, which is particularly selected from nintedanib and pirfenidone

[0044] As already shown, TGF-β3 antibodies (including TGF-β3 specific antibodies) are effective both in vitro and in vivo. Accordingly, one aspect of the present invention relates to stimulating an immune response in a subject by administering one or more TGF-β3 antibodies of the present invention, with or without an antigen molecule, in an amount sufficient to stimulate a favorable immune response in such a subject.

[0045] The present invention includes compositions and / or kits that comprise one or more TGF-β3 antibodies of the present invention together with one or more immunogenic proteins or peptides. The compositions include pharmaceutical compositions and immunological compositions. The antibodies or compositions of the present invention can be administered systemically or in a targeted manner, including administration to an affected or target organ, administration to a tumor, administration in the region or location of a tumor, or direct administration to a tumor, such as intratumoral injection.

[0046] Antibodies, fragments, and recombinant antibodies that comprise the CDR domains according to the present invention can be used in methods for treating or diagnosing a human or animal body, such as methods for treating tumors in a human patient, the methods comprising administering to the patient an effective amount of the antibodies, fragments, and recombinant antibodies of the present invention. Antibodies, fragments, and recombinant antibodies that comprise the CDR domains according to the present invention can be used in methods for stimulating or enhancing an immune response to cancer, tumor cells, or one or more cancer or tumor antigens in a mammal, particularly a human, the methods comprising administering to the mammal an effective amount of the antibodies, fragments, and recombinant antibodies of the present invention. Antibodies, fragments, and recombinant antibodies that comprise the CDR domains according to the present invention can be used in methods for inhibiting or reducing the recurrence or metastasis of cancer in a mammal, particularly a human, the methods comprising administering to the mammal an effective amount of the antibodies, fragments, and recombinant antibodies of the present invention. Antibodies, fragments, and recombinant antibodies that comprise the CDR domains according to the present invention can be used in methods for inhibiting or blocking the stimulation of TGFβ, particularly TGFβ3, in a mammal, particularly a human, in response to radiation or cancer therapy, the methods comprising administering to the mammal an effective amount of the antibodies, fragments, and recombinant antibodies of the present invention. In one aspect of the methods, a TGF-β3 specific antibody, fragment, and recombinant antibody that comprise the CDR domains according to the present invention are administered in combination with or subsequent to radiation therapy and / or cancer therapy to a mammal.

[0047] The therapeutic methods of the present invention are related to the prevention or treatment of cancer, or the stimulation or enhancement of the immune response to cancer, or the inhibition of protection against immune-mediated cancer cells (including melanoma, breast cancer, prostate cancer, and lung cancer). In one aspect of the method, the specific TGF-β3 neutralizing antibodies of the present invention (including their active fragments) are used to stimulate or enhance the immune response to cancer (including melanoma, breast cancer, prostate cancer, and lung cancer). In one aspect, one or more specific neutralizing TGF-β3 antibodies or their active fragments of the present invention stimulate or enhance the immune response via cancer vaccines or cancer immunotherapies or via cell therapies such as cancer-targeted T cell therapies (including radiotherapy).

[0048] The antibodies of the present invention and, in a specific embodiment, antibodies having the sequences represented in Figure 10 , 11 , 12, or 13, or their active fragments, and single-chain, recombinant, or synthetic antibodies derived therefrom (especially those containing Figure 7 and 8 depicted heavy-chain CDR region sequences and light-chain CDR region sequences, including those containing heavy-chain CDR SEQ ID NO:1, 34, and 35 or 30 and light-chain CDR SEQ ID NO:4, 5, and 33) can be expressed in immune cells (including lymphoid cells, including T cells). In one such aspect, the antibodies, or their fragments, and single-chain, recombinant, or synthetic antibodies derived therefrom are expressed and secreted by lymphoid cells (including T cells). Lymphoid cells such as T cells can be prepared and used in therapies, including immunomodulation, and in cancer therapies. In one aspect, lymphoid cells (such as T cells) expressing one or more TGFβ3 antibodies of the present invention can be further engineered to express another / other one or more proteins, such as receptors. In one aspect, lymphoid cells (such as T cells) expressing one or more TGFβ3 antibodies of the present invention can be combined with lymphoid cells (such as T cells) engineered to express another / other one or more proteins (such as receptors). In one aspect, the receptor is a chimeric antigen receptor (CAR). In one aspect, the receptor is a T cell receptor. In one aspect, the other / other one or more proteins are cancer antigens or tumor antigens or tumor antigen antibodies or their fragments or immunomodulatory agents. The present invention provides therapeutic methods that include administering to a mammal lymphoid cells, such as T cells, expressing one or more TGFβ3 antibodies of the present invention. In one aspect, the method relates to treating cancer or preventing the recurrence or metastasis of cancer. In one aspect, the method relates to modulating the immune response, including in cancer or cancer therapies.

[0049] The binding members and antibodies of the present invention and, in a specific embodiment, having Figure 10 ,11 An antibody against the sequence represented in 12 or 13, or an active fragment thereof, and single-chain, recombinant or synthetic antibodies derived therefrom (especially those comprising the heavy-chain CDR region sequences and light-chain CDR region sequences depicted in Figure 7 and 8 , including those comprising heavy-chain CDR SEQ ID NO: 1, 34 and 35 or 30 and light-chain CDR SEQ ID NO: 4, 5 and 33) can be formulated in a pharmaceutical composition for administration, said pharmaceutical composition comprising a suitable vehicle, carrier or diluent or comprising an adjuvant and / or an immunomodulator. Such pharmaceutical compositions may also comprise means for modulating the half-life of the antibody or fragment by methods known in the art (such as pegylation).

[0050] The pharmaceutical or immunogenic compositions of the invention may further comprise additional antibodies or therapeutic agents. In one aspect, such other agents or therapies may be selected from anti-cancer agents or therapies, anti-mitotic agents, apoptotic agents or antibodies, or immunomodulators, or small molecule inhibitors against immunomodulators. More generally, these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g., anti-mitotic agents), inhibitors or signal transduction inhibitors. The compositions may be administered in combination with an immunomodulator (such as an adjuvant). The compositions may also be administered in combination with other anti-TGFβ antibodies, other immunomodulatory antibodies or other anti-tumor antigen antibodies or may comprise a combination therewith.

[0051] The diagnostic utility of the invention extends to the use of the antibodies of the invention in characterizing tumor or cell samples or in assays for screening tumors or cancers (including in vitro and in vivo diagnostic assays). The antibodies of the invention may carry a detectable label or a functional label. The specific binding member may carry a radioactive label, such as an isotope 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I, 111 In, 117 Lu, 211 At, 198 Au, 67 Cu, 225 Ac, 213 Bi,99 Tc and 186 Re. In one aspect, the label can be an enzyme, including where detection can be accomplished by any of a number of colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gas quantification techniques currently in use and known in the art.

[0052] The immunoconjugates or antibody fusion proteins of the invention (where the specific binding members of the invention, particularly antibodies and their fragments, are conjugated or attached to other molecules or agents) further include, but are not limited to, binding members conjugated to chemical ablatives, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents, or drugs.

[0053] The invention includes assay systems, which can be formulated in the form of test kits for quantitative analysis of, for example, the extent of the presence of TGFβ3. The system or test kit can contain a labeled component prepared by one of the radiolabeling techniques and / or enzyme techniques (coupling the label to the antibody) discussed herein and one or more additional immunochemical reagents, where at least one is a free or immobilized component to be determined or one or more of its binding partners.

[0054] Other objects and advantages will be apparent to those skilled in the art from a review of the following detailed description and the appended claims, which are made with reference to the following illustrative drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Depicted are the cloned hybridoma murine 1901 antibody parental VH and VK nucleic acids (designated as _genscript_DNA) (SEQ ID NO:9 and 11) and VH and VK amino acid sequences (designated as _genscript_AA) (SEQ ID NO:10 and 12). The XXXXXX in gray shading is the native signal peptide present in the Genscript sequence.

[0056] Figure 2 Plotted are the ELISA results for LCR1901_phage and LCR1901_soluble scFv, demonstrating that reformatting the murine mAb1901 variable domains into scFv retained binding to and fine specific recognition of TGFb3, as evaluated by ELISA.

[0057] Figure 3Depicts the amino acid sequence of grafted mouse 1901 (SEQ ID NO:13) and the amino acid sequence of LCR1901_Glv1 (SEQ ID NO:14), as well as the grafting of IGKV1-39*01 / IGHV1-69*08. The murine CDR cores are in bold; the Vernier residues are underlined; the scFv linker is in gray italics (CLUSTAL O(1.2.1) multiple sequence alignment). Homology: (*) conserved sequence; (:) conserved mutation; (.) semi-conserved mutation; () non-conserved mutation.

[0058] Figure 4 Plotted the ELISA results of the parental soluble scFv LCR1901_ScFv and LCR1901_Glv1_scFv, and demonstrated that grafting murine CDRs into a human framework retained binding to TGFb3 and fine specific recognition of TGFb3.

[0059] Figure 5 Depicts the ELISA results of the indicated constructs 1901_mouse, 1901_chimeric, 1901_Glv1 (grafted), and 13A1-mouse (13A1 is a murine anti-TGFb1 antibody) against TGFb1, TGFb2, and TGFb3. Reverting the grafted variable domains to a full huIgG1 scaffold retained TGFb3 binding and fine specific recognition of TGFb3.

[0060] Figure 6 Tabulated the CDR / linker VH and VK residues with potential sequence liabilities, their proposed corrections, and the final selected residues based on competitive binding assays. IMGT numbering was used for amino acid residue sequence numbering.

[0061] Figure 7 Provided the VH protein sequences and an overview of LCR-hu1901 CDR grafting for the VH chain. The sequences tabulated are as follows: murine 1901 VH protein sequence (SEQ ID NO:15), LCR1901_Glv1 (grafted onto the huIgHV1-69*09 framework) (SEQ ID NO:16), LCR1901 VH_1G10 (SEQ ID NO:17), LCR1901 VH_1G10m (SEQ ID NO:18), LCR1901 VH_1G10m_Glv1_03 (SEQ ID NO:19), and LCR1901 VH_1G10m_02(J) (SEQ ID NO:36). X = change relative to the original graft; X* = Conserved murine residues; # = Reverted from human to original mouse. IMGT numbering is used for amino acid residue sequence numbering. CDRs (in bold) and Vernier regions are grafted; parental IGHJ is retained.

[0062] ^^A29>T (major hu CDR1 residue); I53 (CDR2 junction) > retain parental; N66, T68, G69 (CDR2 N-glycon) > D66, S68, E69; K72 (CDR2 junction) > Q72; M108, M115 (CDR3 sulfoxide risk) > M108 (preferred), L115

[0063] ^^^S123>L123 (corrected to hu IGHJ4)

[0064] ^^^^huFR2 reverted to murine FR2: R43->K43; A45->R45; Q48->K48.

[0065] Figure 8 The VL(κ) protein sequence and an overview of LCR-hu1901 CDR grafting for the VK chain are provided. The sequences listed in the table are as follows: murine 1901 VL(κ) protein sequence (SEQ ID NO:20), LCR1901_Glv1 (grafted onto the huIgKV1-39*01 framework) (SEQ ID NO:21), LCR1901 VK_Glv1_03(F) (SEQ ID NO:22), and LCR1901VK_Glv1_05(H) (SEQ ID NO:23). X* = Conserved murine residues; # = Reverted from human to original mouse. IMGT numbering is used for amino acid residue sequence numbering.

[0066] ^ Parental CDRs (in bold) / Vernier residues grafted onto the IGKV1-39*01 framework region; L124>V124 (corrected to hu IGKJ4)

[0067] ^^FR2: K48>Q48; A49>S49; FR3: S74>D74; S83>Y83; L89>F89; P96>A96; T101>V101; F99>V99 (not murine; murine = L)

[0068] ^^^FR3: S74>D74; S83>Y83; L89>F89; P96>A96; T101>V101; F99>V99 (not murine; murine = L).

[0069] Figure 9Table provides the global germline alignment homology of the V-region variable domains (IGKV elements; IMGT)^% position identity (position similarity); average of the 3 most highly scoring functional alleles.

[0070] Figure 10 The protein sequence of LCR_1901_VH_1G10m-VK_GLv1_03(F)(1901-1C) is provided. The sequence contains the VH heavy chain amino acids LCR_1901_VH_1G10m (SEQ ID NO:18) and the VLκ light chain amino acids VK_Glv1_03(F) (SEQ ID NO:22). The CDRs are in bold. The constant region CH IgG4(S228P) sequence (SEQ ID NO:24) is shown as well as also the CK IGKC*01 (SEQ ID NO:25).

[0071] Figure 11 The protein sequence of LCR1901_VH_1G10m-VK_GLv1_05(H)(1901-1A) is provided. The sequence contains the VH heavy chain amino acids LCR_1901_VH_1G10m_03(K) (SEQ ID NO:19) and the VLκ light chain amino acids VK_Glv1_03(F) (SEQ ID NO:22). The CDRs are in bold. The constant region CH IgG4(S228P) sequence (SEQ ID NO:24) is shown as well as also the CK IGKC*01 (SEQ ID NO:25).

[0072] Figure 12 The protein sequence of LCR1901_VH_1G10m_03(K)-VK_GLv1_03(F)(1901-1D) is provided. The sequence contains the VH heavy chain amino acids LCR_1901_VH_1G10m (SEQ ID NO:18) and the VLκ light chain amino acids VK_Glv1_05(H) (SEQ ID NO:23). The CDRs are in bold. The constant region CH IgG4(S228P) sequence (SEQ ID NO:24) is shown as well as also the CK IGKC*01 (SEQ ID NO:25).

[0073] Figure 13The protein sequence of LCR1901_VH_1G10m_03(K)-VK_GLv1_05(H)(1901 - 1B) is provided. The sequence contains the VH heavy chain amino acids LCR_1901_VH_1G10m_03(K) (SEQ ID NO:19) and the VLκ light chain amino acids VK_Glv1_05(H) (SEQ ID NO:23). The CDRs are in bold. The constant region CH IgG4(S228P) sequence (SEQ ID NO:24) and also CK IGKC*01(SEQ ID NO:25) are shown.

[0074] Figure 14 Predicted stacking torsion angles are provided (based on the 568PDB structure; PAPS, bioinf.org.uk / abs / paps / ). Although the predicted VH / VK torsion angles are significantly different from the murine parent, the potency of the improved / recovered [K + F] GLv1 reverse mutant pairings is achieved. Both humanized VH and VK predictions contribute to the angle shift. The [K + F] prediction shows a slight angle shift relative to the compromised GLv1 original graft - suggesting a possible explanation for the recovered neutralizing potency in the TMLEC assay. The minimal revertant pairing of VH_1G10m with the newly designed GLv1_05(H) κ - chain maintains the predicted [K + F] angle.

[0075] Figure 15 Depicted is the neutralization of TGFb antibody pairs against TGFb3 - induced luciferase expression in TMLEC. The antibodies evaluated are (A) 1901 parent, (B) 1901 1A (LCR1901_VH_1G10m - VK_GLv1_05(H)), (C) 1901 1B (LCR1901_VH_1G10m_03(K)-VK_GLv1_05(H)), (D) 1901 1C (LCR_1901_VH_1G10m - VK_GLv1_03(F)) and (E) 1901 1D (LCR1901_VH_1G10m_03(K)-VK_GLv1_03(F)) antibodies.

[0076] Figure 16 Inhibition of TGFb3 signaling in the TMLEC assay (TGF - β3 inhibition units / μg) is provided. Compared to the parental murine 1901 antibody, the humanized LCR1901 IgG4 antibody has improved functional neutralization. The values are calculated from the Ab concentration that inhibits 50% TGF - b3 (500 pg / ml) in the TMLEC assay.

[0077] Figure 17Depicts the TGF-β specificity of humanized 1901 constructs (A and B) 1901-A, (C and D) 1901-B, (E and F) 1901-C, and (G and H) 1901D. The left set of panels (A, C, E, and G) shows higher antibody concentrations, while the right set of panels (B, D, F, and H) depicts a closer view of antibody concentrations up to 600 ng / ml.

[0078] Figure 18 Depicts the competitive ELISA binding curves of LCR1901 IgG4 variants 1A, 1B, 1C, and 1D with the parental murine 1901 antibody.

[0079] Figure 19 Depicts the comparative ELISA binding analysis of monovalent humanized 1901 candidates Fab 1B, 1C, and 1D against TGFb1 and TGFb3.

[0080] Figure 20 Provides representative Biacore single-cycle kinetic sensorgrams of monovalent humanized 1901 candidate Fab against a reference for immobilized TGFb3. TGFb3 was directly immobilized; Fab was used as the soluble analyte (0 - 2.5 nM). The single-cycle kinetics were as follows: contact: 360 s; dissociation: 500 s; flow rate: 30 μl / min.

[0081] Figure 21 provides the SEC profiles of purified humanized 1901 candidate IgG4 antibodies (A) VH_1G10m-VK_Glv1_05 (1901-1A), (B) VH_1G10m_03-VK_Glv1_05 (1901-1B), (C) VH_1G10m-VK_Glv1_03 (1901-1C), and (D) VH_1G10m_03-VK_Glv1_03 (1901-1D).

[0082] Figure 22 shows the representative DSF melting temperatures of purified humanized 1901 candidate IgG4 antibodies (A) VH_1G10m-VK_Glv1_05 (1901-1A), (B) VH_1G10m_03-VK_Glv1_05 (1901-1B), (C) VH_1G10m-VK_Glv1_03 (1901-1C), and (D) VH_1G10m_03-VK_Glv1_03 (1901-1D).

[0083] Figure 23 Lists the DSF unfolding transitions and temperatures in a table.

[0084] ^Fab transition overlaps with CH2 unfolding - no discrete Tm2.

[0085] Figure 24 Provided are (A) the relative crude protein expression yields of the humanized 1901 candidate IgG4 antibody and (B) SDS-PAGE (denaturing) of 1901 candidate antibodies 1a, 1B, 1C, and 1D. Grow 2 ml transfection cultures in triplicate; load 10 μl of supernatant.

[0086] Figure 25 Depicted is the in vitro rescue of TGFb3-mediated inhibition of anti-MSLN CAR-T target cell killing by the TGFb3-specific antibody mAb 1901-1B. Effector: Primary human T cells transfected with anti-mesothelin CAR (hP4; US 2014301993 A1). Target: H-226 human lung cancer (Meso++); E:T ratio 5:1, Added: TGFb3 (1 ng / ml), 1901_1B TGFb3-selective huIgG4, Readout: Incucyte: Cytotox Red accumulation caused by dead target cells (6 - 20 h kill slope; total red image integrated intensity per image).

[0087] Figure 26 Depicted is the in vitro rescue of TGFb3-mediated inhibition of anti-EGFR CAR-T target cell killing by the TGFb3-specific antibody mAb 1901-1B. Effector: Primary human T cells (CD3 / CD28-activated and expanded PBMCs) transfected with anti-EGFR CAR (reverse-engineered scFv panitumumab). Target: MDA-MB-231 human breast cancer (EGFR+); E:T ratio 5:1. Added: TGFb3 (1 ng / ml), 1901_1B TGFb3-selective huIgG4 (500 ng / ml). Readout: Incucyte: Time-dependent change in the number of pre-stained (CytolightRED) target cells.

[0088] Figure 27 Depicted is the 1901-1B antibody secreted by T cells relative to immobilized TGFβ3. The binding of medium supernatants from Jurkat T cell cultures expressing and secreting the TGFβ3 antibody to TGFβ3 was evaluated. Iso ctrl is an isotype control antibody. The 1901-1B antibody was added as a binding control in one sample. Supernatants from 1901-1B transfected T cells were added and also serially diluted up to 50-fold, and binding to immobilized TGFβ3 was demonstrated, which was comparable to the added 1901-1B antibody control. Detailed Description

[0089] In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art can be used. Such techniques are well explained in the literature. See, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology", Volumes I - III [Ausubel, R.M. ed. (1994)]; "Cell Biology: A Laboratory Handbook", Volumes I - III [J.E. Celis ed. (1994)]; "Current Protocols in Immunology", Volumes I - III [Coligan, J.E. ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames and S.J. Higgins eds. (1984)]; "Animal Cell Culture" [R.I. Freshney ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[0090] Accordingly, if used herein, the following terms shall have the definitions set forth below.

[0091] A. Terms

[0092] The terms "TGF-β3", "TGFb3", and "TGF-Beta3" refer to and include both the human and murine transforming growth factor β isoform 3 proteins. Exemplary full-length amino acid sequences of human and murine TGF-β3 are provided herein.

[0093] The antibody "1901-1A" or "1A" is also designated as LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H). Antibody 1901-1A comprises the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23).

[0094] The antibody "1901-1B" or "1B" is also designated as LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H). Antibody 1901-1B comprises the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23).

[0095] The antibody "1901-1C" or "1C" is also designated as LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F) (1901-1C). Antibody 1901-1C comprises the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22).

[0096] The antibody "1901-1D" or "1D" is also designated as LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F). Antibody 1901-1D comprises the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22).

[0097] The term "specific binding member" describes a member of a pair of molecules that have binding specificity for one another. The members of a specific binding pair can be of natural origin or produced synthetically, in whole or in part. One member of the pair of molecules has a region or cavity on its surface that specifically binds to the specific spatial and polar organization of the other member of the pair of molecules and is thus complementary thereto. Accordingly, the members of the pair have the property of specifically binding to one another. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application relates to antigen-antibody type reactions.

[0098] The term "antibody" describes immunoglobulins, whether natural or produced, in whole or in part, synthetically. The term also encompasses any polypeptide or protein having a binding domain that functions as an antibody binding domain or is homologous to an antibody binding domain. The term also contemplates CDR-grafted antibodies. An "antibody" is any immunoglobulin that binds specifically to an epitope and includes antibodies and fragments thereof. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the latter being described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567. The term "one or more antibodies" includes wild-type immunoglobulin (Ig) molecules, which typically comprise four full-length polypeptide chains (two heavy (H) chains and two light (L) chains), or equivalent Ig homologues thereof (e.g., camel antibodies, which contain only heavy chains); includes full-length functional mutants, variants, or derivatives thereof that retain the requisite epitope-binding characteristics of the Ig molecule, and includes bispecific, bivalent, multispecific, and dual variable domain antibodies; the immunoglobulin molecule can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Also included within the meaning of the term "antibody" are any "antibody fragments".

[0099] "Antibody fragment" means a molecule comprising at least one non-full-length polypeptide chain, including (i) a Fab fragment, which is a monovalent fragment consisting of variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CH1) domains; (ii) an F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the heavy chain portion of a Fab(Fd) fragment, which consists of VH and CH1 domains; (iv) a variable fragment (Fv), which consists of the VL and VH domains of a single arm of an antibody; (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E.S. et al., Nature 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a single-chain Fv fragment, in which the VH domain and the VL domain are linked by a peptide linker that permits the two domains to associate to form an antigen-binding site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to permit pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on the other chain and form two antigen-binding sites (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions; (xi) a multivalent antibody fragment (scFv dimer, trimer and / or tetramer (Power and Hudson, J Immunol Methods 242:193-204, 9 (2000)); (xii) a minibody, which is a bivalent molecule consisting of an scFv fused to a constant immunoglobulin domain CH3 or CH4, where the constant CH3 or CH4 domain serves as a dimerization domain (Olafsen T et al. (2004) Prot Eng Des Sel 17(4):315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9):1126-1136); and (xiii) other non-full-length portions of the heavy and / or light chain or mutants, variants or derivatives thereof, alone or in any combination.

[0100] Since antibodies can be modified in various ways, the term "antibody" shall be construed to cover any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents, and homologs of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Thus, chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Patent Nos. 4,816,397 and 4,816,567.

[0101] The "antibody binding site" is the antigen-specific binding structural portion of the antibody molecule constituted by the light or heavy chain and the variable and hypervariable regions of the light chain.

[0102] The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both the intact immunoglobulin molecule and the immunologically active portions of the immunoglobulin molecule.

[0103] Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of the immunoglobulin molecule containing the complementarity determining regions, including those portions known in the art as Fab, Fab’, F(ab’)2, and F(v), which portions are preferably used in the therapeutic methods described herein.

[0104] Antibodies can also be bispecific, where one binding domain of the antibody is a specific binding member of the present invention and the other binding domain has a different specificity, e.g., to recruit effector functions and the like. The bispecific antibodies of the present invention include those in which one binding domain of the antibody is a specific binding member of the present invention, including its fragments, and the other binding domain is a different antibody or its fragment, including the binding domain of a different anti-cancer or anti-tumor specific antibody. The other binding domain can be an antibody that recognizes or targets a specific cell type, such as in a neuron- or glia-specific antibody. In the bispecific antibodies of the present invention, one binding domain of the antibody of the present invention can be combined with other binding domains or molecules that recognize a specific cell receptor and / or modulate the cell in a specific manner, such as, for example, immunomodulators (e.g., one or more interleukins), growth regulators, or cytokines or toxins (e.g., ricin) or anti-mitotic or apoptotic agents or factors. Thus, the TGFβ-3 antibodies of the present invention can be used to direct or target agents, labels, other molecules or compounds, or antibodies in indications such as wound healing, inflammation, cancer, or tumors.

[0105] The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only antibody-binding sites capable of immunoreacting with a specific antigen. Thus, a monoclonal antibody typically exhibits a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody can also contain an antibody molecule having multiple antibody-binding sites, each of which is immunospecific for a different antigen; for example, a bispecific (chimeric) monoclonal antibody.

[0106] The term "antigen-binding domain" describes a portion of an antibody that includes regions that specifically bind and are complementary to a part or all of an antigen. In cases where the antigen is large, the antibody can bind only to a specific part of the antigen, which is called an epitope. The antigen-binding domain can be provided by one or more antibody variable domains. Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0107] The immunoconjugates or antibody fusion proteins of the present invention (wherein the antibodies, antibody molecules, or fragments thereof used in the present invention are conjugated or attached to other molecules or agents) further include, but are not limited to, such antibodies, molecules, or fragments conjugated to a chemical ablator, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, antimicrobial agent or peptide, cell wall and / or cell membrane, or a drug.

[0108] The term "one or more adjuvants" describes substances, compounds, agents or materials that can be used to improve the immune response or the stimulation of immune cells or components, and can in some cases be combined with any specific antigen in an immunological composition, a pharmaceutical composition or a vaccine composition. Adjuvants can be used to increase the amount of antibodies and effector T cells produced and to reduce the amount and frequency of injection of antigens or immunostimulants or modulators. Although some antigens are administered without an adjuvant, there are many such antigens that lack sufficient immunogenicity to stimulate a useful immune response in the absence of an effective adjuvant. Adjuvants also improve the immune response from "self-sufficient" antigens, since the immune response obtained can be increased or the amount of antigen administered can be reduced. Adjuvants can serve as a tissue depot for slow release of antigens and also as activators of the lymphoid system that non-specifically enhance the immune response (Hood et al., Immunology, 2nd edition, 1984, Benjamin / Cummings: Menlo Park, California, p. 384). In a preferred aspect, the adjuvant is physiologically and / or pharmaceutically acceptable in mammals, particularly humans. Standard adjuvants for experimental animals are Freund's adjuvants. Freund's complete adjuvant (FCA) is an emulsion containing mineral oil in saline and heat-killed mycobacteria. Freund's incomplete adjuvant (FIA) omits the mycobacteria. Both FIA and FCA induce good humoral (antibody) immunity, and FCA additionally induces a high level of cell-mediated immunity. However, due to side effects, neither FCA nor FIA is acceptable for clinical use. In particular, mineral oil is known to cause granulomas and abscesses, and Mycobacterium tuberculosis is the agent that causes tuberculosis. Previously known and used adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponins, mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Mineral salt adjuvants include, but are not limited to: aluminum hydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide, and calcium hydroxide. Preferably, the adjuvant composition further comprises lipids of a fat emulsion, the emulsion comprising about 10% (by weight) vegetable oil and about 1%-2% (by weight) phospholipid.Preferably, the adjuvant composition further optionally comprises an emulsion form having oily particles dispersed in a continuous aqueous phase, having an amount of polyol forming the emulsion from about 0.2% (by weight) to about 49% (by weight), optionally up to 15% (by weight) of a metabolizable oil in an amount forming the emulsion, and optionally up to about 5% (by weight) of an ethylene glycol ether-based surfactant in an amount stabilizing the emulsion. There have been many substances that have been tried as adjuvants, such as the lipid A portion of Gram-negative bacterial endotoxin and trehalose dimycolate of mycobacteria. Phosphatidyl lysophosphatidylcholine exhibits adjuvant activity (Arnold et al., Eur. J Immunol. 9:363-366, 1979). Some synthetic surfactants exhibit adjuvant activity, including dimethyldioctadecylammonium bromide (DDA) and certain linear polyoxypropylene polyoxyethylene (POP-POE) block polymers (Snippe et al., Int. Arch. Allergy Appl. Immunol. 65:390-398, 1981; and Hunter et al., J. Immunol. 127:1244-1250, 1981).

[0109] The term "specific" can be used to refer to a situation in which one member of a specific binding pair will not show any significant binding to molecules other than one or more of its specific binding partners. The term is also applicable, for example, in the case where an antigen-binding domain is specific for a particular epitope carried by a number of antigens, in which case the specific binding member carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope.

[0110] The term "comprise" is generally used in the sense of "include", that is, allowing the presence of one or more features or components.

[0111] The term "consisting essentially of" refers to a product having a defined number of residues, particularly a peptide sequence, which is not covalently attached to a larger product. In the case of the peptides of the present invention mentioned above, those skilled in the art should understand, however, that minor modifications to the N or C terminus of the peptide can be considered, such as chemical modifications to the termini to add protecting groups, etc., for example, amidation of the C terminus.

[0112] The term "isolated" refers to a state in which the specific binding member of the present invention or the nucleic acid encoding such a binding member will be in accordance with the present invention. The member and the nucleic acid will be free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids found with them in their natural environment or in the environment in which they are prepared (e.g., cell culture), where such preparation is carried out by recombinant DNA techniques practiced in vitro or in vivo. The member and the nucleic acid can be formulated with a diluent or adjuvant and still be considered isolated for practical purposes - for example, the member, if used to coat a microtiter plate for an immunoassay, will typically be mixed with gelatin or other carriers, or when used for diagnosis or therapy, will be mixed with a pharmaceutically acceptable carrier or diluent.

[0113] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" means microgram, "mg" means milligram, "ul" or "μl" means microliter, "ml" means milliliter, and "l" means liter.

[0114] The terms "antibody", "anti-TGFβ3 antibody", "TGFβ3 antibody", "TGF-β3 antibody", "humanized TGFβ3 antibody", "TGFb3 antibody", and any variants not specifically listed may be used interchangeably herein and, as used throughout this application and the claims, refer to a proteinaceous material comprising a single or multiple proteins and extending to those proteins having the amino acid sequence data presented in Figure 7 、 8 、10, 11, 12, and 13 and the activity profiles set forth herein and in the claims. Exemplary such TGFβ3 antibodies provided herein include antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D as provided and characterized herein. The antibodies provided herein extend to those having the activity profiles described herein and Figure 7 、 8, the amino acid sequence data presented in 10, 11, 12, and 13 (including the CDR sequences SEQ ID NO:1, 34, 35, 28 - 30 and 4, 5, 6, 31 - 33, and including the variable region heavy chain sequences SEQ ID NO:18 and 19 and 36 and the variable region light chain sequences SEQ ID NO:22 and 23) and the antibodies or proteins of the activity profiles set forth herein and in the claims, including antibody fragments. Accordingly, proteins exhibiting substantially equivalent or altered activity are also contemplated. Such modifications may be intentional, e.g., such as those obtained by site-directed mutagenesis, or may be accidental, such as those obtained by mutation in a host that is a producer of the complex or its named subunits. Additionally, the terms "antibody"; "anti-TGFβ3 antibody"; "TGFβ3 antibody"; "TGF-β3 antibody"; "humanized TGFβ3 antibody"; and the exemplary antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D are intended to include within their scope the proteins specifically enumerated herein and all substantially homologous analogs and allelic variants.

[0115] In one aspect of the invention, and specifically provided herein are antibodies that are specific for the TGF-β isoform TGF-β3. In a particular aspect, the antibodies of the invention are humanized, including where the antibody has been modified to increase its similarity to human naturally-occurring antibody variants. Such specific TGF-β3 antibodies bind and recognize the TGF-β3 isoform and do not or do not significantly bind or recognize alternative TGF-β isoforms, particularly TGF-β1 and TGF-β2. The TGF-β3-specific antibodies of the invention are exemplified by antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D.

[0116] The amino acid residues described herein are preferably in the "L" isomer form. However, any L-amino acid residue can be replaced with a residue in the "D" isomer form, provided that the polypeptide retains the desired immunoglobulin-binding functional properties. NH2 refers to the free amino group present at the amino terminus of the polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of the polypeptide. Consistent with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), the following correspondence table shows the abbreviations for amino acid residues:

[0117] Corresponding Table

[0118]

[0119]

[0120] It should be noted that all amino acid residue sequences are represented herein by formula in their left - to - right orientation in the conventional direction from the amino - terminus to the carboxyl - terminus. In addition, it should be noted that dashes at the beginning or end of an amino acid residue sequence indicate a peptide bond of a sequence that is linked to one or more additional amino acid residues. The above table is presented to correlate the three - letter and one - letter notations that may occur alternately herein.

[0121] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous DNA replication unit in vivo (i.e., is capable of replicating under its own control).

[0122] A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment can be attached so as to cause the replication of the attached segment.

[0123] A "DNA molecule" refers to a polymer form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either its single - stranded form or double - stranded helix. This term refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, this term includes double - stranded DNA found especially in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. When discussing the structure of a particular double - stranded DNA molecule, sequences can be described herein according to the usual convention of giving only the sequence along the non - transcribed strand of the DNA in the 5' to 3' direction (i.e., the strand having a sequence homologous to the mRNA).

[0124] A "replication origin" refers to those DNA sequences that participate in DNA synthesis.

[0125] A DNA "coding sequence" is a double - stranded DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed and translated into a polypeptide in vivo. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. The coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and a transcription termination sequence will usually be located at the 3' end of the coding sequence.

[0126] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, etc., which provide for the expression of a coding sequence in a host cell.

[0127] A "promoter sequence" is a DNA regulatory region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For the purposes of defining the present invention, the promoter sequence is bounded at its 3' end by the transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. The transcription start site (conveniently defined by nuclease S1 mapping) and the protein-binding domains (consensus sequences) responsible for binding RNA polymerase will be found within the promoter sequence. Eukaryotic promoters will usually but not always contain a "TATA" box and a "CAT" box. In addition to the -10 and -35 consensus sequences, prokaryotic promoters also contain the Shine-Dalgarno sequence.

[0128] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. When RNA polymerase transcribes a coding sequence into mRNA, the coding sequence is "under the control" of the transcription and translation control sequences in the cell, and the mRNA is then translated into the protein encoded by the coding sequence.

[0129] A "signal sequence" may be included before the coding sequence. This sequence encodes a signal peptide located at the N-terminus of the polypeptide, which communicates with the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the culture medium, and this signal peptide is removed by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of native proteins in prokaryotes and eukaryotes.

[0130] As used herein, the term "oligonucleotide" when referring to the probes of the present invention is defined as a molecule composed of two or more, preferably more than three ribonucleotides. Its exact size will depend on many factors, which in turn depend on the ultimate function and use of the oligonucleotide.

[0131] As used herein, the term "primer" refers to an oligonucleotide (whether naturally occurring as in purified restriction digests or synthetically produced) that is capable of serving as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (i.e., in the presence of nucleotides and an inducer such as DNA polymerase and at a suitable temperature and pH). The primer can be single-stranded or double-stranded and must be long enough to prime the synthesis of the desired extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, primer source, and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide primers typically contain 15 - 25 or more nucleotides, although it can contain fewer nucleotides.

[0132] The primers of the present invention are selected to be "substantially" complementary to different strands of a specific target DNA sequence. This means that the primers must be sufficiently complementary to hybridize to their corresponding strands. Thus, the primer sequences need not reflect the exact sequence of the template. For example, non-complementary nucleotide fragments can be attached to the 5' end of the primer, and the remainder of the primer sequence is complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed within the primer, provided that the primer sequence has sufficient complementarity to the sequence of the strand to hybridize thereto and thereby form a template for synthesis of an extension product.

[0133] As used herein, the terms "restriction endonuclease" and "restriction enzyme" refer to bacterial enzymes that each cut double-stranded DNA at or near a specific nucleotide sequence.

[0134] When such DNA has been introduced inside a cell, the cell has been "transformed" with exogenous or heterologous DNA. The transforming DNA may or may not integrate (covalently link) into the chromosomal DNA that makes up the cell genome. For example, in prokaryotes, yeast, and mammalian cells, the transforming DNA can remain on an episomal element such as a plasmid. For eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into the chromosome such that it is inherited by daughter cells via chromosomal replication. The ability of eukaryotic cells to establish cell lines or clones consisting of populations of daughter cells containing the transforming DNA demonstrates this stability. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of primary cells that is capable of stable growth in vitro for many generations.

[0135] Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90% or 95%) of the nucleotides match over a defined length of the DNA sequences. Substantially homologous sequences are identified by comparing the sequences using standard software available in sequence databases or in Southern hybridization experiments under stringent conditions defined, for example, for that particular system. Defining appropriate hybridization conditions is within the skill in the art.

[0136] It should be understood that also within the scope of the present invention are DNA sequences encoding the specific binding members (antibodies) of the present invention, said DNA sequences encoding, for example, having an amino acid sequence as provided in Figure 10 , 11 , 12 or 13 or comprising herein or Figure 7 , 8An antibody with a CDR domain region sequence listed in 0, 10, 11, 12 or 13, but degenerate thereto. "Degenerate to" means using different three-letter codons to specify a particular amino acid. As is well known in the art, the following codons can be used interchangeably to encode each particular amino acid:

[0137]

[0138]

[0139] It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have T in place of U.

[0140] In the sequence encoding Figure 7 , 8 , 10, 11, 12 and / or 13, amino acids, antibody fragments, CDR region sequences (in particular, heavy chain CDR SEQ ID NO: 1, 34, 35, 30, 28, 29 and / or light chain CDR SEQ ID NO: 4, 5, 6, 33, 31, 32), mutations can be made such that a particular codon becomes a codon encoding a different amino acid. Such mutations are typically made by making the fewest possible nucleotide changes. Such substitution mutations can be made so as to change the amino acids in the resulting protein in a non-conservative manner (e.g., by changing an amino acid from a group of amino acids belonging to a particular size or characteristic to an amino acid belonging to another group) or in a conservative manner (e.g., by changing an amino acid from a group of amino acids belonging to a particular size or characteristic to an amino acid belonging to the same group). Such conservative changes generally result in less change in the structure and function of the resulting protein. Non-conservative changes are more likely to alter the structure, activity or function of the resulting protein. The present invention includes sequences containing amino acid changes and substitutions (including conservative changes) that do not significantly alter the activity or binding characteristics of the resulting protein.

[0141] The following is an example of various amino acid groupings:

[0142] Amino Acids with Nonpolar R - Groups

[0143] Alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine

[0144] Amino Acids with Uncharged Polar R - Groups

[0145] Glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine

[0146] Amino acids with a charged polar R group (negatively charged at pH 6.0)

[0147] Aspartic acid, glutamic acid

[0148] Basic Amino Acids (Positively charged at pH 6.0)

[0149] Lysine, arginine, histidine (at pH 6.0)

[0150] Another grouping could be those amino acids with a phenyl group:

[0151] Phenylalanine, tryptophan, tyrosine

[0152] Another grouping could be based on molecular weight (i.e., the size of the R group):

[0153]

[0154]

[0155] Particularly preferred substitutions are:

[0156] - Lys substitutes for Arg and vice versa, so that the positive charge can be maintained;

[0157] - Glu substitutes for Asp and vice versa, so that the negative charge can be maintained;

[0158] - Ser substitutes for Thr, so that the free - OH can be maintained; and

[0159] - Gln substitutes for Asn, so that the free NH2 can be maintained.

[0160] Exemplary and preferred conservative amino acid substitutions include any of the following:

[0161] Glutamine (Q) substitutes for Glutamic acid (E) and vice versa; Leucine (L) substitutes for Valine (V) and vice versa; Serine (S) substitutes for Threonine (T) and vice versa; Isoleucine (I) substitutes for Valine (V) and vice versa; Lysine (K) substitutes for Glutamine (Q) and vice versa; Isoleucine (I) substitutes for Methionine (M) and vice versa; Serine (S) substitutes for Asparagine (N) and vice versa; Leucine (L) substitutes for Methionine (M) and vice versa; Lysine (L) substitutes for Glutamic acid (E) and vice versa; Alanine (A) substitutes for Serine (S) and vice versa; Tyrosine (Y) substitutes for Phenylalanine (F) and vice versa; Glutamic acid (E) substitutes for Aspartic acid (D) and vice versa; Leucine (L) substitutes for Isoleucine (I) and vice versa; Lysine (K) substitutes for Arginine (R) and vice versa.

[0162] Amino acid substitutions can also be introduced to replace amino acids with particularly preferred properties. For example, Cys can be introduced at potential sites to obtain a disulfide bridge with another Cys. His can be introduced as a particularly "catalytic" site (i.e., His can act as an acid or a base and is the most common amino acid in biochemical catalysis). Pro may be introduced due to its particularly planar structure, which induces a β-turn in the protein structure.

[0163] Two amino acid sequences are "highly homologous" or "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90% or 95% or 98% or 99%) are identical or represent conservative substitutions. The CDR regions of two antibodies are substantially homologous when one or more or one or several or one to three or one or two amino acids are replaced by similar or conservative amino acids, and one or more of the antibodies has / have the binding and activity profiles of one or more of the antibodies disclosed herein, particularly one or more of antibodies 1901-1A, 1901-1B, 1901-1C, and / or 1901-1D. Antibodies can be substantially homologous where one, two, or three amino acids, or up to three amino acids, in the CDR domain region, where one, two, three, or four or up to four amino acids, are replaced by another amino acid, and where the antibody retains the antibody binding and activity profiles.

[0164] The present disclosure provides exemplary CDR domain region amino acid substitutions. Thus, according to the present invention, antibody CDR domain sequences, particularly the 1901 murine antibody CDR domain sequences, have been modified to provide amino acid substitutions and variant CDR domain sequences therein in the antibodies herein. According to the present invention, the antibodies of the present invention comprising novel, variant, or altered CDR domain sequences from the murine 1901 antibody significantly retain TGFB-3 binding, specificity, and neutralization, and have further improved properties, including sequences having increased similarity to human naturally-occurring antibody variants. Thus, according to the present invention, there are provided TGF-β3 antibodies, particularly TGF-β3 specific antibodies, having a heavy chain variable region comprising a CDR1 sequence of SSWIH (SEQ ID NO:1), a CDR2 sequence of RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence of RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30); a CDR1 sequence of SSWIH (SEQ ID NO:1), a CDR2 sequence of RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence of RMITTQAALDY (SEQ ID NO:35); or a CDR1 sequence of GYTFSSSWIH (SEQ ID NO:28), a CDR2 sequence of WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and a CDR3 sequence of ARRMITTQAAL (SEQ ID NO:30). In a further aspect, the antibodies of the present invention may comprise a light chain variable region sequence comprising a CDR1 sequence of KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence of YASNRYT (SEQ ID NO:5), and a CDR3 sequence of QQDYSSPY (SEQ ID NO:33); a CDR1 sequence of KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence of YASNRYT (SEQ ID NO:5), and a CDR3 sequence of QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6); or a CDR1 sequence of KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence of LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence of QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0165] In one aspect, antibodies against TGF-β3 are provided, which comprise a heavy chain variable region, the heavy chain variable region comprising CDRs, the CDRs including a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence comprising the sequence RMITTQAAL (SEQ ID NO:37). In one such aspect, the antibody comprises a heavy chain variable region, the heavy chain variable region comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence comprising RMITTQAAL (SEQ ID NO:37). In one aspect, the antibody comprises a heavy chain variable region, the heavy chain variable region comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAAL (SEQ ID NO:37). In a further aspect, antibodies are provided, which further comprise light chain variable region CDRs, the light chain variable region CDRs including a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33). In yet a further aspect, antibodies are provided, which comprise light chain variable region CDRs, the light chain variable region CDRs including a CDR1 sequence KASQSVINAVA (SEQ ID NO:4) or KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence YASNRYT (SEQ ID NO:5) or LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0166] The "heterologous" region of a DNA construct is an identifiable DNA segment within a larger DNA molecule that is not found in nature associated with the larger molecule. Thus, when a heterologous region encodes a mammalian gene, the gene will typically be flanked by DNA that is not flanking the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a cDNA in which the genomic coding sequence contains introns, or a synthetic sequence having codons different from the native gene). Allelic variations or naturally occurring mutational events do not give rise to heterologous regions of DNA as defined herein.

[0167] When expressing that an expression control sequence controls and regulates the transcription and translation of a DNA sequence, the DNA sequence is "operably linked" to the expression control sequence. The term "operably linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to allow the expression of the DNA sequence under the control of the expression control sequence and the production of the desired product encoded by the DNA sequence. If the gene to be inserted into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

[0168] The term "agent" means any molecule including polypeptides, antibodies, polynucleotides, chemical compounds, and small molecules. In particular, the term agent includes compounds such as test compounds or drug candidate compounds.

[0169] The term "agonist" refers to a ligand that stimulates the binding of a receptor ligand in the broadest sense.

[0170] The term "assay" means any method for measuring a specific property of a compound. A "screening assay" means a method for characterizing or selecting compounds from a collection of compounds based on their activity.

[0171] The term "preventing" or "prevention" refers to reducing the risk that a subject will acquire or develop a disease or disorder (i.e., preventing at least one clinical symptom of the disease from developing), where the subject may have been exposed to a pathogen or be predisposed to the disease prior to the onset of the disease.

[0172] The term "prophylaxis" is related to the term 'prevention' and is subsumed within the term 'prevention', and refers to measures or procedures aimed at preventing rather than treating or curing a disease. Non-limiting examples of prophylactic measures can include administering a vaccine; administering low molecular weight heparin to hospitalized patients, for example, who are at risk of thrombosis due to immobilization; and administering an antimalarial agent such as chloroquine prior to visiting a geographical area where malaria is endemic or where the risk of contracting malaria is high.

[0173] "Therapeutically effective amount" means an amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit a biological or medical response of a subject that is sought by a doctor or other clinician. In particular, with respect to Gram-positive bacterial infections and the growth of Gram-positive bacteria, the term "effective amount" is intended to include an effective amount of a compound or agent that will cause an increase in the amount or degree of biologically significant tumor regression and / or an increase in the length of the survival period or disease-free period or remission period of the subject. The phrase "therapeutically effective amount" as used herein means an amount sufficient to prevent and preferably reduce by at least about 30 percent, more preferably at least 50 percent, most preferably at least 90 percent, a clinically significant change in the growth or amount of tumor size, or enhance the survival period or disease-free period by at least about 30 percent, more preferably at least 50 percent, most preferably at least 90 percent.

[0174] In one embodiment, the terms "treating" or "treatment" of any disease or infection refer to ameliorating the disease or infection (i.e., arresting the growth of the disease or infection agent or bacteria or reducing the manifestation, degree, or severity of at least one clinical symptom thereof). In another embodiment, "treating" or "treatment" refers to ameliorating at least one physical parameter that may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or infection physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In one other embodiment, "treating" or "treatment" involves slowing the progression of the disease or reducing the infection.

[0175] As used herein, the term "one or more fibrotic disorders" or "one or more fibrotic diseases" refers to and includes disorders or diseases characterized by excessive or persistent scar formation, particularly due to excessive or abnormal production and deposition of the extracellular matrix, and that are associated with abnormal accumulation of cells and / or fibronectin and / or collagen and / or increased fibroblast recruitment, and includes, but is not limited to, fibrosis of individual organs or tissues such as the heart, kidney, liver, joints, lung, pleural tissue, peritoneal tissue, skin, cornea, retina, musculoskeletal, and digestive tract. In certain aspects, the term fibrotic disease refers to idiopathic pulmonary fibrosis (IPF), cystic fibrosis, other diffuse parenchymal lung diseases of diverse etiologies (including iatrogenic drug-induced fibrosis, occupational and / or environmental-induced fibrosis), granulomatous diseases (sarcoidosis, hypersensitivity pneumonitis), collagen vascular diseases, alveolar proteinosis, Langerhans cell granulomatosis, lymphangioleiomyomatosis, genetic diseases (Hermansky-Pudlak syndrome, tuberous sclerosis, neurofibromatosis, metabolic storage disorders, familial interstitial lung disease), radiation-induced fibrosis, chronic obstructive pulmonary disease (COPD), scleroderma, bleomycin-induced pulmonary fibrosis, chronic asthma, silicosis, asbestos-induced pulmonary fibrosis, acute respiratory distress syndrome (ARDS), renal fibrosis, tubulointerstitial fibrosis, glomerulonephritis, focal segmental glomerulosclerosis, IgA nephropathy, hypertension, Alport syndrome, intestinal fibrosis, liver fibrosis, cirrhosis, alcohol-induced liver fibrosis, toxic / drug-induced liver fibrosis, hemochromatosis, non-alcoholic steatohepatitis (NASH), biliary injury, primary biliary cirrhosis, infection-induced liver fibrosis, virus-induced liver fibrosis, autoimmune hepatitis, corneal scarring, hypertrophic scarring, Dupuytren's disease, keloid, skin fibrosis, scleroderma of the skin, systemic sclerosis, spinal cord injury / fibrosis, myelofibrosis, vascular restenosis, atherosclerosis, arteriosclerosis, Wegener's granulomatosis, and Peyronie's disease.

[0176] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to a human and typically do not produce an allergic reaction or a similar adverse reaction (such as gastric upset, dizziness, etc.).

[0177] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" means microgram, "mg" means milligram, "ul" or "μl" means microliter, "ml" means milliliter, and "l" means liter.

[0178] B. Detailed disclosure.

[0179] The present invention provides antibodies against transforming growth factor β3 (TGF-β3) for diagnostic and therapeutic purposes. In particular, antibodies specific for TGF-β3 are provided, wherein the antibodies recognize and are capable of binding to both human and murine TGF-β3 and do not recognize or bind to other TGFβ forms, in particular the antibodies do not recognize or bind to TGF-β1 or TGF-β2. In particular, the antibodies of the present invention are humanized and are modified or contain amino acid substitutions to increase their similarity to naturally occurring human antibody variants while retaining or enhancing their TGF-β3 specificity and neutralizing activity, including to enhance their suitability, acceptability, and effectiveness in the human body and for human diseases and disorders. Exemplary such TGF-β3 antibodies are provided herein. Exemplary antibodies include antibody 1901-1A, 1901-1B, 1901-1C, and 1901-1D. Exemplary antibodies include antibodies comprising a heavy chain sequence of SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:36. Exemplary antibodies include antibodies comprising a heavy chain sequence of SEQ ID NO:18 or SEQ ID NO:19. The present invention particularly provides antibodies or active fragments thereof that recognize and neutralize TGF-β3, particularly wherein the antibodies or active fragments do not recognize or neutralize TGF-β1 or TGF-β2.

[0180] In general aspects, the present invention provides TGF-β3 antibodies against human and murine TGF-β3 that neutralize TGF-β3 activity. In one aspect, such antibodies comprise a heavy chain variable region comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35) or ARRMITTQAAL (SEQ ID NO:30); a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35); or a CDR1 sequence GYTFSSSWIH (SEQ ID NO:28), a CDR2 sequence WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and a CDR3 sequence ARRMITTQAAL (SEQ ID NO:30). In a further aspect, the antibodies of the present invention may comprise a light chain variable region sequence comprising a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33); a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6); or a CDR1 sequence KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6). In one such aspect, the present invention provides a TGF-β3 antibody comprising a heavy chain sequence SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:36. In one such aspect, the present invention provides a TGF-β3 antibody comprising a heavy chain sequence SEQ ID NO:18 or SEQ ID NO:19. Exemplary antibodies are provided, including antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D. The present invention provides TGF-β3 antibodies against human and murine TGF-β3 that do not cross-react or bind with TGF-β1 and / or TGF-β2 and specifically neutralize TGF-β3 activity.In a particular aspect, the antibodies of the invention block TGF-β3-mediated signal transduction and / or TGF-β3-mediated cellular responses or cell proliferation. In a particular aspect, the invention provides anti-TGF-β3 specific antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D. In another particular aspect, the invention provides a TGF-β3 specific antibody capable of specifically binding and neutralizing TGF-β3, which comprises a heavy chain amino acid sequence listed in SEQ ID NO:18 or 19 or 36 and. Figure 10 , 11 , 12 or 13.

[0181] In another aspect, the invention provides an antibody against TGF-β3, the antibody comprising a heavy chain variable region sequence and a light chain variable region, the heavy chain variable region sequence comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAALDY (SEQ ID NO:35) or a CDR1 sequence GYTFSSSWIH (SEQ ID NO:28), a CDR2 sequence WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and a CDR3 sequence ARRMITTQAAL (SEQ ID NO:30), the light chain variable region comprising a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQID NO:6); or a CDR1 sequence KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0182] In one aspect, the present invention provides an antibody against TGF-β3, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising CDRs, the CDRs including a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence comprising the sequence RMITTQAAL (SEQ ID NO:37). In one such aspect, the antibody comprises a heavy chain variable region, the heavy chain variable region comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence comprising RMITTQAAL (SEQ ID NO:37). In one aspect, the antibody comprises a heavy chain variable region, the heavy chain variable region comprising a CDR1 sequence SSWIH (SEQ ID NO:1), a CDR2 sequence RIYPGDGDTDYSEKFQ (SEQ ID NO:34), and a CDR3 sequence RMITTQAAL (SEQ ID NO:37). In a further aspect, an antibody is provided, the antibody further comprising light chain variable region CDRs, the light chain variable region CDRs including a CDR1 sequence KASQSVINAVA (SEQ ID NO:4), a CDR2 sequence YASNRYT (SEQ ID NO:5), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33). In yet a further aspect, an antibody is provided, the antibody comprising light chain variable region CDRs, the light chain variable region CDRs including a CDR1 sequence KASQSVINAVA (SEQ ID NO:4) or KASQSVINAVAWY (SEQ ID NO:31), a CDR2 sequence YASNRYT (SEQ ID NO:5) or LLIYYASNRYT (SEQ ID NO:32), and a CDR3 sequence QQDYSSPY (SEQ ID NO:33) or QQDYSSPYT (SEQ ID NO:6).

[0183] In another aspect of the invention, provided herein is one or more antibodies or one or more fragments thereof that bind to the same epitope of TGF-β3, such as, in particular, human TGF-β3, as one or more of the antibodies described herein. In another embodiment, provided herein is one or more antibodies or one or more antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3). In a specific embodiment, provided herein is one or more antibodies or one or more antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3) to the extent that the antibodies or antigen-binding fragments thereof described herein compete with themselves for binding to TGF-β3 (e.g., human TGF-β3).

[0184] The unique specificities and affinities of the antibodies and fragments of the present invention provide diagnostic and therapeutic uses for identifying, characterizing, and targeting conditions associated with TGF-β3 expression, activity, or activation. In particular, the antibodies of the present invention that target TGF-β3 can be used to modulate the immune response. In one aspect, the antibodies of the present invention that target TGF-β3 can be used to modulate the immune response against cancer, cancer cells, or tumor cells, as well as cancer antigens or tumor antigens. The antibodies can be applied to the therapeutic treatment or management of cancer. The antibodies can be applied to enhance the anti-cancer immune response and enhance cancer vaccines. The antibodies can be applied to enhance the therapeutic effects of one or more radiotherapy treatments, including anti-cancer and / or anti-cell effects. In a particular aspect, the antibodies of the present invention can be applied to the treatment, management, and / or prevention of cancer, including cancer recurrence and metastasis. Applicable conditions include infectious diseases, cancer, host immune responses (including in transplantation), and immune diseases or disorders (such as autoimmune diseases or inflammatory conditions). Applicable cancers include adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, anorectal cancer, anal canal cancer, appendiceal cancer, childhood cerebellar astrocytoma, basal cell carcinoma, cutaneous cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urothelial bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brainstem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, gastrointestinal cancer, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasms, mycosis fungoides, Seziary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer (gastric cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumor (endocrine pancreas), Kaposi sarcoma, kidney cancer, renalcancer), laryngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous neck cancer, oral cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disease, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorder, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, low malignant potential ovarian tumors, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family of sarcoma tumors, Kaposi sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer (gastric cancer), supratentorial primitive neuroectodermal tumors, testicular cancer, laryngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, corpus cancer, vaginal cancer, vulvar cancer, and Wilm's Tumor. In one aspect, the applicable cancers include or are selected from breast cancer, melanoma, prostate cancer, and lung cancer. In one aspect, the TGF-β3 antibody of the present invention can be applied to treat or modulate breast cancer, melanoma, prostate cancer, or lung cancer.

[0185] Evidence of TGFβ production by tumor cells and myeloid-derived suppressor cells and TGFβ immunosuppressive activity at the tumor site supports that blocking TGFβ, particularly specifically blocking TGF-β3, can enhance antigen uptake, presentation, and activation of the anti-tumor immune response, including where the anti-tumor response is T cell-mediated directed by cancer antigens or other antigens and / or mediated by therapeutic vaccines. In one aspect of the invention, one or more TGF-β3 antibodies, particularly one or more TGF-β3 neutralizing antibodies, can be combined with or administered in a composition of one or more cancer antigens and one or more adjuvants, including administration to a patient to promote a more robust initiation and activation of the adaptive anti-tumor response to enhance immunotherapy against cancer. Additional inhibitors of TGFβ activity, such as small molecules, antisense, or aptamers, can also be used to inhibit TGFβ activity, including or particularly TGF-β3.

[0186] Potent anti-tumor immunity requires modulation of multiple arms of the host immune response and targeting of pathways that contribute to tumor cell growth and survival. Combinatorial agents that modulate the immune response and block tumor growth and progression can generate anti-cancer immunity and block tumor growth to improve clinical outcomes (Vanneman, M (2012) Nature Reviews Cancer (12):237-251). Thus, in one aspect of the invention, the one or more anti-TGF-β3 antibodies can be administered alone or in combination with other treatments, therapies, or agents, simultaneously or sequentially, depending on the condition to be treated. Immunomodulators can be included in a composition having one or more TGF-β3 antibodies or administered with and / or at different times from one or more TGF-β3 antibodies to enhance immunomodulation and / or cancer therapy, including immunotherapy against cancer. Immunomodulators can be adjuvants. Suitable immunomodulators include IDO, TDO (Platten M (2012) Cancer Research 72(21):5435-40), α-galactosylceramide and its analogs (such as threitol ceramide (ThrCer) and ThrCer 6), TLR ligands (such as poly I:C (TLR3), MPL (TLR4), imiquimod (TLR7), R848 (TLR8), or CpG (TLR9)), iCOS, CTLA-4, PD1, PD1 ligand, OX40 and OX40 ligand, Lag3, GITR, GITR ligand interleukin, tumor necrosis factor (TNF), or other growth factors, colony-stimulating factors, T cell modulators (including CD8 +T cell modulators), cytokines or hormones (Mellman I (2011) Nature (480):480-489). Additional immunomodulators are small molecules, antagonist antibodies or agonist antibodies that target applicable immunomodulators, including IDO, TDO, the Toll-like receptor family or iCOS, CTLA-4, PD1, PD1 ligand, OX40 and OX40 ligand, interleukin, tumor necrosis factor (TNF), or other growth factors, colony stimulating factors, T cell modulators (including CD8 + T cell modulators), cytokines.

[0187] Additional immunomodulators (including TLR ligands such as poly I:C (TLR3), MPL (TLR4), imiquimod (TLR7), R848 (TLR8) or CpG (TLR9))) can be used in combination with a TGF-β3 specific neutralizing antibody to produce enhanced immune stimulation and provide protection against conditions (such as infectious diseases or cancer) in which an effective immune response of the immune system is desired.

[0188] One or more TGF-β3 specific antibodies can also be used as one or more immunostimulants or one or more adjuvants, in combination with antigenic materials such as, but not limited to, proteins, peptides or nucleic acids, in order to produce a protective immune response, such as a B cell and IgG antibody response against the administered antigen. One or more TGF-β3 specific antibodies can also be used as one or more immunostimulants or one or more adjuvants, in combination with antigenic materials such as, but not limited to, proteins, peptides or nucleic acids, in order to produce a protective immune response, such as a T cell or CTL response against the administered antigen.

[0189] Such an antigenic material can be and can include any material suitable for preventing or treating one / the specific disease. Specifically, with regard to cancer, examples of tumor-associated peptides and protein antigens that can be administered to induce or enhance an immune response are derived from tumor-associated genes and the encoded proteins, including MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, tyrosinase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7. For example, antigenic peptides characteristic of tumors include those listed in the published PCT application WO 00 / 20581 (PCT / US99 / 21230).

[0190] Other TGF-β related, particularly TGF-β3 related disorders, diseases or conditions that would benefit from treatment with anti-TGF-β3 antibodies include diseases characterized by extracellular matrix accumulation, diseases caused by locally activated TGF-β3 or circulating TGF-β3, conditions caused by immunosuppression due to endogenous TGF-β3 production, acute immunodeficiencies caused by severe injury, burns and illnesses such as viral or bacterial infections, multi-organ systemic illnesses caused by TGF-β3 production or overproduction, and tumors that produce TGF-β3. Non-limiting specific examples include neuronal, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocoel disorders; fibrosis; scarring; tissue damage such as that caused by radiation; and adhesions during wound healing; fibrotic skin disorders (such as scleroderma); CNS pathological scar tissue; dermal scars; keloid scar formation; and neural scar formation; fibrotic diseases of the peritoneal cavity, lung, liver and kidney, such as chronic liver fibrosis, acute liver injury, interstitial lung and kidney fibrosis, and cirrhosis; cystic fibrosis; vascular disorders, such as myocardial fibrosis; arterial injury, such as atherosclerosis and arteriosclerosis; vascular diseases; vascular pathologies; kidney diseases; systemic sclerosis; infections, such as macrophage pathogen infections and viral infections (such as hepatitis C and HIV); immunological disorders, angiogenesis disorders and inflammatory disorders and deficiencies (such as rheumatoid arthritis); ocular disorders, particularly those involving ocular fibrosis, including proliferative retinopathy, retinal detachment, and after glaucoma drainage surgery (such as the neurosensory retina, retinal pigment epithelium-choroid and vitreous of the human eye), and cataracts; osteoporosis; adult respiratory distress syndrome; restenosis after myocardial infarction, angioplasty; glomerulonephritis; diabetes related conditions, such as hyperglycemia, diabetes, diabetic kidney disease, diabetic nephropathy, diabetic neuropathy or retinopathy; and macrophage deficiency diseases.

[0191] In another specific embodiment, provided herein is a first antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3), wherein the first antibody or antigen-binding fragment thereof competes for binding in an assay comprising the steps of: (a) incubating a TGF-β3-coated ELISA plate with the first antibody or antigen-binding fragment thereof in unlabeled form; (b) adding the labeled antibody or antigen-binding fragment thereof described herein to the TGF-β3-coated ELISA plate and incubating the TGF-β3-coated ELISA plate; and (c) detecting the binding of the antibody or antigen-binding fragment thereof described herein to TGF-β3. In one aspect, the binding of antibody 1901-1A or an antigen-binding fragment thereof, antibody 1901-1B or an antigen-binding fragment thereof, antibody 1901-1C or an antigen-binding fragment thereof, or antibody 1901-1D or an antigen-binding fragment thereof is detected after incubation with the first antibody or antigen-binding fragment thereof. In one aspect, the binding of an antibody or antigen-binding fragment thereof comprising a heavy-chain variable region sequence of SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:36 is detected after incubation with the first antibody or antigen-binding fragment thereof. In one aspect, provided herein is a first antibody or an antigen-binding fragment thereof that competes with the antibodies or antigen-binding fragments thereof described herein for binding to TGF-β3 (e.g., human TGF-β3), wherein the first antibody or antigen-binding fragment thereof competes for binding in an assay comprising the steps of: (a) incubating a TGF-β3-coated ELISA plate with the first antibody or antigen-binding fragment thereof in unlabeled form; (b) adding the biotinylated antibody or antigen-binding fragment thereof described herein to the TGF-β3-coated ELISA plate and incubating the TGF-β3-coated ELISA plate; and (c) detecting the binding of the antibody or antigen-binding fragment thereof described herein to TGF-β3. In this one aspect, the labeled or biotinylated antibody or antigen-binding fragment thereof is selected from antibody 1901-1A or an antigen-binding fragment thereof, antibody 1901-1B or an antigen-binding fragment thereof, antibody 1901-1C or an antigen-binding fragment thereof, or antibody 1901-1D or an antigen-binding fragment thereof. In one aspect, in the presence of the first antibody or antigen-binding fragment thereof in unlabeled form, the binding of one or more of the antibodies or antigen-binding fragments of 1901-1A, 1901-1B, 1901-1C, or 1901-1D is reduced, particularly significantly reduced.

[0192] In another specific embodiment, provided herein is a first antibody or an antigen-binding fragment thereof that competes with the antibody or an antigen-binding fragment thereof described herein for binding to TGF-β3 (e.g., human TGF-β3), wherein the competition is exhibited as a reduction of more than 60% (e.g., 65%, 70%, 75%, 85%, 90%, 95%, or 98%, or between 60% and 65%, 65% and 70%, 70% and 75%, 75% and 80%, 80% and 85%, 85% and 95%, or 95% and 100%) in the binding of the first antibody or an antigen-binding fragment thereof to TGF-β3 (e.g., human TGF-β3). In another specific embodiment, provided herein is a first antibody or an antigen-binding fragment thereof that competes with the antibody or an antigen-binding fragment thereof described herein for binding to TGF-β3 (e.g., human TGF-β3), wherein the competition is exhibited as a reduction of more than 60% (e.g., 65%, 70%, 75%, 85%, 90%, 95%, or 98%, or between 60% and 65%, 65% and 70%, 70% and 75%, 75% and 80%, 80% and 85%, 85% and 95%, or 95% and 100%) in the binding of one or more of the antibodies or antigen-binding fragments of 1901-1A, 1901-1B, 1901-1C, or 1901-1D in the presence of and / or following the binding of the first antibody or an antigen-binding fragment thereof.

[0193] In a specific aspect, provided herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to TGF-β3 (e.g., human TGF-β3) with an antibody comprising (i) a VL domain comprising the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 that comprise the VL CDR of the antibodies provided in Figure 10 , 11 , 12, or 13; and (ii) a VH domain comprising the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 that comprise the CDR of the antibodies provided in Figure 10 , 11 , 12, or 13.

[0194] In a particular embodiment, provided herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to TGF-β3 (e.g., human TGF-β3) with an antibody comprising the VH CDR of antibody 1901-1A, 1901-1B, 1901-1C, or 1901-1D. In a particular embodiment, provided herein are antibodies that compete for specific binding to TGF-β3 (e.g., human TGF-β3) with an antibody comprising the VH and VL CDR of antibody 1901-1A, 1901-1B, 1901-1C, or 1901-1D.

[0195] In a specific aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that immunospecifically binds to an epitope that is the same as the epitope to which an antibody (e.g., 1901-1A, 1901-1B, 1901-1C, or 1901-1D) containing the amino acid sequences described herein (see, e.g., Figure 7 , 8 , 10, 11, 12, 13) specifically binds to TGF-β3 (e.g., human TGF-β3). Assays known to those of skill in the art or described herein (e.g., X-ray crystallography, ELISA assays, etc.) can be used to determine whether two antibodies bind to the same epitope.

[0196] A panel of monoclonal antibodies that recognize human and murine TGF-β3 can be screened for various properties (i.e., isotype, epitope, affinity, etc.). Of particular interest are antibodies that mimic the activity of the exemplary antibodies 1901-1A, 1901-1B, 1901-1C, or 1901-1D, have an affinity for human and murine TGF-β3, do not react with TGF-β1 or TGF-β2, and directly affect the activity of TGF-β3, particularly by neutralizing TGF-β3.

[0197] The monoclonal antibodies of the present invention can comprise a heavy chain variable region, such as those exemplified in SEQ ID NO:18 or SEQ ID NO:19, and optionally a light chain variable region. Generally, CDR regions containing amino acid sequences substantially as listed in Figure 7 and 8 , particularly the heavy chain CDRs of SEQ ID NO:1, 34, 35, 30, 28, 29, and optionally having light chain CDRs SEQ ID NO:4, 5, 33, 6, 31, 32, will be carried in a structure that allows the CDR regions to bind to TGF-β3 and particularly to human and murine TGF-β3.

[0198] "Substantially as listed in..." means that the variable region sequences and / or particularly the CDR sequences of the present invention will be the same as or highly homologous to the specified regions of Figure 7 , 8 , 10, 11, 12, and / or 13. "Highly homologous" contemplates that only few substitutions can be made in the variable region sequences and / or in the CDR sequences, preferably from 1 to 8, preferably from 1 to 5, preferably from 1 to 4, or from 1 to 3, or 1 or 2 substitutions. The term substantially as listed in... includes particularly conservative amino acid substitutions that do not substantially or significantly affect the specificity and / or activity of the antibodies of the present invention. For variable region sequences as well as for CDR region sequences, both conservative and non-conservative amino acid substitutions are contemplated herein.

[0199] Substitutions can be made in the variable region sequences outside the CDRs to preserve the CDR sequences. Thus, changes in the variable region sequences or alternative non-homologous or grafted variable region sequences can be introduced or used such that the CDR sequences are maintained and the remainder of the variable region sequences can be substituted.

[0200] Alternatively, substitutions can be made specifically in the CDRs. Exemplary CDR sequences for the antibodies of the invention, particularly for antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D, are listed and described herein (including in Figure 10 , 11 , 12, and 13 and in SEQ ID NO:1, 34, 35, 28, 29, 30, 4, 5, 6, 31, 32, and 33). Exemplary CDR sequences include substitutions in the CDR sequences, particularly where the amino acids in the CDR regions of the murine 1901 antibody have been altered or substituted. The antibodies of the invention having the substitutions as described above and considered are selected to maintain activity and specificity commensurate with the exemplary antibodies (including antibodies 1901-1A, 1901-1B, 1901-1C, and / or 1901-1D) and to have the characteristics listed herein and in the claims.

[0201] There are several recognized and known methods and ways to determine the CDRs in antibodies. The most commonly used CDR identification methods currently are Kabat (Wu TT, Kabat EA (1970) J Exp Med 132:211 - 250; Kabat EA et al. (1983) Sequence of Proteins of Immunological Interest. Bethesda: National Institute of Health), IMGT (Lefranc MP et al. (2003) Dev Comp Immunol 27:55 - 77) and Chothia (Chothia C, Lesk AM (1987) J Mol Biol 196:901 - 917; Chothia C et al. (1989) Nature 342:877 - 883; Lefranc MP et al. (2003) Dev Comp Immunol 27:55 - 77). Each of these methods has designed a unique residue numbering scheme, according to which the hypervariable region residues are numbered, and then the start and end of each of the six CDRs are determined based on certain key positions. IMGT is generally used in the studies of the present invention. Although these different ways can identify slightly offset CDR sequences, they generally provide overlapping sequences and amino acids, and can be combined to identify the amino acids that should be maintained or conserved and the amino acids that may be suitable for variation or alteration while maintaining binding.

[0202] A substantial portion of the immunoglobulin variable domain will contain at least three CDR regions together with the framework regions therebetween. Preferably, the portion will also contain at least about 50% of one or both of the first framework region and the fourth framework region, where the 50% is the C - terminal 50% of the first framework region and the N - terminal 50% of the fourth framework region. Additional residues at the N - terminus or C - terminus of the substantial portion of the variable domain can be those that are not normally associated with the naturally occurring variable domain regions. For example, the construction of the specific binding members of the present invention made by recombinant DNA techniques can result in the introduction of N - terminal or C - terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include introducing linkers to link the variable domains of the present invention to other protein sequences as provided herein and / or known to those skilled in the art, including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies) or protein tags.

[0203] Although in a preferred aspect of the present invention, comprising based on Figure 7 、 8, specific binding members of a pair of binding domains of the sequences substantially listed in 10, 11, 12 or 13 are preferred, but single binding domains based on these sequences, particularly based on the heavy and light chain CDRs, form other aspects of the present invention. Based on Figure 7 , 8 , in the case of binding domains of the sequences substantially listed in 10, 11, 12 or 13, such binding domains can be used as targeting agents against TGF-β3, since it is known that immunoglobulin VH domains are capable of binding target antigens in a specific manner.

[0204] Portions or domains of the antibodies of the present invention are contemplated and incorporated, including any portion or domain, including those that are modified or fused to a reagent, label, or other domain or fragment, wherein the portion or domain retains the characteristics of the antibody herein, including TGF-β3 specific binding, and optionally including TGF-β3 specific neutralization, as exemplified by antibodies 1901-1A, 1901-1B, 1901-1C, and 1901-1D herein. Antibodies and antibody fragments of the present invention include smaller recombinant antibody fragments (e.g., classical monovalent antibody fragments (Fab, scFv) and engineered variants (diabodies, triabodies, minibodies, and single domain antibodies), which retain the targeting specificity of the whole antibody (mAb) (for a review, see Hollinger P and Hudson PJ (2005) Nature Biotech 23(9):1126-1136). They include, for example, domain antibody (dAb) fragments, which contain a single variable domain (Ward, E.S. et al., Nature 341, 544-546 (1989)); camelid antibodies; isolated complementarity determining regions (CDRs); single-chain Fv fragments, wherein the VH domain and the VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); diabodies, which are bivalent bispecific antibodies wherein the VH domain and the VL domain are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with the complementary domains on the other chain and generating two antigen-binding sites (WO 94 / 13804; P. Holliger et al. Proc. Natl. Acad. Sci. USA 906444-6448, (1993)); linear antibodies, which contain a pair of tandem Fv segments (VH-CH1-VH-CH1), which pair with a complementary light chain polypeptide to form a pair of antigen-binding regions; multivalent antibody fragments (scFv dimers, trimers, and / or tetramers (Power and Hudson, J Immunol. Methods 242:193-204 9(2000)); and minibodies, which are composed of a constant immunoglobulin domain CH3 or CH4 (e.g., IgG1(C H 3) and IgE(C H4)) A bivalent molecule composed of a fused scFv, where the constant CH3 or CH4 domain serves as a dimerization domain (Olafsen T et al. (2004) Prot Eng Des Sel 17(4):315 - 323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9):1126 - 1136). These smaller antibodies and engineered variants or fragments can be produced more economically and can have other unique and superior properties for a range of diagnostic and therapeutic applications. For example, scFV2 - Fc can accumulate at high abundance in tumors or tissues, and microantibodies are approximately 80 kD and can be ideally used for therapy due to their higher uptake in tissues, with a faster clearance rate than intact immunoglobulins (150 kDa) or Fab’2 (110 kDa) and a better tissue - to - blood ratio. Antibody fragments can be engineered into multivalent and multispecific reagents that are linked to therapeutic payloads such as radionuclides, toxins, enzymes, liposomes, and viruses and engineered to enhance therapeutic efficacy. Recently, single - antibody domains have been engineered and selected as targeting reagents against immunologically silent cavities in enzymes, receptors, and infectious agents to date.

[0205] The specific binding member of the present invention can further comprise an antibody constant region or a part thereof. For example, a specific binding member based on Figure 7 , 8 , 10, 11, 12, or 13 can be attached at its C - terminus to the constant domain of an antibody light chain, including the human Cκ or Cλ chain, preferably the Cλ chain. Similarly, a specific binding member based on Figure 7 , 8 , 10, 11, 12, or 13 can be attached at its C - terminus to all or a part of an immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE, IgD, and IgM) and any isotype subclass (especially IgG1, IgG2b, and IgG4). IgG1 is preferred.

[0206] An antibody or any fragment thereof can be conjugated or recombinantly fused to any cytotoxin, bacterium, or other (e.g., pseudomonas) exotoxin, ricin, or diphtheria toxin. The toxin moiety used can be the whole toxin or any specific domain of the toxin. Such antibody-toxin molecules have been successfully used for targeting and treating different types of cancer, see, e.g., Pastan, Biochim Biophys Acta. Oct 24, 1997; 1333(2):C1-6; Kreitman et al., N Engl J Med. Jul 26, 2001; 345(4):241-7; Schnell et al., Leukemia. Jan 2000; 14(1):129-35; Ghetie et al., Mol Biotechnol. Jul 2001; 18(3):251-68.

[0207] Bispecific and trispecific multimers can be formed by associating different scFv molecules and have been designed as crosslinking reagents for recruiting T cells to tumors (immunotherapy), crosslinking reagents for virus retargeting (gene therapy), and as hemoglobin agglutination reagents (immunodiagnosis), see, e.g., Todorovska et al., J Immunol Methods. Feb 1, 2001; 248(1-2):47-66; Tomlinson et al., Methods Enzymol. 2000; 326:461-79; McCall et al., J Immunol. May 15, 2001; 166(10):6112-7.

[0208] Fully human antibodies can be prepared by immunizing transgenic mice that carry most of the human immunoglobulin heavy and light chains. Such mice are well known in the art, and examples of such mice are Xenomouse TM (Abgenix, Inc.) (U.S. Patent Nos. 6,075,181 and 6,150,584), HuMAb-Mouse TM (Medarex, Inc. / GenPharm) (U.S. Patent 5545806 and 5569825), TransChromo Mouse TM (Kirin) and KM Mouse TM(Medarex / Kirin). Antibodies can then be prepared, for example, by standard hybridoma techniques or by phage display. These antibodies will then contain only fully human amino acid sequences. Fully human antibodies can also be generated from libraries using phage display. Phage display can be carried out using techniques well known to the person skilled in the art and as provided, for example, in Hoogenboom et al. and Marks et al. (Hoogenboom HR and Winter G. (1992) J Mol Biol. 227(2):381-8; Marks JD et al. (1991) J Mol Biol. 222(3):581-97; and also U.S. Patents 5885793 and 5969108).

[0209] The antibodies of the present invention can be labeled with a detectable label or a functional label. Detectable labels include, but are not limited to, radioactive labels such as isotopes 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I, 111 In, 117 Lu, 211 At, 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc and 186 Re. The radioactive label can be attached to the antibody of the present invention using conventional chemical methods known in the art of antibody imaging. Labels also include fluorescent labels (e.g., fluorescein, rhodamine, Texas Red) and labels conventionally used in MRI-CT imaging in the art. They also include enzyme labels such as horseradish peroxidase, β-glucuronidase, β-galactosidase, urease. Labels further include chemical moieties such as biotin that can be detected by binding to a specific homologous detectable moiety (e.g., labeled avidin). Functional labels include substances designed to target tumor sites to cause destruction of tumor tissue. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin; and enzymes capable of converting a prodrug to an active drug at the tumor site, such as bacterial carboxypeptidase or nitroreductase.

[0210] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope), or an epitope can, for example, collectively derive from two or more non-contiguous regions of one or more polypeptides (conformational, non-linear, discontinuous or non-continuous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled to mass spectrometry (e.g., MALDI mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping).

[0211] In certain aspects, competitive binding assays can be used to determine whether an antibody is, for example, competitively blocked by another antibody in a dose-dependent manner, e.g., when two antibodies recognize the same or spatially overlapping epitopes in a competitive binding assay that can be configured in all number of different forms using a labeled antigen or a labeled antibody (such as a competitive ELISA assay), the antibody binds to the same epitope or an overlapping epitope as a reference antibody. In a particular embodiment, an antibody can be tested in a competitive binding assay with the antibodies described herein (e.g., 1901-1A, 1901-1B, 1901-1C, 1901-1D).

[0212] In addition, antibodies that recognize and bind to the same or overlapping epitopes of TGF-β3 (e.g., human TGF-β3) can be identified using conventional techniques (such as immunoassays) (e.g., by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen, i.e., a competitive binding assay). Competitive binding assays can also be used to determine whether two antibodies have similar binding specificities for an antigen or an epitope (including a specific epitope on an antigen or protein target). Competitive binding can be determined in an assay in which the immunoglobulin being tested inhibits the specific binding of another antibody to a common antigen or a target antigen. In one aspect, in competitive binding, the binding of an antibody or an antigen-binding fragment of the invention (including the TGFβ3 antibodies described herein, e.g., including 1901-1A, 1901-1B, 1901-1C or 1901-1D) is reduced in the presence of the immunoglobulin being tested, and thus competitive binding is evaluated and determined and / or confirmed.

[0213] In some aspects, competitive binding assays can be used to determine whether an antibody is, for example, competitively blocked by another antibody in a dose-dependent manner. For example, when two antibodies recognize the same or spatially overlapping epitopes in a competitive binding assay (such as a competitive ELISA assay) that can be configured in all manner of different forms using a labeled antigen or a labeled antibody, the antibody binds to an epitope that is substantially the same as or overlapping with the epitope to which a reference antibody binds. In a particular embodiment, an antibody described herein (such as including 1901-1A, 1901-1B, 1901-1C, or 1901-1D) can be used to test an antibody in a competitive binding assay. Competitive binding assays are well known in the art. Exemplary competitive binding assays are provided herein. For example, competition is demonstrated between the antibodies herein, including as described in the Examples and the Figures.

[0214] In a specific aspect, provided herein is an antibody or an antigen-binding fragment thereof that binds to an epitope that is the same as the epitope to which an antibody (such as any one of antibodies 1901-1A, 1901-1B, 1901-1C, or 1901-1D) that comprises an amino acid sequence described herein (see, for example, Figure 7 , 8 , 10, 11, 12, or 13) specifically binds to TGF-β3 (such as human TGF-β3). In a specific aspect, provided herein is an antibody or an antigen-binding fragment thereof that binds to an epitope that overlaps with the epitope to which an antibody (such as any one of antibodies 1901-1A, 1901-1B, 1901-1C, or 1901-1D) that comprises an amino acid sequence described herein (see, for example, Figure 7 , 8 , 10, 11, 12, or 13) specifically binds to TGF-β3 (such as human TGF-β3). Assays known to those of skill in the art or described herein (such as X-ray crystallography, ELISA assays, etc.) can be used to determine whether two antibodies bind to the same epitope. A Biacore assay can be used to evaluate and determine competitive binding as well as epitope binding. A Biacore can be used to determine the extent to which different antibodies interact with a single antigen or epitope to evaluate protein-protein or antibody-protein interactions and to determine binding affinity.

[0215] TGF-β3 plays an important role in controlling the immune system and is both a tumor promoter and a tumor suppressor. Studies of TGF-β3 in cancer provide a rationale for the therapeutic effect of blocking TGF-β3 signaling in human cancers. Overexpression of TGF-β ligands has been reported in most cancers, including tumors resistant to conventional chemotherapy, and high levels of TGF-β ligands in tumor tissue and / or serum are associated with early metastatic recurrence and / or poor patient outcome (Teicher, B. A. et al. (1997) In Vivo 11:463-472; Wojtowicz-Praga, S. (2003) Invest New Drugs 21:21-32; Ito, N., et al. (1995) Cancer Lett 89:45-48; Shariat, S. F., et al. (2001) Cancer 92:2985-2992; Shariat, S. F., et al. (2001) J Clin Oncol 19:2856-2864; Tsushima, H., et al. (2001) Clin Cancer Res 7:1258-1262; Rich, J. N. (2003) Front Biosci 8:e245-e260). Animal studies with pan-TGF-β antibodies have shown inhibition of tumor recurrence or metastasis in fibrosarcoma, colon cancer, and breast cancer (Terabe M et al. (2003) J Exp Med 198:1741-1752; Nam J-S et al. (2008) Cancer Res 68(10):3835-3843) and reduced acceleration of radiation-induced metastatic breast cancer (Biswas S et al. (2007) 117:1305-1313). The evidence to date strongly supports that blocking TGFβ can enhance antigen uptake, presentation, and activation of the anti-tumor immune response mediated by therapeutic vaccines. Indeed, recent studies have demonstrated that blocking TGF-β using the murine TGF-β pan-antibody ID11, which recognizes TGF-β1, TGF-β2, and TGF-β3, occurs via CD8 +T cells synergistically enhance tumor vaccines in animal models (Terabe M et al. (2009) Clin Cancer Res 15:6560-6569; Takaku S et al. (2010) Int J Cancer 126(7):1666). Radiotherapy has the potential to convert irradiated tumors into in-situ vaccines (Formanti SC et al. (2012) Int J Radiat Oncol Biol Phys 84:870-880). In a recent study, in a preclinical model of metastatic breast cancer, administration of a non-specific TGF-β neutralizing antibody (1D11) during radiotherapy increased the ability of the therapy to induce a T cell response to endogenous tumor antigens (Vanpoille-Box C et al. (2015) Cancer Res 75(11):2232-2242). Additional PD-1 blockade enhanced the effectiveness of radiotherapy with the TGF-β antibody.

[0216] TGF-β antibodies have been generated, and specific examples designated as 1D11 and its humanized counterpart GC1008 have been evaluated in animal models and early human clinical trials and are provided and disclosed in patent applications, including in WO2007076391, WO 2005097832, WO 2006086469, and 5,571,714. However, the antibody 1D11 and its humanized counterpart are general TGF-β antibodies that recognize all TGF-β forms, including TGF-β1, TGF-β2, and TGF-β3. Thus, the antibody 1D11 and its humanized counterpart do not provide specific and directed regulation of TGF-β3.

[0217] Monoclonal antibodies obtained from a species other than human (such as a mouse) by hybridoma technology can be humanized, which means that the non-human antibodies are genetically engineered to be more human-like in order to avoid HAMA when infused into humans. Methods for humanizing antibodies are well known in the art, and more common methods include complementarity determining region (CDR) grafting and veneering (also known as surface resurfacing). These methods have been widely described in the literature and patents, see for example, King “Applications and Engineering of Monoclonal Antibodies” Taylor and Francis, 1998; U.S. Patents 5,225,539, 5,530,101, 5,585,089, 5,859,205 and 6,797,492, each of which is incorporated herein by reference. Another common method is the veneering (v) technique (Daugherty et al. (1991). Nucleic Acids Res. 19(9), 2471-6; U.S. Patent 6,797,492; Padlan, E.A. (1991) Mol. Immunol. 28(4-5), 489-98; European Patent No. 519596). In which substitutions are made of surface-exposed residues in the framework regions (different from those typically found in human antibodies) in order to minimize the immunogenicity of the antibody variable domain while retaining ligand-binding properties.

[0218] Antibodies (including fragments thereof) can have certain diagnostic applications and can be used, for example, for the purpose of detecting and / or measuring conditions such as cancer, pre-cancerous lesions, conditions associated with or caused by hyperproliferative cell growth, etc.

[0219] Radioactively labeled specific binding members, especially antibodies and their fragments, can be used in in vitro diagnostic techniques and in vivo radioimaging techniques as well as in radioimmunotherapy. In the case of in vivo imaging, the specific binding members of the present invention can be conjugated with imaging agents other than one or more radioisotopes, said imaging agents including but not limited to magnetic resonance image enhancing agents, where for example, antibody molecules are loaded with a large number of paramagnetic ions via chelating groups. Examples of chelating groups include EDTA, porphyrins, polyamine crown ethers, and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanum, holmium, and fermium. In a further aspect of the present invention, radioactively labeled specific binding members, especially antibodies and their fragments, especially radioimmunoconjugates can be used in radioimmunotherapy, especially as radioactively labeled antibodies for cancer therapy. In yet a further aspect, radioactively labeled specific binding members, especially antibodies and their fragments can be used in radioimmunoguided surgical techniques, where they can identify and indicate the presence and / or location of such cells before, during, or after surgery to remove cancer cells, pre-cancerous cells, tumor cells, and hyperproliferative cells.

[0220] The immunoconjugates or antibody fusion proteins of the present invention (wherein the specific binding members of the present invention, especially antibodies and their fragments are conjugated or attached to other molecules or agents) further include but are not limited to binding members conjugated with chemical ablating agents, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents, or drugs.

[0221] Radioimmunotherapy (RAIT) has entered the clinic and has demonstrated the efficacy of using various antibody immunoconjugates. It has been evaluated in colorectal cancer 131 I-labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 (Behr TM et al. (2002) Cancer 94(4 Suppl):1373-81), and it has been evaluated in medullary thyroid cancer with 90The same antibodies against the Y label (Stein R et al. (2002) Cancer 94(1):51-61). Radioimmunotherapy using monoclonal antibodies has been evaluated and reported for non-Hodgkin lymphoma and pancreatic cancer (Goldenberg DM (2001) Crit Rev Oncol Hematol 39(1-2):195-201; Gold DV et al. (2001) Crit Rev Oncol Hematol 39(1-2)147-54). Methods of radioimmunotherapy with specific antibodies are also described in U.S. Patents 6,306,393 and 6,331,175. Radioimmunoguided surgery (RIGS) has also entered clinical practice and demonstrated efficacy and usefulness, including the use of anti-CEA antibodies and antibodies against tumor-associated antigens (Kim JC et al. (2002) Int J Cancer 97(4):542-7; Schneebaum S et al. (2001) World J Surg 25(12):1495-8; Avital S et al. (2000) Cancer 89(8):1692-8; McIntosh DG et al. (1997) Cancer Biother Radiopharm 12(4):287-94).

[0222] Adoptive cell transfer (ACT) is emerging as a new pillar of cancer therapy based on collecting and using the patient's own immune cells to treat cancer. There are several types of ACT, including TIL, TCR, and CAR (Haanen et al. (2018) J Immunother Cancer 474:449-461). One approach uses immune cells that have infiltrated the environment within and around the tumor, called tumor-infiltrating lymphocytes (TIL). Another approach to ACT involves engineering the patient's T cells to express a specific T cell receptor (TCR) to recognize tumor cell antigens (Mackall et al. (2019) Nature Medicine 25:1341-1355). Chimeric antigen receptors (CAR) use the portion of a synthetic antibody that targets a specific surface cell antigen, and CAR T cell therapy has made significant progress in clinical development. In CAR therapy, T cells are isolated from the patient and genetically engineered to produce a CAR, so that the T cells recognize and attach to the specific Antigen ... on tumor cells. CAR T cell therapy targeting the B cell antigen CD19 has proven successful in children and young adults with ALL and also in lymphoma patients.

[0223] The TGF-β3 antibody or fragment thereof of the present invention can further be used to construct a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a co-stimulatory signaling region, and a signaling domain of the TGF-β3 antibody. In these and other embodiments, the antigen-binding domain can be a Fab or scFv of the TGF-β3 antibody. In yet another other embodiment, TGF-β3 is present in the tumor microenvironment or on cells in the tumor microenvironment. In still other embodiments, the co-stimulatory signaling region comprises the intracellular domain of a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.

[0224] T cells that are modified to express a chimeric antigen receptor (CAR) and administered alone are inhibited in the hostile tumor microenvironment. By way of non-limiting example, further modification of these cells to express a secreted scFv (e.g., PD-1, PDL-1, or CTLA-4) (referred to as an "Armored" CAR) improves anti-tumor function due to its ability to modulate the tumor microenvironment and resist inhibitory factors (e.g., as described in U.S. Patent 10,124,023 and Brentjens et al. (2018) Nat Biotechnol 36(9):847-856). In one such embodiment, an Armored CAR expressing one or more TGF-β3 antibodies (including one or more of its scfvs) of the present invention is contemplated, wherein the TGF-β3 antibody enhances CAR cell activity and blocks immunosuppression, including being inhibited by endogenous TGF-β. In another embodiment, the TGF-β3 antibody of the present invention can be used in adoptive cell therapy (ACT), wherein the TGF-β3 antibody or fragment thereof will be genetically introduced into T cells (preferably but not limited to tumor-infiltrating lymphocytes (TILs)) isolated from a cancer patient, and then, such T cells are expanded and delivered back to the patient, whereby the T cells will target the tumor and express and secrete the TGF-β antibody or fragment thereof in the local tumor microenvironment to counter the immunosuppressive environment there. In another embodiment, an exogenous TGF-β3 antibody or fragment thereof can be added to the expanded T cell population when delivered back into the patient for ACT. By way of non-limiting example, an exogenous TGF-β3 antibody or fragment thereof can be added to the expanded T cell population as described in WO 2019 / 086711 before being delivered back into the patient for ACT.

[0225] The TGF-β3 antibody of the present invention can also be used in adoptive cell therapy (ACT), wherein the TGF-β3 antibody or its fragment will be genetically introduced into T cells isolated from a cancer patient, and then, such T cells are expanded and delivered back to the patient, whereby the T cells will target the tumor and express the TGF-β3 antibody or its fragment in the local tumor microenvironment to counter the immunosuppressive environment there. Preferably, the T cells used will be tumor infiltrating lymphocytes (TIL).

[0226] One of ordinary skill in the art can use in vivo cancer animal models or animal xenograft studies to further or alternatively screen, evaluate, and / or validate the specific binding members and antibodies or their fragments of the present invention, including further evaluating TGF-β3 regulation and inhibition in vivo and inhibition of tumor progression, recurrence, metastasis, or the immune response to tumor cells or the response to an antigen or vaccine (including tumor or cancer antigen or vaccine). Such animal models include, but are not limited to, models of immune response, immunomodulation, vaccination, cancer, and cancer metastasis. Models of cancers whose recurrence or metastasis is associated with elevated TGF-β3 levels are particularly susceptible to and targeted by the antibodies of the present invention. Such cancers include melanoma, breast cancer, lung cancer, and prostate cancer. Exemplary and suitable models are known to those of ordinary skill in the art and are readily available, and include those cited and / or described herein and those known in the art. For example, the antibodies or their fragments of the present invention can be evaluated in breast cancer models, including the tumorigenicity of human breast cancer cells in athymic mice (Arteaga CL et al. (1993) Cell Growth Diff 4:193-201) or in Neu-induced mammary tumors (Muraoka-Cok RS et al. (2004) Cancer Res 64:2002-2011), or the metastasis of transgenic mammary tumors can be evaluated (Siegel PM et al. (2003) Proc Natl Acad Sci USA 100:8430-8435). Additionally, for example, a method similar to that reported by Takaku et al. (Takaku S et al. (2010) Int J Cancer 126(7):1666) can be used to examine the anti-tumor effect of TGF-β3 antibodies in the prevention of CT26 colon cancer tumors injected in syngeneic mice with a whole cell vaccine.

[0227] The antibodies of the present invention can be administered to a patient in need of treatment via any suitable route, including by injection (including intraperitoneal, intramuscular, subcutaneous, intravenous) into the bloodstream or CSF or directly into the tumor site or by intratumoral administration or intratumoral injection. The exact dose will depend on many factors, including whether the antibody is for diagnostic or therapeutic use, the size and location of the tumor, the exact nature of the antibody (whether whole antibody, fragment, diabody, etc.), and the nature of the detectable or functional label attached to the antibody. In the case of therapies using radionuclides, a suitable single dose can be from about 45 mCi / m 2 to a maximum of about 250 mCi / m 2 . Preferred doses are in the range of 15 to 40 mCi, and further preferred doses are in the range of 20 to 30 mCi or 10 to 30 mCi. Such therapies may require bone marrow or stem cell replacement. Typical antibody doses for tumor imaging or tumor treatment will be in the range of from 0.5 to 40 mg, preferably from 1 to 4 mg, of antibody in F(ab’)2 form. Naked antibodies are preferably administered at a dose of 20 to 1000 mg protein / dose, or 20 to 500 mg protein / dose, or 20 to 100 mg protein / dose. This is the dose for a single treatment in an adult patient and can be adjusted proportionally for children and infants and also proportionally for other antibody forms such as in relation to molecular weight. Depending on the physician's determination, treatment can be repeated at intervals of once a day, twice a week, once a week, or once a month.

[0228] Pharmaceutical Compositions and Therapeutic Compositions

[0229] The antibodies and fragments of the present invention will generally be administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the specific binding member. Thus, in addition to the active ingredient, the pharmaceutical compositions according to the present invention and for use according to the present invention may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection (e.g., intravenous), or by deposition at the tumor site.

[0230] The binding members and antibodies of the present invention and in one particular embodiment having Figure 7 , 8 , 10, 11, 12 the sequences represented, or fragments thereof, and single-chain, recombinant, or synthetic antibodies derived therefrom, and particularly comprising Figure 7 and Figure 8 in or Figure 10 , 11An antibody with a heavy chain CDR region sequence and a light chain CDR region sequence depicted in SEQ ID NO: 1, 34, 35, 28, 29, 30, 37, 4, 5, 6, 31, 32, 33 or 12, 13 can be formulated in a pharmaceutical composition that includes a suitable vehicle, carrier or diluent, or includes an adjuvant and / or immunomodulator for administration in instances where therapy is appropriate, such as for treating cancer or stimulating or enhancing an immune response, including an immune response against cancer. Such pharmaceutical compositions may also include means for modulating the half-life of the binding member, antibody or fragment by methods known in the art, such as polyethylene glycolylation. Such pharmaceutical compositions may further include additional antibodies or therapeutic agents.

[0231] The compositions of the invention can be administered alone or in combination with other treatments, therapies or agents (simultaneously or sequentially, depending on the condition to be treated). Additionally, the invention contemplates and includes compositions that contain the binding members described herein (particularly antibodies or fragments thereof) and other agents or therapies, such as anti-cancer agents or therapies, anti-mitotic agents, apoptotic agents or antibodies, or immunomodulators, or small molecule inhibitors against immunomodulators. More generally, these anti-cancer agents can be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g., anti-mitotic agents), inhibitors or signal transduction inhibitors. Other treatments or therapies can include administration of a suitable dose of a pain relieving drug, such as a non-steroidal anti-inflammatory drug (e.g., aspirin, paracetamol, ibuprofen or ketoprofen) or an opiate (such as morphine or an anti-emetic). Additionally, the compositions can be administered with an immunomodulator, such as α-galactosylceramide, interleukin, tumor necrosis factor (TNF), or other growth factors, colony stimulating factors, cytokines or hormones that stimulate an immune response and the reduction or elimination of cancer cells or tumors. The compositions can be administered with an immunomodulator, such as an adjuvant. The compositions can also be administered with or can include a combination with other anti-TGFβ antibodies, other immunomodulatory antibodies or other anti-tumor antigen antibodies. In one aspect, the compositions are administered in combination with another antibody, particularly an anti-tumor antigen antibody.

[0232] The invention also includes antibodies and fragments thereof covalently attached or otherwise associated with other molecules or agents. These other molecules or agents include, but are not limited to, molecules with different recognition characteristics (including antibodies or antibody fragments), toxins, ligands, and chemotherapeutic agents. In an additional aspect, the antibodies or fragments of the invention can be used to target or direct therapeutic molecules or other agents, such as targeting a molecule or agent to TGFβ-expressing cells or TGFβ-responsive cells, particularly TGF-β3-expressing or responsive cells, such as cells at a wound site, tumor site, inflammatory region or cancerous lesion.

[0233] The pharmaceutical composition for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions generally contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oils. Saline solutions, glucose or other sugar solutions or diols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[0234] For intravenous injection or injection at the site of disease, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the relevant art can well prepare suitable solutions using, for example, isotonic media such as sodium chloride injection, Ringer's Injection, lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as required.

[0235] The composition can be administered alone or in combination with other treatments, therapies or agents (simultaneously or sequentially, depending on the condition to be treated). Additionally, the present invention contemplates and includes compositions comprising the binding members described herein (particularly antibodies or fragments thereof) and other agents or therapies such as anti-cancer agents or therapies, hormones, anti-mitotic agents, anti-apoptotic agents, antibodies, or immunomodulators. More generally, these anti-cancer agents can be, but are not limited to, tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational regulators, cell growth or division inhibitors (e.g., anti-mitotic agents), or signal transduction inhibitors. Other treatments or therapies can include the administration of a suitable dose of pain-relieving drugs such as non-steroidal anti-inflammatory drugs (e.g., aspirin, paracetamol, ibuprofen or ketoprofen) or opiates (such as morphine or anti-emetics). The composition can be administered in combination (sequentially (i.e., before or after) or simultaneously) with tyrosine kinase inhibitors (including but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine and / or other chemotherapeutic agents. Thus, these agents can be specific anti-cancer agents or immunocyte response regulators, or can be more general anti-cancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine or lomustine. Additionally, the composition can be administered with: hormones (such as dexamethasone), immunomodulators such as interleukins, tumor necrosis factor (TNF), or other growth factors, colony stimulating factors, cytokines, agonists or antagonist antibodies to immunomodulators that stimulate, enhance or inhibit the immune response and the reduction or elimination of cancer cells or tumors. The composition can also be administered with or can comprise a combination with other anti-tumor antigen antibodies.

[0236] Additionally, the present invention contemplates and includes therapeutic compositions for use in combination of one or more antibodies or fragments with conventional radiotherapy.

[0237] The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of the present invention. The subject therapeutic compositions comprise a mixture of pharmaceutically acceptable excipients (carriers) and one or more of the specific binding members or antibodies, polypeptide analogs thereof, or fragments thereof as active ingredients as described herein. In one embodiment, the composition comprises an antigen capable of modulating the specific binding of the binding member / antibody of the present invention to target cells. In one embodiment, the composition comprises an antigen or a vaccine formulation, particularly a tumor antigen or a cancer vaccine.

[0238] The preparation of therapeutic compositions containing polypeptides, analogs, or active fragments as active ingredients is well known in the art. Typically, such compositions can be prepared as injectable solutions or suspensions in liquid form. However, solid forms suitable for dissolution or suspension in a liquid prior to injection can also be prepared. The formulations can also be emulsions. Generally, the active therapeutic ingredient is mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Additionally, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the effectiveness of the active ingredient.

[0239] The polypeptides, analogs, or active fragments can be formulated into therapeutic compositions in the form of neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), and the acid addition salts are formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed from free carboxyl groups can also be derived from inorganic bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0240] Alternative paradigms for cancer therapy using intratumoral immunomodulation and intratumoral immunity have been described and reviewed (Marabelle A et al. (2014) Clin Cancer Res 20(7):1747-1756). Such paradigms are particularly effective for drug therapies (including immune-stimulatory monoclonal antibodies) designed to interact with molecules that play a role in the activation of immune cells to reverse cancer-induced immune tolerance and promote an anti-tumor immune response (Marabelle A et al. (2014) Clin Cancer Res 20(7):1747-1756; Mellman I et al. (2011) Nature 480:480-489). Application of this paradigm to the antibodies and active fragments of the present invention is an aspect of the present invention. Delivery of immune-stimulatory monoclonal antibodies directly into tumors to generate or promote a systemic anti-tumor immune response (including a more potent anti-tumor response with less autoimmune toxicity or other side effects) and a need for less drug as compared to systemically administered drugs or antibodies, delivery of antibodies in an adjuvant around established tumors (delivery of anti-CTLA-4 Ab in a water-in-oil emulsion adjuvant (Montadine ISA51) around colon cancer tumors) eradicated local tumors and prevented significant tumor development (Fransen MF et al. (2013) Cancer Res 19:5381-5389).

[0241] The composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, the ability of the subject's immune system to utilize the active ingredient, and the degree of the desired peptide / MHC or tumor antigen binding ability. The precise amount of active ingredient required for administration depends on the judgment of the practitioner and is specific to each individual. Suitable regimens for initial and subsequent administrations are also variable and may include an initial administration followed by repeated doses by subsequent injection or other administration at intervals of one or more hours. Alternatively, continuous intravenous infusion sufficient to maintain an appropriate and adequate concentration in the blood or at the desired site of treatment is contemplated.

[0242] Diagnostic Assays

[0243] The present invention also relates to a variety of diagnostic applications, including methods for detecting the expression or presence of elevated TGF-β3, TGF-β3-mediated cancer, or more generally cancer, by assessing the presence or amount of TGF-β3-responsive cells by reference to their ability to be recognized by one or more specific binding members of the present invention. Peptide complexes can be identified, targeted, labeled, and / or quantified on cells including immune cells and / or tumor cells.

[0244] Diagnostic uses of the specific binding members of the invention, particularly antibodies and fragments thereof, include in vitro and in vivo uses that are well known and standard to those skilled in the art and are based on the present specification. Diagnostic assays and kits for in vitro assessment and evaluation of tumor and cancer status, as well as tumor response or immune response, can be used to diagnose, evaluate, and monitor patient samples, including those known to have or suspected of having cancer, pre-cancerous conditions, conditions related to hyperproliferative cell growth, or from tumor samples. Assessment and evaluation of cancer, tumor, and metastatic disease status can also be used to determine a patient's suitability for a drug clinical trial or for administration of a particular chemotherapeutic agent or specific binding member of the invention, particularly an antibody (including combinations thereof), relative to different agents or binding members. This type of diagnostic monitoring and assessment is already in practice using antibodies against the HER2 protein in breast cancer (Hercep Test, Dako), where the assay is also used to evaluate patients for antibody therapy using Herceptin. In vivo uses include tumor imaging or assessment of an individual's cancer status, including radiological imaging.

[0245] Preferably, the antibodies for the diagnostic methods of the invention are murine antibodies, human antibodies, humanized antibodies, or recombinant antibodies. Preferably, the antibodies are single-chain antibodies or domain antibodies. Additionally, the antibody molecules used herein can be in the form of Fab, Fab’, F(ab’)2, or F(v) portions of the whole antibody molecule, particularly Fab.

[0246] The presence of TGF-β3 or TGF-β3-responsive cells or TGF-β3-responsive genes or proteins in cells can be determined by conventional in vitro or in vivo immunological procedures applicable to such determinations. Many useful procedures are known. The procedures and their applications are familiar to those skilled in the art and can thus be used within the scope of the present invention.

[0247] In another embodiment of the invention, commercial test kits suitable for use by medical professionals can be prepared to determine the presence or absence of aberrant expression (including but not limited to amplified TGF-β3) in suspected target cells. Depending on the test techniques discussed above, one class of such kits will contain at least a labeled or its binding partner, such as an antibody specific thereto, and of course instructions, depending on the method selected. The kits can also contain peripheral reagents, such as buffers, stabilizers, etc.

[0248] Accordingly, test kits can be prepared for demonstrating the presence or elevated levels of TGF-β3 or TGF-β3-responsive elements or proteins, said test kits comprising:

[0249] (a) A predetermined amount of at least one labeled immunoreactive component obtained by directly or indirectly attaching a specific binding member of the present invention or its specific binding partner to a detectable label;

[0250] (b) Other reagents; and

[0251] (c) Instructions for using the kit.

[0252] A test kit can be prepared for demonstrating the presence of TGF-β3-mediated cancers (especially selected from breast cancer, lung cancer, liver cancer, prostate cancer, bladder cancer), said test kit comprising:

[0253] (a) A predetermined amount of at least one labeled immunoreactive component obtained by directly or indirectly attaching a specific binding member of the present invention or its specific binding partner to a detectable label;

[0254] (b) Other reagents; and

[0255] (c) Instructions for using the kit.

[0256] According to the above, an assay system can be prepared for screening potential drugs that effectively regulate the presence or activity of TGF-β3 and / or the activity or binding of the antibodies of the present invention. An antigenic peptide or a binding member or antibody can be introduced into the test system, and an expected drug can also be introduced into the resulting cell culture, and the subsequent culture is examined to observe any changes in cell activity, antibody binding, or the amount and degree of TGF-β3 due to the addition of the individual expected drug or due to the effect of the addition amount of one or more known agents.

[0257] Nucleic Acids

[0258] The present invention further provides an isolated nucleic acid encoding the specific binding member of the present invention. The nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid that encodes the polypeptide of the present invention as defined above, including the polypeptides listed in Figure 7 、 8 、10, 11, 12 and / or 13, including the polypeptides of SEQ ID NO:18, 19, 36, 22 and / or 23; or capable of encoding its CDR regions, including SEQ ID NO:1, 34, 35, 30, 28, 29, 4, 5, 6, 33, 31 and / or 32.

[0259] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes, which comprise at least one of the above polynucleotides. The present invention also provides recombinant host cells, which comprise one or more of the above constructs. Nucleic acids encoding any of the specific binding members provided herein themselves form an aspect of the present invention, as do methods of producing specific binding members, which methods include expression from their encoding nucleic acids. Expression can be conveniently achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. After production by expression, the specific binding member can be isolated and / or purified using any suitable technique and then used as appropriate.

[0260] Specific binding members and encoding nucleic acid molecules and vectors according to the present invention can be provided, which are isolated and / or purified, for example, in substantially pure or homogeneous form from their natural environment or, in the case of nucleic acids, in a form free or substantially free of nucleic acids or genetic material of origin other than the sequence encoding a polypeptide having the desired function. Nucleic acids according to the present invention can comprise DNA or RNA and can be wholly or partially synthetic.

[0261] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Suitable vectors can be selected or constructed, which contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Optionally, the vector can be a plasmid (viral, such as phage) or a phagemid.

[0262] Accordingly, a further aspect of the present invention provides a host cell containing a nucleic acid as disclosed herein. A still further aspect provides a method, which includes introducing such a nucleic acid into a host cell. The introduction can employ any available technique. After introduction, expression from the nucleic acid can be induced or permitted, for example, by culturing the host cell under conditions that express the gene. The present invention also provides methods, which include using the constructs as stated above in an expression system in order to express a specific binding member or polypeptide as above.

[0263] Another feature of the present invention is the expression of the DNA sequences disclosed herein. As is well known in the art, a DNA sequence can be expressed by operably linking the DNA sequence to an expression control sequence in a suitable expression vector and using the expression vector to transform a suitable unicellular host. A wide variety of host / expression vector combinations can be used in expressing the DNA sequences of the present invention. Useful expression vectors, for example, can consist of segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, such as the E. coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNA, such as numerous derivatives of phage λ, such as NM989, and other phage DNA, such as M13 and filamentous single-stranded phage DNA; yeast plasmids, such as the 2μ plasmid or its derivatives; vectors useful for eukaryotic cells, such as vectors useful for insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to use phage DNA or other expression control sequences; and so on.

[0264] Any of a variety of expression control sequences--sequences that control the expression of a DNA sequence operably linked thereto--can be used in these vectors to express the DNA sequences of the present invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma, or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase (e.g., Pho5), the promoter of yeast α-mating factor, and other sequences known to control the gene expression of prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.

[0265] A wide variety of unicellular host cells can also be used to express the DNA sequences of the present invention. These hosts can include well-known eukaryotic and prokaryotic hosts in tissue culture, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces; fungi, such as yeast; and animal cells, such as CHO, YB / 20, NSO, SP2 / 0, Rl.l, B-W and L-M cells, African green monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (e.g., Sf9), and human and plant cells.

[0266] It should be understood that not all vectors, expression control sequences, and hosts will function equally well in expressing the DNA sequences of the present invention. In the case of the same expression system, not all hosts will function equally well. However, without departing from the scope of the present invention, those skilled in the art will be able to select appropriate vectors, expression control sequences, and hosts without undue experimentation to achieve the desired expression.

[0267] As mentioned above, the DNA sequence encoding the specific binding member can be synthetically prepared rather than cloned. The DNA sequence can be designed with appropriate codons for the amino acid sequence of the specific binding member. Generally, if the sequence is to be used for expression, one will select preferred codons for the desired host. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. The synthetic DNA sequence allows for the convenient construction of genes that will express analogs or "mutant proteins" of the specific binding member. Alternatively, the DNA encoding the mutant protein can be made by site-directed mutagenesis of the native specific binding member gene or cDNA, and the mutant protein can be directly prepared using conventional polypeptide synthesis.

[0268] The present invention can be better understood by reference to the following non-limiting examples, which are provided as illustrations of the present invention. The following examples are presented to more fully illustrate the preferred embodiments of the present invention, but should in no way be construed as limiting the broad scope of the present invention.

[0269] Example 1

[0270] Mouse TGFβ-3 antibody

[0271] Mature mouse and human TGF-β3 share 100% homology at the protein level, which makes it extremely difficult to generate mouse antibodies against both human and mouse proteins due to immune tolerance. The mouse and human TGF-β3 amino acid sequences are as follows:

[0272] Mouse

[0273] NCBI Reference Sequence: NP_033394.2 (SEQ ID NO:7)

[0274]

[0275] Human

[0276] UniProtKB / Swiss-Prot: P10600.1 (SEQ ID NO:8)

[0277]

[0278] Previously, a self-vaccination protocol was used to successfully generate TGF-β3 antibodies in mice. A set of murine anti-TGF-β3 antibodies was isolated: MTGF-β3-9 / 8 (also designated as MTGF-β3-9), MTGF-β3-1203 / 11 (also designated as MTGF-β3-12), MTGF-β3-1679 / 2 (also designated as MTGF-β3-16), MTGF-β3-1719 / 13 (also designated as MTGF-β3-17), MTGF-β3-1901 / 16 (also designated as MTGF-β3-19). All of the antibodies are IgG class antibodies; MTGF-β3-19 is an IgG1 antibody, antibodies MTGF-β3-9, MTGF-β3-16, and MTGF-β3-17 are IgG2a antibodies, and antibody MTGF-β3-12 is IgG2b. The antibodies, their specificity for TGF-β3 binding and neutralization, as well as their sequence descriptions are provided in PCT / US2016 / 036965, published as WO 2016 / 201282 and USSN 15 / 580,746, published as US 2018-0148501 A1, which patents are incorporated herein by reference.

[0279] The murine monoclonal antibody MTGF-β3-1901 / 16 (also designated as MTGF-β3-19) was selected and is specifically designated herein as antibody 1901 for further development as well as chimerization and humanization, as described in the following examples. The TGFβ3 antibody 1901 (MTGF-β3-1901 / 16) has a heavy chain variable region sequence and a light chain variable region, the heavy chain variable region sequence comprising the CDR1 sequence SSWIH (SEQ ID NO:1), the CDR2 sequence RIYPGDGDTNYTGKFKG (SEQ ID NO:2), and the CDR3 sequence RMITTQAAMDY (SEQ ID NO:3), and the light chain variable region comprising the CDR1 sequence KASQSVINAVA (SEQ ID NO:4), the CDR2 sequence YASNRYT (SEQ ID NO:5), and the CDR3 sequence QQDYSSPT (SEQ ID NO:6).

[0280] Example 2

[0281] TGFβ signaling in the tumor microenvironment, particularly TGF-β3 signaling, is involved in the negative regulation of effective immune responses to cancer through multiple mechanisms. TGFβ blockade can overcome this immunosuppression, especially when combined with other checkpoint-targeted drugs. This is an emerging and promising therapeutic modality in immuno-oncology. A series of murine monoclonal antibodies against TGFβ were previously generated by immunizing mice with recombinant murine TGF protein isoforms TGFβ1, TGFβ2, and TGFβ3. Murine monoclonal antibody clone 1901 was selected for further development based on its high selective specificity for both human and murine TGFβ3 but not for TGFβ1 and TGFβ2 and its ability to block TGFβ3 signaling in in vitro and in vivo potency assays (US 2018 / 0148501 A1).

[0282] Initiate a project to generate a humanized form of murine TGFb3 antibody 1901 that has similar or improved functional in vitro potency to the original murine mAb. Humanized antibodies may be more acceptable and useful in therapeutic modalities for humans. Using CDR / FR grafting and sequence mutagenesis techniques driven by functional potency assays, we engineered a series of humanized forms based on murine mAb1901. Briefly, the antibody was first expressed as an scFv in E. coli for initial screening of binding to TGFb3, and then as a full-length human IgG1 or IgG4 antibody in a transient mammalian cell system. Purified antibodies were screened by ELISA for their ability to block the binding of murine 1901 to TGFb3 and their in vitro potency (TMLEC assay) to inhibit TGFb-induced signaling in a reporter cell line. Functional potency assays were performed independently in two laboratories. From this screening, four IgG4 antibodies with significantly higher potency to functionally inhibit TGFb3 signaling in TMLEC were selected: LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F)(1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H)(1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F)(1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H)(1901-1B) for extended in vitro characterization, including binding specificity, binding kinetics, structure, and physicochemical characteristics such as small-scale expression yield, SEC profile, and thermal stability. Notably, these antibodies are more potent than the original murine 1901 antibody and also have different CDR sequences compared to the murine 1901 antibody. Thus, new antibodies have been generated that are humanized and have novel and unique heavy and light chain CDRs and that are specific for TGF-β3. Based on the characterization profiles, antibody LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H)(LCR1901-1B) and antibody LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F)(LCR1901-1C) were selected for further development.

[0283] The following provides the detailed materials and methods used in this example and the following examples:

[0284] Materials and Methods

[0285] Clone LCR1901 as a scFv into the pCHV101 phage display vector

[0286] The VH and VK chains of the parental murine LCR1901 anti-TGFb3 mAb and its humanized CDR-grafted GLv1 variant were designed and synthesized as a single open reading frame scFv and cloned into the phagemid vector (pCHV101).

[0287] Digest the scFv host vector as follows to release the scFv insert:

[0288]

[0289] Digest the pCHV101 phagemid vector similarly:

[0290]

[0291]

[0292] Incubate the digest at 37 °C for 1 h and electrophorese on a 1% agarose gel. Gel extract the insert band (approx. 770 bp) and the linearized pCHV101 vector (approx. 4830 bp) using the Zymoclean Gel DNA Recovery Kit (Zymo Research, catalog number D4002) into 20 μl of warm (preheated at 65 °C) 0.2x kit elution buffer (EB) diluted in PCR-grade H2O.

[0293] Perform the ligation using the following, with an insert:vector ratio of 3:1:

[0294]

[0295] Incubate the ligation at room temperature for 20 minutes, then transfer 5 μl of the ligation mixture into 50 μl of E.Cloni competent cells. Incubate the cell / DNA mixture on ice for 10 min, then add 75 μl of 2xTYG and streak 125 μl of the diluted cells on a solid 2xTYAG selective agar plate. For the agar plate, the name T refers to the added tryptone, Y refers to the added yeast extract, G refers to the added glucose, and A refers to the added ampicillin antibiotic, which uses standard technical formulations. Incubate the plate overnight at 37 °C.

[0296] Pick colonies into 3 ml of liquid 2xTYAG medium and incubate at 37 °C. Prepare the cloned vector DNA using the Vacuum PureYield plasmid miniprep system Quickprotocol (Promega, catalog number FB093), and sequence using the primers pCHV101_SeqFOR1 (5'-CTGAAAGGAAGGATATAGAATGTGC-3') (SEQ ID NO:38) and PD1-2 (5'-GTCGTCTTTCCAGACGTTAG-3') (SEQ ID NO:39).

[0297] Phage / scFv ELISA

[0298] Electroporate the miniprep clones containing the correct scFv sequence into Escherichia coli TG1 cells (Lucigen, catalog number 60502-1), plate on selective 2xTYAG plates, and incubate at 30 °C for 18 - 20 h.

[0299] Pick ampicillin-resistant colonies into 96-well U-bottom plates (Greiner bio-one, catalog number 650201) filled with 200 μl of 2xTYAG and incubate overnight at 30 °C, 750 rpm, 70% humidity. Then use the cultures for scFv and phage ELISA according to the following protocol:

[0300] Expression of soluble scFv

[0301] From the overnight plates, transfer 4 μl of culture / well to 96-well U-bottom plates containing 170 μl / well of TB medium supplemented with 0.1% glucose and 100 μg / ml ampicillin. Grow the bacteria at 30 °C, 750 rpm, 70% humidity for 4 h. Induce scFv expression by adding 20 μl of IPTG (diluted to 1 mM in TB medium) to obtain a final concentration of 100 μM. Carry out the expression of soluble scFv overnight at 30 °C, 750 rpm, 70% humidity for 16 - 18 h.

[0302] Rescue of scFv-phage

[0303] From the overnight plate, transfer 3.5 μl of the culture / well to a 96-well U-bottom plate containing 100 μl / well of 2xTYAG. Grow the bacteria at 30 °C, 750 rpm, 70% humidity for 4 h. To rescue the phage particles, add 100 μl of 2xTYAG / M13KO7 helper phage (Invitrogen, catalog number 18311019) (200 μl of M13KO7 (Invitrogen, catalog number 18311019) diluted to 10 ml of 2xTYAG) to each well, and let the plate stand at 37 °C for 1 h. Transfer the infected culture (5 μl / well) to a new 96-well U-bottom plate filled with 2xTY medium supplemented with 50 μg / ml kanamycin and 100 μg / ml ampicillin at 200 μl / well. Rescue the phage displaying scFv overnight at 30 °C, 750 rpm, 70% humidity for 16 - 18 h.

[0304] ELISA

[0305] For ELISA, coat a 96-well maxisorp Nunc-immunoplate (Thermo Scientific, catalog number 2022-10) with 50 μl / well of human recombinant TGF-β1 (Acrobiosystems, catalog number TG1-H4212), TGF-β2 (R&D Systems, catalog number 302-B2-010), and human recombinant TGF-β3 (Shenandoah Biotechnology, catalog number 100-109) at 500 ng / ml in PBS overnight at 4 °C. Block the wells by directly adding 230 μl of blocking solution (5% non-fat milk / 0.05% Tween 20 in PBS) without first washing the plate. Incubate for 45 min with gentle stirring. Meanwhile, block the bacterial cultures containing the expressed scFv and rescued phage by directly adding 120 μl of blocking solution. Wash the blocked maxisorp plate 3 times with PBST (PBS + 0.1% Tween 20), then apply 150 μl of the blocked scFv or phage culture to the wells. Incubate for 1 h at room temperature with gentle stirring, then wash the wells with 4x PBST.

[0306] For scFv ELISA, 100 μl of the primary mouse 9E10 anti-cmyc Ab diluted 1 / 1000 in PBS + 1% BSA was added to the wells and then incubated for 1 h at room temperature with gentle agitation. The plates were washed 4 times with PBST and then 100 μl of the secondary goat anti-mouse IgG(Fab')2 HRP conjugate (Sigma, catalog number A9917) diluted 1 / 10,000 in PBS + 1% BSA was added. The plates were incubated for 1 h at room temperature with gentle agitation. The ELISA was developed by adding 100 μl / well of the TMB substrate (Thermo Fisher Scientific, catalog number 34021), stabilized with 50 μl / well of the stop solution (2 N sulfuric acid), and the absorbance signal was read at 450 nm and 620 nm.

[0307] For phage ELISA, 100 μl of the primary rabbit anti-fd bacteriophage Ab (Sigma, catalog number B7786) diluted 1 / 1000 in PBS + 1% BSA was added to the wells and then incubated for 1 h at room temperature with gentle agitation. The plates were washed 4 times with PBST and then 100 μl of the secondary mouse anti-rabbit IgG (γ-chain specific) HRP conjugate (Sigma, catalog number A1949) diluted 1 / 15,000 in PBS + 1% BSA was added. The plates were incubated for 1 h at room temperature with gentle agitation and then washed 5 times with PBST and then 2 times with PBS. The ELISA was developed as for the scFv plates above.

[0308] Clonal variant competitive screening (GLv2 VH library)

[0309] A small DNA library of LCR1901_GLv1 VH CDR variants was designed based on analysis of homology to public germline and proprietary databases. The VH DNA library was synthesized by a commercial vendor and, after excision of the GLv1 VH chain, was cloned en masse into the pCHV101 LCR1901_GLv1 vector. The resulting library containing GLv1 VK and the mutant VH CDR library (designated pCHV101 LCR1901_GLv2) was electroporated into Escherichia coli TG1 cells together with the parental pCHV101 LCR1901_GLv1 graft vector. The cells were plated onto 2xTYAG and incubated at 30 °C for 18 - 20 h.

[0310] On the second day, fresh TG1 colonies containing the pCHV101-LCR1901_GLv2 library clones were picked into columns 2-11 of a 96-well U-bottom plate (Greiner bio-one, catalog number 650201) filled with 200 μl / well of 2xTYAG medium. Similarly, TG1 colonies containing the graft parent pCHV101-LCR1901_GLv1 vector were picked into column 1 (no-competition control). Column 12 (background control) contained only 200 μl / well of 2xTYAG medium. The plate was incubated overnight at 30 °C, 750 rpm, 70% humidity to generate a clone master plate.

[0311] On the second day, a 96-well expression plate with cells from the master plate was incubated using a 96-pin replicator. The expression plate contained 170 μl / well of TB supplemented with 0.1% glucose and 100 μg / ml ampicillin. The cultures were grown at 30 °C, 750 rpm, 70% humidity for 6 h and then scFv expression was induced by adding 50 μl of IPTG (0.45 mM in TB medium) to obtain a final concentration of 100 μM. Expression of the scFv was allowed to proceed overnight at 30 °C, 750 rpm, 70% humidity. Meanwhile, a 96-well maxisorp plate (Thermo Scientific, catalog number 2022-10) was coated overnight at 4 °C with 50 μl / well of human recombinant TGF-β3 (Shenandoah Biotechnology, catalog number 100-109) at a concentration of 250 ng / ml in PBS.

[0312] On the second day, the scFv expression plate was centrifuged to pellet the cells and 75 μl of the scFv supernatant was transferred from each well to the corresponding wells of a fresh non-binding polypropylene 96-well U-bottom plate. To these samples, 75 μl of LCR1901_GLv1 mAb (IgG1) competitor antibody at a concentration of approximately 200 ng / ml (prepared in PBS containing 0.2% Tween 20 and 2% BSA) was added to give a final volume of 150 μl with 100 ng / ml of the competitor mAb. This plate was called the 'competitor dilution plate'.

[0313] After a 15-min equilibration period, 230 μl of blocking solution (0.05% Tween 20, 5% non-fat dry milk) was added directly to the wells. Blocking was allowed to proceed for 45 min with gentle agitation.

[0314] The TGF-b3-coated maxisorp ELISA plates were washed three times with PBST (PBS + 0.1% Tween 20), and 100 μl / well of the scFv / GLv1 mAb mixture was added from the competitor dilution plate. Incubation was allowed to proceed for 1 h at room temperature.

[0315] The maxisorp ELISA plates were washed five times with PBST, and the ELISA plates were developed by adding 100 μl / well of a 1 / 5000 dilution (1% BSA in PBS + 0.1% Tween 20) of the secondary goat anti-human IgG-Fc-HRP conjugate antibody (SinoBiological, catalog number SSA001). After incubation for 1 h at room temperature with gentle stirring, 100 μl / well of the TMB substrate (Thermo Fisher Scientific, catalog number 34021) was added. The development was stopped by adding 50 μl / well of 2N sulfuric acid, and the absorbance was read at 450 nm and 620 nm.

[0316] Clonal variant competition screening (VK revertant framework library)

[0317] The scFv clone containing the LCR1901_1G10m VH chain paired with the LCR1901_GLv1 VK chain was used as the starting point. For each chain, the contribution of the parental LCR1901 murine framework (Fr) regions was investigated. For each chain, three variants were synthesized in which Fr1, Fr2, or Fr3 was replaced with the murine parental regions. These six chains were randomly combined by sequential batch cloning into pCHV101 to generate a small population of scFv DNA clones containing one or two murine Fr regions. After electroporation into TG1, the scFv was expressed, and 80 clones randomly selected from 2xTYAG plates were competitively screened as described above, in this case using the LCR1901 murine parental mAb as the ELISA competitor and an anti-mouse Fc-HRP secondary development reagent.

[0318] Re-conversion of scFv VH and VK chains to IgG1 / IgG4

[0319] The VH chain contained in the pCHV101 scFv vector was amplified using the following PCR primer mixture (working stock solution of 10 μM total oligonucleotides).

[0320] Nco1 FOR Pool*

[0321]

[0322] Sal1 REV Pool

[0323]

[0324] The VK chain contained in the pCHV101 scFv vector was amplified using the following PCR primer mixture (working stock solution is 10 μM total oligonucleotide).

[0325] BssH11 FOR Pool*

[0326]

[0327] Not1 REV Pool (κ)

[0328]

[0329] The PCR reaction was set up as follows:

[0330] VH – Each Reaction (50 μl )

[0331] 1 μl scFv miniprep (20 - 30 ng / μL)

[0332] 2 μl VH_Switch_FOR pool (10 μM total oligonucleotide)

[0333] 2 μl HJSal_REV pool (10 μM total oligonucleotide)

[0334] 20 μl PCR-grade H2O

[0335] 25 μl 2x LongAmp taq master mix (NEB; #M0287)

[0336] VK PCR - Each Reaction (50 ul)

[0337] 1 μl pCHV101 scFv miniprep (20 - 30 ng / μl)

[0338] 2 μl VLK_Switch_FOR pool (10 μM total oligonucleotide)

[0339] 2 μl KJNot_REV pool (10 μM total oligonucleotide)

[0340] 20 μl PCR-grade H2O

[0341] 25 μl 2x LongAmp taq master mix (NEB; #M0287)

[0342] PCR conditions: Initial denaturation at [94°C - 30 s], followed by 25 cycles of [94°C - 30 s], [60°C - 30 s], [65°C - 1 min]; and a final extension step of [65°C - 5 min]. Purify the PCR product using a DNA Clean & Concentrator - 5TM (DNAclean&concentrator-5TM) (Zymo Research; #D4003) into 12 μl of warm (preheated at 65°C) 0.2x kit elution buffer (EB) diluted in PCR-grade H2O.

[0343] Digest the purified PCR as follows:

[0344]

[0345]

[0346]

[0347] Digest the IgG expression vector as follows:

[0348]

[0349]

[0350]

[0351] Perform the digestion at 37°C for 1 - 2 h. Purify the PCR product using a DNA Clean & Concentrator - 5TM (kit from Zymo Research; #D4003) into 20 μl of warm (preheated at 65°C) 0.2x kit elution buffer (EB) diluted in PCR-grade H2O. Electrophorese the digested vector on a 0.9% agarose gel and gel extract the corresponding linearized vector band using the Zymoclean Gel DNA Recovery Kit (Zymo Research, catalog number D4002) into 30 μl of diluted EB.

[0352] Ligate at an insert:vector molar ratio of 3:1,

[0353]

[0354]

[0355] Ligation was carried out at room temperature for 2 h and overnight at 16 °C. The ligation mixture (2 μl) was combined with 50 μl of E. Cloni competent cells (prepared in-house) and incubated on ice for 10 min. After dilution in 75 μl of 2xTYG, the transformed cells were streaked onto selective 2xTYAG plates and incubated overnight at 37 °C. Typically, 4 colonies of each construct were picked into 3 ml of 2xTYAG medium and grown overnight at 37 °C. Plasmid minipreps were prepared using the vacuum PureYield plasmid miniprep system rapid protocol (Promega, catalog number FB093), and the inserts were determined by sequencing using primer PD1-5 (5’-GAGGATTTGATATTCACCTGG-3’) (SEQ ID NO:54) for the VH chain and primer PD1-91 (5’-GAATTCGATCAGGACTGAACAGAG-3’) (SEQ ID NO:55) for the VK chain. For correct expression vector clone chains, a 50 ml overnight culture grown in 2xTYAG at 37 °C and the ZymoPURE TM Plasmid Midiprep Kit (Promega, catalog number D4201) was used to prepare vector midipreps for HEK cell transfection.

[0356] Antibody expression - General

[0357] Protein expression was accomplished by transient transfection using the HEK293-6E / pTT transient expression system (National Research Council of Canada; obtained under license). Cells were grown in unbaffled Erlenmeyer flasks (TriForest, catalog number FPC0125S-K) at 120 rpm, 37 °C, and 5% CO2. F17 medium containing 4 mM GlutaMAX, 0.1% F-68 and 25 μg / ml G418 was used to grow the transfected cells to a cell density of 1×10 6 cells / ml.

[0358] The transfection procedure was as follows: 45 ml of cells at a density of approximately 1.0x 10 6HEK293-6E cells (viability > 97%) at [X] cells / ml were transferred to a 250 ml flask. For each desired expression culture, 25 μg of mid-prepared DNA of the expression vector (12.5 μg for each VK and VH chain pairing) was added to a 15 ml Falcon tube. A volume of transfection medium (F17 medium only) was added to the tube to give a final volume of 5 ml, and the solution was gently mixed by pipetting. 37.5 μl of pure [pure] FectoPRO TM ( SA, catalog number 116 - 010) was added to a separate empty 15 ml Falcon tube. 5 ml of the diluted DNA was carefully added to the FectoPRO TM reagent, and the solution was mixed by pipetting. After incubation at room temperature for 20 min, the DNA-FectoPRO TM mixture was added to the 250 ml flask containing the cells, and the flask was gently rotated to mix. The flask was immediately transferred to a 37 °C humidified shaking incubator (120 rpm) containing 5% CO2. After 5 days, the expression supernatant was harvested by centrifuging the cells at 2500 g for 3 min and transferring the clarified medium to a fresh 50 ml Falcon tube.

[0359] IgG Antibody Purification

[0360] IgG1 and IgG4 antibodies were purified on an AKTA Pure protein purification system 25L (GE Healthcare) using a 5 ml HiTrap Mab Select protein A column (GE Healthcare, catalog number 11003494). The column was equilibrated with PBS. After loading the sample at a flow rate of 5 ml / min, the column was washed with PBS to remove unbound proteins. The antibodies were eluted with 0.1 M citrate pH 3.2 at a flow rate of 3 ml / min and neutralized with Tris, then dialyzed overnight against PBS (15 ml Slide-A-Lyzer G2 dialysis cassette, 10K MWCO; Thermo Fisher Scientific, catalog number 87731). The protein was then concentrated by centrifugation (Vivaspin 20, 50KDa MWCO; GE Healthcare, catalog number 28932362).

[0361] Antibody Expression - 2 ml Scale [Yield Analysis]

[0362] The IgG4 antibody yield analysis was determined using 2 ml transfection cultures as follows: Approximately 1.0x10 6Cells (viability > 97%) were transferred into each well of a 12-well plate, and F17 complete medium was added to give a final volume of 1 ml. For each expression culture, 0.5 μg of DNA prepared in medium amounts in the expression vector (0.25 μg per strand) was transferred into a 1.5 ml Eppendorf tube, and then 100 μl of F17 medium was added. The solution was thoroughly mixed by pipetting and immediately transferred into a separate Eppendorf tube containing 0.75 μl of pure [purified] FectoPro. The transfection mixture was incubated at room temperature for 20 minutes and then carefully added to the wells containing the cells. The samples were gently swirled for mixing, and the plate was immediately transferred to a humidified shaking incubator at 37 °C with 5% CO2 (120 rpm). The expression medium was harvested after 5 days. Each antibody was transfected separately in triplicate to evaluate the comparative crude expression yield in the system.

[0363] Fab antibody purification

[0364] The His-tagged Fab antibody was purified using a 5 ml HisTrap Excel column (GE Healthcare, catalog number 17-3712-05) on an AKTA pure protein purification system 25L (GE Healthcare). The column was equilibrated with IMAC washing buffer pH 7.5 (50 mM Tris, 0.5 mM NaCl, 10 mM imidazole). After loading the sample at a flow rate of 1 ml / min, the column was first washed with the IMAC washing buffer and then with IMAC elution buffer pH 7.5 (50 mM Tris, 0.5 mM NaCl, 300 mM imidazole). The protein was eluted at a flow rate of 1 ml / min and then dialyzed overnight using PBS (15 ml Slide-A-Lyzer G2 dialysis cassette, 10K MWCO; Thermo Fisher Scientific, catalog number 87731). The protein was then concentrated by centrifugation (Vivaspin 20, 10KDa MWCO; GE Healthcare, catalog number 28932360).

[0365] Size exclusion chromatography and SDS-PAGE gel analysis

[0366] The monodispersity / aggregation state of the antibody was examined by qualitative size exclusion chromatography (SEC) using a Superdex 200 Increase 5 / 150 GL column (GE Healthcare, catalog number 28-9909-45). The column was equilibrated in PBS and then 100 μl of the sample was loaded at a flow rate of 0.3 ml / min.

[0367] By reducing and non-reducing SDS-PAGE using A 4%-12% Bis-Tris protein gel (Thermo Fisher Scientific, catalog number NP0321BOX) was used to examine the size and quality of the antibodies. MOPS buffer was used as the running buffer (Thermo Fisher Scientific catalog number). 5 μg of each antibody was mixed with 5 μl of LDS-sample buffer (Thermo Fisher Scientific catalog number) + / - 2 μl of sample reducing buffer. The samples were heated at 70 °C for 10 minutes before loading. The gel was stained with InstantBlue (Expedeon, catalog number ISB1L) for visualization.

[0368] Thermal shift assay

[0369] The thermal stability of the anti-TGF-β3 antibody was evaluated using the Protein Thermal Shift Assay with a 7500 Fast Real-Time PCR Instrument (Applied Biosystems). The assay involved mixing the antibody with a Protein Thermal Shift TM (protein thermal shift TM ) dye and applying a controlled heating ramp. As the protein begins to denature, the dye interacts with the exposed hydrophobic regions and emits stronger fluorescence, thereby establishing one or more transition temperatures.

[0370] Samples were prepared as follows:

[0371]

[0372] Experimental setup on the 7500 Fast RT-PCR machine:

[0373]

[0374] The Tm was calculated using the graph of the first derivative with respect to temperature through the 7500 instrument software.

[0375] TMLEC functional neutralization

[0376] This assay was based on TMLEC cells (transfected mink lung epithelial cells) containing a luciferase reporter gene, as described by Abe M et al. Analytical Biochemistry 1994, 216:276-284. The cell line was subcloned several times in the laboratory, and subclone 20 was used in these assays. The cells were cultured in DMEM supplemented with 10% FCS and AAG (0.55 mM L-arginine, 0.24 mM L-asparagine, 1.5 mM L-glutamine, and 400 μg / ml G418) under 8% CO2.

[0377] Incubate the TGFb± antibody dilution in a 96-well plate at 37 °C for 4 hours. Then transfer 100 ul of this <<mixture>> to a 96-well flat-bottom opaque ELISA plate suitable for luciferase activity counting containing 5 x 10 x 4 TMLEC cells / well) in 100 ul of the above medium, and culture in the wells for at least 1 h.

[0378] All TGFβ isotypes were used at a final concentration of 500 pg / ml. Antibody dilutions generally started at 30 ug / ml. Incubate the plates for 20 - 24 h. Then carefully remove 100 ul of the well contents and replace with 100 ul of luciferase substrate diluted in the lysis buffer of the Perkin-Elmer Ultra-brite luciferase kit. Immediately quantify luciferase activity in a bioluminescence ELISA reader.

[0379] Competitive binding of IgG4 mAb (relative to biotinylated murine LCR1910 parent)

[0380] Coat Nunc Maxisorb ELISA plates overnight at 8 °C with 0.5 μg / ml hTGF-β1 (Peter Sun, NIH) or hTGF-β3 (Shenandoah Biotech) (0.5 μg / ml in 40 mM glycine buffer pH 9). Wash the plates and block for 1 h with 10% FCS at 37 °C. In separate low-binding Greiner Bio-One ELISA plates, mix various concentrations of the competing Ab with a constant concentration of biotinylated parent 13A1 or 1901 (200 ng / ml in PBS + BSA (10 mg / ml)) and then transfer to the TGF-β-coated Nunc Maxisorb ELISA plates. After incubation at 37 °C for 2 h, wash the plates, add avidin-HRP, and incubate at 37 °C for 1 h. Wash the plates again, add TMB substrate, and quantify the bound biotinylated antibody by measuring the color development at a wavelength of 450 nm in an ELISA reader.

[0381] Example 3

[0382] Rescue and graft murine 1901 antibody into scFv

[0383] Rescue murine antibody 1901 VH and VL into scFv (LCR1901_scFv), express in E. coli and verify binding specificity

[0384] Synthesize as Figure 1The nucleotide sequences of the murine antibody 1901 VH and VL domains (SEQ ID NO: 9 and 11, respectively) were expressed as scFv in Escherichia coli. The scFv was converted to the orientation VK-linker-VH. Minor point mutations (VK: 52-54 AGG>CGA and 181-183 CGC>CGG; VH: 118-120 AGG>CGT and 292-297 AGAAGG>CGTCGC) were incorporated at this stage to exchange rare Arg codons in the reading frame, potentially improving expression in Escherichia coli. Subsequently, the scFv was cloned into the proprietary phagemid vector pCHV101, which allows dual expression and secretion of the molecule from Escherichia coli as a free scFv (LCR1901_scFv) or as a fusion with the pIII coat protein of filamentous bacteriophage (LCR1901_phage). Both expression formats retained strong and specific binding to surface-immobilized recombinant human TGFb3 in ELISA ( Figure 2 ).

[0385] Human VH was selected for grafting

[0386] IGHV1-69*08 was selected from the IMGT reference directory (imgt.org) and the internal IgM / D sequence database as the closest global human germline VH sequence.

[0387] Human VL was selected for grafting

[0388] IGKV1-39*01 was selected from the IMGT reference directory (imgt.org) and the internal IgM / D sequence database as the closest global human germline VL sequence.

[0389] Mouse CDRs were grafted onto human VH and VL frameworks, expressed as scFv and binding specificity was verified

[0390] Six murine CDRs (light chain CDRs: CDR1 KASQSVINAVA (SEQ ID NO:4), CDR2 YASRNYT (SEQ ID NO:5), and CDR3 QQDYSSPYT (SEQ ID NO:6); and heavy chain CDRs: CDR1 SSSWIH (SEQ ID NO:26), CDR2 RIYPGDGDTNYTGKFK (SEQ ID NO:27), and CDR3 RMITTQAAMD (SEQ ID NO:3)) were transplanted onto the corresponding human VH and VK frameworks and Venier regions and initially expressed as scFv. The scFv was converted to the orientation VK-linker-VH. The scFv nucleotide sequence was synthesized by GeneArt (Thermo Fisher Scientific) and cloned into the pCHV101 phagemid, and then the scFv (1901_GLv1_scFv) was expressed in E. coli. Figure 3 An amino acid sequence alignment of the murine and transplanted 1901 scFv constructs (SEQ ID NOs: 13 and 14, respectively) is shown. The six CDRs are shown in bold. Transplanting the murine CDR regions onto the selected human VK and VH framework regions retained the binding specificity for TGFb3 ( Figure 4 ).

[0391] The scFv was re-converted to human IgG1, expressed in HEK293 cells and the binding specificity was verified

[0392] The human VK and VH chains were re-converted by fusing them with their respective heavy and light chain human construct domains contained in discrete pTT5-based mammalian expression vectors. The resulting IgG1 heterodimer was expressed into the medium by transient co-transfection using a small-scale HEK293-6E suspension cell culture. The supernatants containing the transplanted antibody as well as the murine chimeric (mouse variable domains fused to human IgG1 Fc) antibody were subjected to TGFβ-specific ELISA together with the original purified parental murine antibody 1901. Both the chimeric and transplanted human IgG1 1901 antibodies retained the binding specificity for TGFb3 ( Figure 5 ).

[0393] Example 4

[0394] Sequence analysis and introduced mutations

[0395] Potential CDR sequence disadvantages in LCR1901_GLv1 were identified and corrected by mutagenesis

[0396] The LCR1901_GLv1 CDR sequences of both the VH and VK chains were subjected to BLAST homology analysis against both the IMGT variable domain reference set (imgt.org) and a proprietary database containing approximately 6 million translated IgM / IgD VH reads obtained from the peripheral blood of healthy donors. A series of residues were identified that may include potential physicochemical development risks or are mismatches / rare when considered together with human residues (human consensus residues) at the same position in the alignment of the highest scoring CDR / J homology( Figure 6 ). A small permutation library for the proposed CDR VH variants was constructed and the competitive binding of the scFv constructs to LCR1901_GLv1 was tested in ELISA. Additionally, a small library of mutated VK mutants was generated and combined with the hits of the above VH mutant library. Figure 6 Based on competitive binding assays, the proposed CDR and linker residues with potential sequence disadvantages, their proposed human consensus residue corrections, and the validation of the finally selected residues are summarized. The resulting VH is designated LCR1901_VH_1G10 (SEQ ID NO:17)( Figure 7 ). The CDRs in LCR1901_VH_1G10 contain changes relative to the original 1901 antibody and correspond to the following VH CDRs: CDR1 GY T FSSSWIH (SEQ ID NO:28), CDR2 WIGRIYPGDGDT D Y SE KF Q SEQ ID NO:29) and CDR3 ARRMITTQAA L (SEQ ID NO:30) (amino acid residues in the CDRs that are changed relative to the parental 1901 antibody are underlined).

[0397] Additional mutations in LCR1901_VH_1G10

[0398] The residue S123 in the linker region of FR4 was mutated to L123 to correct to hu IGHJ4. The resulting VH is designated LCR1901_VH_1G10m (SEQ ID NO:17)( Figure 7 ). The FR2 in the VH chain of LCR1901 was identified as crucial for maintaining the neutralizing potency of the antibody. Subsequently, three residues in FR2 were reverted to the original murine residues (R43>K43; A45>R45; and Q48>K48), and the resulting VH is designated LCR1901_VH_1G10m_GLv1_03(K) (SEQ ID NO:19)( Figure 7)。In the alternative VH sequence, only one residue in the FR2 region reverted to the original murine residue (Q48>K48), and the resulting Vh was designated LCR1901_VH-1G10m_02(J) (SEQ ID NO:36). The LCR1901_VH_1G10m and LCR1901_VH_1G10m_GLv1_03(K) (and also LCR1901_VH-1G10m_02(J)) heavy chains retained the alterations of LCR1901_VH_1G10 and the variant CDRs (SEQ ID NOs:28 - 30 indicated above).

[0399] Additional mutations in the LCR1901_VK_GLv1 FR regions

[0400] The residue L124 in the linker region of FR4 was mutated to V124 to correct for hu IGKJ4. The resulting VK was designated LCR1901_VK_GLv1 (SEQ ID NO:21)( Figure 8 )。The FR3 in the VK chain of LCR1901 and to a lesser extent FR2 were identified as crucial for maintaining the neutralizing potency of the antibody. The combination of FR reverse point mutations and VH-VK chain pairing experiments identified VK chains with higher TGFb3 neutralizing potency than LCR1901_VK_GLv1( Figure 8 ),which are described below:

[0401] LCR1901_VK_GLv1_03(F) (SEQ ID NO:22)( Figure 8 ) has two revertant mutations to the original murine residues (K48>Q48 and A49>S49) in FR2 and five revertant mutations to the original murine residues (S74>D74; S83>Y83; L89>F89; P96>A96 and T101>V101) in FR3. Additionally, in FR3, F99 was adjusted to V99 to obtain a better human "local" match (IGKV4-1*01) than the "global" IGKV1-39*01 engraftment.

[0402] LCR1901_VK_GLv1_05(H) (SEQ ID NO:23)( Figure 8 ) has five revertant mutations to the original murine residues (S74>D74; S83>Y83; L89>F89; P96>A96 and T101>V101) in FR3. Additionally, in FR3, F99 was adjusted to V99 to obtain a better human local match (IGKV4-1*01) than the "global" IGKV1-39*01 engraftment.

[0403] LCR1901_VK_GLv1, LCR1901_VK_GLv1_03(F), and LCR1901_VK_GLv1_05(H) retain the 1901VL CDRs that are shown in bold in Figure 8 : CDR1 KASQSVINAVAWY (SEQ ID NO:31), CDR2 LLIYYASNRYT (SEQ ID NO:32), and CDR3 QQDYSSPY (SEQ ID NO:33).

[0404] Germline homology of the LCR1901 VH+VK variants

[0405] Global alignment analysis of LCR1901 VH and VK was performed using the IMGT database (imgt.org), and the corresponding germline homology was determined for each chain. The germline homology of each chain was greater than 80% (range 81% - 87%) ( Figure 9 ).

[0406] Example 5

[0407] Expression of antibody variants in the form of human IgG4

[0408] The LCR1901 antibody variants were re-engineered into human IgG4(S228P) antibodies and expressed in the form of human IgG4. For this purpose, human IGHG4*01 was selected as the human Ig constant heavy chain and further modified to accommodate the S228P mutation, which has been recognized and shown in the art to stabilize antibodies with potential Fab-arm exchange (Silva et al., J Biol Chem. February 27, 2015; 290(9):5462-9). Human CK*01 was selected as the human Ig constant light chain. The IgG4 heterodimer was expressed into the culture medium by transient co-transfection of heavy and light chain vectors using small-scale HEK293-6E suspension cell cultures and then purified by protein A affinity chromatography.

[0409] The protein sequences of the four LCR1901 IgG4 variants are as follows Figures 10 - 13 shown

[0410] Figure 10 LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F), also designated 1901-1C, is provided in. The 1901-1C antibody contains the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22).

[0411] Figure 11 There is provided LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H), also designated as 1901-1A. The 1901-1A antibody comprises the heavy chain sequence LCR1901_VH_1G10m (SEQ ID NO:18) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23).

[0412] Figure 12 There is provided LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F), also designated as 1901-1D. The 1901-1D antibody comprises the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_03(F) (SEQ ID NO:22).

[0413] And Figure 13 There is provided LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H), also designated as 1901-1B. The 1901-1B antibody comprises the heavy chain sequence LCR1901_VH_1G10m_03(K) (SEQ ID NO:19) and the light chain sequence LCR1901_VK_GLv1_05(H) (SEQ ID NO:23).

[0414] Example 6

[0415] Predicted stacking angles of variants

[0416] The stacking torsion angles of the corresponding antibodies of each of the LCR1901 VH and VK variants were predicted by computer simulation using PAPS (bioinf.org.uk / abs / paps). The predicted values of the initial CDR grafts (1901_VH_GLv1 / 1901_VK_GLv1) and subsequent highly active mutant variants were very similar and significantly different from the original 1901 murine antibody( Figure 14 ).

[0417] Example 7

[0418] Potency of the antibody to neutralize TGFB signaling

[0419] The neutralizing potency of the LCR1901 IgG4 antibody against TGFβ signaling was determined using TMLEC reporter cells by assessing the neutralization of TGFβ isotype-specific signaling in the TMLEC reporter cell assay. Four IgG4 variants, LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F)(1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H)(1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F)(1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H)(1901-1B), were highly potent in neutralizing the expression of the luciferase reporter gene product induced by TGFβ3 in a dose-dependent manner. All four LCR1901 variants were more potent than the parental murine antibody 1901 in neutralizing TGFβ3 signaling in TMLEC cells( Figure 15 and Figure 16 ).

[0420] In addition, an LCR1901 variant antibody with VH sequence LCR1901_VH_1G10m_02(J)(SEQ ID NO:36) and VL sequence LCR1901_VK_GLv1_03(F)(SEQ ID NO:22) was generated, and its TGFβ3 signaling in TMLEC cells was tested and found to be effective in neutralization (data not shown).

[0421] Example 8

[0422] TGFB isotype specificity

[0423] The TGFβ isotype specificity of the LCR1901 IgG4 variants was evaluated in the TMLEC reporter assay. Four IgG4 variants, LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F)(1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H)(1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F)(1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H)(1901-1B), were tested and showed highly selective neutralization of TGFβ3 isotype-induced signaling but not the expression of the luciferase reporter gene product induced by TGFβ1 and TGFβ2 isotypes(Figure 17 )。

[0424] Example 9

[0425] Competitive Binding and Binding Kinetics

[0426] The potency of the humanized LCR1901 IgG4 antibody variants to competitively bind to TGFb3 with the parental murine antibody was evaluated by ELISA. Four IgG4 variants, LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F)(1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H)(1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F)(1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H)(1901-1B), competitively bound to TGFb3 with the parental murine 1901 antibody in a dose-dependent manner ( Figure 18 )。

[0427] Characterization and Binding Kinetics of LCR1901 Monovalent Fab Antibodies

[0428] Four IgG4 variants, LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F)(1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H)(1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F)(1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H)(1901-1B), were expressed as stable Fabs and the binding kinetics to TGFb were evaluated using surface plasmon resonance (Biacore). The Fab binding ELISA data are summarized in Figure 19 and representative Biacore binding profiles are shown in Figure 20 . The binding affinities of the Fabs are in the pM range.

[0429] Example 10

[0430] Additional Characterization of the Humanized Antibody

[0431] SEC Profile Analysis:The molecular integrity (not shown) of four IgG4 variants, LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F) (1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H) (1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F) (1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H) (1901-1B), was evaluated using analytical size-exclusion chromatography (SEC). The four LCR1901 IgG4 antibodies exhibited similar elution behavior. No signs of fragmentation or peak broadening were observed, and the aggregation level was low, indicating that the IgG4 antibodies were stable and molecularly homogeneous. A typical SEC profile is shown in Figure 21.

[0432] Thermal Stability: The relative molecular stability of four IgG4 variants, LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F) (1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H) (1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F) (1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H) (1901-1B), to heat stress was evaluated using differential scanning fluorimetry (DSF). The four LCR1901 IgG4 variants showed 2-transition non-cooperative domain melting curves with a characteristic earlier IgG4 CH2 domain unfolding (Tm1). The unfolding of the Fab domain is represented by transition 2 (Figure 22 and Figure 23 )

[0433] Scaled - up Expression Yield:The suitability of four IgG4 variants, LCR1901_VH_1G10m-LCR1901_VK_GLv1_03(F)(1901-1C), LCR1901_VH_1G10m-LCR1901_VK_GLv1_05(H)(1901-1A), LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_03(F)(1901-1D), and LCR1901_VH_1G10m_03(K)-LCR1901_VK_GLv1_05(H)(1901-1B), for potential larger-scale expression was evaluated using transient expression in a single mammalian cell system (2 ml scale). These four LCR1901 IgG4 variants were found to be highly expressed in the transient single mammalian cell system. The expression yields are summarized in Figure 24 below.

[0434] Example 11

[0435] The use and application of genetically engineered T cells expressing chimeric antigen receptors (CARs) directed against cell surface proteins in cancer therapy provides a new and improved approach for various cancers. In some cases, T cells modified to express only the chimeric antigen receptor (CAR) are inhibited in the hostile tumor microenvironment, which may limit the efficacy of CAR T cells in some clinical indications. For example, immunosuppressive cytokines such as IL-6, Il-10, and TGF-β can provide a favorable environment for tumor growth and also inhibit the antitumor activity of endogenous T cells as well as CAR T cells. Studies have shown that inhibition of the TGFβ receptor can enhance CAR T cell activity by inhibiting the immunosuppressive effect of TGFβ on the microenvironment (Vong Q et al. (2017) Blood 130:1791).

[0436] We sought to evaluate the role and efficacy of a TGFb3-specific antibody in counteracting TGFb3-mediated inhibition of CAR T cell activity. CAR T cells directed against mesothelin were evaluated against human lung cancer cells. The killing of human breast cancer cells by CAR T cells directed against the epidermal growth factor receptor (EGFR) was evaluated.

[0437] In lung cancer research, primary human T cells were transfected with anti-mesothelin CAR (hP4; described in US 2014301993 A1). T cells and TGFβ3 were added, and killing of target Meso+H-226 human lung cancer cells was evaluated. The effect of the addition of TGFβ3 antibody 1901-1B on cell killing was assessed. CAR T cells alone killed Meso+ lung target cells. In the presence of TGFβ3, cell killing was significantly inhibited. After addition of the TGFβ3-specific antibody, TGFβ3-mediated inhibition of cell killing disappeared. Antibody 1901-1B was highly potent even at low concentrations (≤50 ng / ml). The results are depicted in Figure 25 which shows rescue of TGFβ3-mediated inhibition of anti-MSLN CAR-T target cell killing by humanized mAb 1901-1B in vitro.

[0438] Similar studies were performed with CAR T cells against EGFR and human breast cancer cells. Primary human T cells (CD3 / CD28-activated and expanded peripheral blood mononuclear cells (PBMC)) were transfected with anti-EGFR CAR (scFv panitumumab). CAR T cells and TGFβ3 were added, and killing of target EGFR+MDA-MB-231 human breast cancer cells was evaluated. The effect of the addition of TGFβ3 antibody 1901-1B on cell killing was assessed. CAR T cells alone killed EGFR+ breast cancer target cells. In the presence of TGFβ3, cell killing was significantly inhibited. After addition of the TGFβ3-specific antibody 1901-1B, TGFβ3-mediated inhibition of cell killing disappeared. The results are depicted in Figure 26 which shows rescue of TGFβ3-mediated inhibition of anti-EGFR CAR-T target cell killing by humanized mAb 1901-1B in vitro.

[0439] Thus, it has been demonstrated in two different cancer cell model systems that inhibition of primary CAR T cell killing by TGFβ3 (representing expected endogenous TGFβ3 inhibition of cell therapy) can be reversed by administration of the TGFβ3 isotype-specific mAb 1901-1B.

[0440] Method

[0441] Target cell lines and primary effector cells: The human endogenous antigen-positive target cell lines H226 (lung cancer, MSLN + , CRL-5826 TM ) and MDA-MB-231 (breast cancer, EGFR + , HTB-26 TM)Maintained in RPMI-1640 Glutamax (Gibco BRL Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum (FBS) and Pen / Strep at a concentration of 100 IU / ml. The cells were maintained in a humidified atmosphere containing 5% carbon dioxide (CO2) at 37 °C.

[0442] Buffy coats were obtained from healthy volunteers from the (Interregional Blood transfusion SRC, Switzerland) blood bank. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats by density centrifugation using Lymphoprep (Axonlab).

[0443] Generation of CAR-modified T cells: The CAR expression cassette was harbored in a pRRL lentiviral vector backbone. The organization of the elements is typical of a second-generation CAR in which the scFv is appended to hCD28 [extracellular spacer, TM, and signaling domain] and hCD3ζ. Monomeric GFP was incorporated by direct fusion downstream of CD3ζ to allow direct assessment of transfection efficiency. The hP4 scFv for the anti-MSLN CAR was generated based on patent US2014301993 A1. The anti-EGFR scFv was reverse-engineered from the published panitumumab sequence (WO 2012138997).

[0444] Virus production was carried out by transient co-transfection of HEK293T producer cells with pRRL-CAR and packaging plasmids (pCMVR8.74 and pMD2.G; Didier Trono Laboratory, EPFL) using the Turbofect transfection reagent (Life Technologies). The virus-containing supernatant was harvested after 48 h and concentrated by ultracentrifugation.

[0445] PBMCs were plated (0.5x 10 6 / well) in a non-tissue culture-treated 24-well plate pre-coated with anti-human antibodies OKT3 (1 μg / mL; Thermo Fisher) and CD28 clone CD28.2 (2 μg / mL; Thermo Fisher). Cells were cultured in complete medium RPMI-1640 + GlutaMAX (Thermo Fisher) supplemented with 10% FBS and human recombinant IL-2 (50 IU / mL, Glaxo IMB) for 2 days. On day 3, freshly prepared lentiviral supernatant was used to transduce CD3 / CD28-activated PBMCs, and they were maintained in complete medium supplemented with human recombinant IL-2 (50 IU / mL, Glaxo IMB) for an additional 2 days. On day 5, complete medium containing IL-7 and IL-15 (Miltenyi Biotec) at 10 ng / ml was added to the expanded PBMCs and incubated at 37 °C, 5% CO2 for 1 - 2 weeks until they were used for the cytotoxicity assay. Subsequently, cells were split and fed every 2 - 3 days with fresh medium plus IL-7 / IL-15. Based on the GFP + percentage of cells, transfection efficiencies of approximately 40% (anti-MSLN CAR) and approximately 50% (anti-EGFR CAR) were determined.

[0446] Kinetic cytotoxicity assay: H226 target cells (100 μL / well) were seeded in 96-well plates at a density of 0.015x10 6 cells / mL in complete medium. The next day, the cell density had reached 0.02x10 6 cells / well. The supernatant was removed, and 50 μL (500 nM) of CytotoxRED dead cell staining reagent (Incucyte, Essen Bioscience) was added to each well. Effector anti-MSLN CAR cells (2x10 6 cells / mL) in complete medium were combined with 1 μL of stock TGFβ3 (MILAN Analytica AG; 500 ng / mL) to give a concentration of 2 ng / mL or left untreated. Treated / untreated CAR cells (50 μL) were added to each well, resulting in a 5:1 E:T ratio + / - 1 ng / mL TGFβ3. The anti-TGFβ3 neutralizing (reversing) potential of the hIgG4 1901_1B antibody was determined by its co-inclusion at a final concentration of 500 ng / mL. The plates were returned to the incubator for 30 min to allow the combined cells and dye to settle and equilibrate, then transferred to the Incucyte system for 3 days. Cell death was monitored as an increase in red fluorescence, and with Analysis was performed using the Incucyte, ZOOM 2016A software. The killing slope was determined using the total red image integrated intensity of each image data.

[0447] Anti-EGFR CAR-mediated killing against MDA-MB-231 was determined similarly, however, in this case, the target cells were pre-stained 24 h prior to plating with 1 μM of the Cytolight rapidRED cytoplasmic staining dye (Essen Bioscience). The stained cells were seeded in 96-well plates at a density of 0.015x10 6 cells / mL in complete medium. The next day, the cell density had reached 0.02x10 6 cells / well. The supernatant was removed and 100 μL / well of effector anti-EGFR CAR cells (2x10 6 cells / mL) + / - 1 ng / mL TGFβ3 was added to achieve a 5:1 E:T ratio. The anti-TGFβ3 neutralizing (reverse) potential of the hIgG4 1901_1B antibody was determined by its co-incubation at a final concentration of 500 ng / mL. The plates were returned to the incubator for 30 min for equilibration and then transferred to the Incucyte system for 3 days. Cell death was monitored as a decrease in the red fluorescence count of the stained target cells and analyzed using the Incucyte, ZOOM 2016A software. The kinetic progress curve was determined using the normalized Total Red Object Area (μm2 / well) data.

[0448] Example 12

[0449] Studies were conducted to demonstrate that variant TGF-β3 antibodies can be transduced into T cells and secreted. T cells were transfected with a vector encoding an anti-TGF-β3 antibody with an N-terminal signal sequence for secretion. T cells (Jurkat) expressed and secreted an antibody that was active and bound target TGF-β3.

[0450] Methods

[0451] Jurkat transfection with anti-TGFβ scFv-Fc fusion construct: To evaluate potential T cell secretion, Jurkat T cells were transfected with a pRRL-based lentiviral vector (Origin: Didier Trono laboratory, EPFL) containing the open reading frame of anti-TGFβ3 (1901_1B) scFv-Fc (hIgG4) with an N-terminal secretory pathway signal sequence. Controls included untransfected or irrelevant isotype control transfected scFv-Fc. One week after transfection, the cells were seeded at 2x 106 Plated at a density of 6 cells / ml in 6-well plates and cultured at 37 °C, 5% CO2 in RPMI containing 10% FBS for 24 h. Then the cells were gently pelleted by centrifugation and the supernatant was collected for subsequent ELISA.

[0452] Enzyme-linked immunosorbent assay (ELISA): A 96-well plate (Nunc Maxisorp, Thermo Fisher Scientific, #442404) was coated overnight at 4 °C with 100 μl of TGFβ3 (Milan Analytica, #002003) at a concentration of 0.5 μg / ml in PBS. The next day, the plate was blocked directly for 1 h with 230 μl of 5% non-fat milk / PBST (0.1% Tween in PBS) with gentle agitation. After blocking, the plate was washed 3 times with PBST and 100 μl of Jurkat supernatant (diluted with 1% BSA in PBST) was added to allow any secreted anti-TGFβ3 scFv-Fc to bind to the TGFβ3 antigen bound to the plate. Incubated for 1 h at room temperature with gentle agitation. The wells were washed 3 times with PBST and 100 μl of HRP-conjugated secondary antibody goat anti-human IgG-Fc-HRP (Sino Biological, #SSA001-200) was added at a concentration of 0.2 μg / ml with gentle agitation for 1 h. Then, the plate was washed 5 times with PBST and 2 times with PBS. After the washing steps, the colorimetric reading was developed with TMB substrate reagent (Biolegend, #34029) and stabilized with 2N sulfuric acid. Absorbance was measured at 450 nm and 620 nm on a BioTek Synergy plate reader.

[0453] Results of Jurkat-secreted anti-TGFβ3 antibody (1901_1B) relative to non-immobilized TGFβ3 are depicted in Figure 27 Figure []. No binding was observed in the case of the isotype control antibody. Serial dilutions of the secreted 1901-1B antibody bound TGF-β3 and were at comparable levels to the added exogenous 1901-1B antibody. These results demonstrate that the variant TGFβ3 antibody can be expressed and secreted by lymphoid cells, including T cells, and that the secreted antibody effectively binds TGFβ3. Lymphoid cells engineered to express the TGFβ3 antibody of the present invention can be used in a variety of applications, including cancer therapy and immunomodulation.

[0454] Without departing from the spirit or essential characteristics of the present invention, the present invention may be embodied in other forms or carried out in other ways. Therefore, it is considered that the present disclosure is illustrative in all respects and not restrictive, and the scope of the present invention is indicated by the appended claims, and all changes within the meaning and range of equivalents are intended to be included therein.

[0455] Throughout the specification, various references are cited, and each reference is incorporated herein by reference in its entirety. Sequence Listing <110> G. Ritter S. Dunn <120> Humanized and Variant TGF-β3-Specific Antibodies and Their Methods and Uses <130> 2745-4PCT <140> Not Specified <141> 2019-11-04 <150> US 62 / 755,840 <151> 2018-11-05 <160> 55 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Mus musculus <400> 1 Ser Ser Trp Ile His 1 5 <210> 2 <211> 17 <212> PRT <213> Mus musculus <400> 2 Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Thr Gly Lys Phe Lys 1 5 10 15 Gly <210> 3 <211> 11 <212> PRT <213> Mus musculus <400> 3 Arg Met Ile Thr Thr Gln Ala Ala Met Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Mus musculus <400> 4 Lys Ala Ser Gln Ser Val Ile Asn Ala Val Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5 Tyr Ala Ser Asn Arg Tyr Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Mus musculus <400> 6 Gln Gln Asp Tyr Ser Ser Pro Thr 1 5 <210> 7 <211> 112 <212> PRT <213> Mus musculus <400> 7 Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn Cys Cys 1 5 10 15 Val Arg Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp Lys Trp 20 25 30 Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly Pro Cys 35 40 45 Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu Gly Leu 50 55 60 Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Pro 65 70 75 80 Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg Thr Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Val Val Lys Ser Cys Lys Cys Ser 100 105 110 <210> 8 <211> 112 <212> PRT <213> Homo sapiens <400> 8 Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn Cys Cys 1 5 10 15 Val Arg Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp Lys Trp 20 25 30 Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly Pro Cys 35 40 45 Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu Gly Leu 50 55 60 Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Pro 65 70 75 80 Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg Thr Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Val Val Lys Ser Cys Lys Cys Ser 100 105 110 <210> 9 <211> 417 <212> DNA <213> Mus musculus <400> 9 atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag 60 gttctcctgc agcagtctgg acctgagctg gtgaagcctg gggcctcagt gaagatttcc 120 tgcaaggctt ctggctacgc attcagtagc tcctggatac actgggtgaa gcagaggcct 180 ggaaagggtc ttgagtggat tggacggatt tatccgggag atggagatac taactatact 240 gggaagttca agggcaaggc cacacttact gcagacaaat cctccagcac agcctacatg 300 caactcagca gcctgacatc tgaggactct gcggtctact tctgtgcaag aaggatgatt 360 acgactcagg cggctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 417 <210> 10 <211> 120 <212> PRT <213> Mus musculus <400> 10 Gln Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Thr Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 11 <211> 381 <212> DNA <213> Mus musculus <400> 11 atgaagtcac agacccaggt cttcgtattt ctactgctct gtgtgtctgg tgctcatggg 60 agttttgtga tgacccagac tcccaaattc ctgcttgtat cagcaggaga cagggttacc 120 ataacctgca aggccagtca gagtgtgatt aatgctgtag cttggtacca acagaagcca 180 gggcagtctc ctaaactgct gatatactat gcatccaatc gctacactgg agtccctgat 240 cgcttcactg gcaatggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 300 gaagacctgg cagtttattt ctgtcagcag gattatagct ctccgtacac gttcggaggg 360 gggaccaagc tggaaataaa a 381 <210> 12 <211> 107 <212> PRT <213> Mus musculus <400> 12 Ser Phe Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Asn Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 13 <211> 256 <212> PRT <213> Artificial Sequence <220> <223> Mouse 1901 VL-VH Transplanted Construct <400> 13 Ser Phe Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Asn Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Arg Gln Glu Gly Gly Ser Gly Glu Gly Gly Ser Gly Glu Ser Asn Ala 115 120 125 Ala Ala Gln Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 130 135 140 Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser 145 150 155 160 Ser Ser Trp Ile His Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu 165 170 175 Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Thr Gly 180 185 190 Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr 195 200 205 Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 210 215 220 Phe Cys Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Ala Ser 245 250 255 <210> 14 <211> 256 <212> PRT <213> Artificial Sequence <220> <223> LCR1901_Glv1 VL-VH Transplanted Construct <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Arg Gln Glu Gly Gly Ser Gly Glu Gly Gly Ser Gly Glu Ser Asn Ala 115 120 125 Ala Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 130 135 140 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser 145 150 155 160 Ser Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 165 170 175 Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Thr Gly 180 185 190 Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr 195 200 205 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 210 215 220 Tyr Cys Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Ala Ser 245 250 255 <210> 15 <211> 120 <212> PRT <213> Mus musculus <400> 15 Gln Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Thr Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 16 <211> 120 <212> PRT <213> Synthetic Sequence <220> <223> LCR1901_Glv1 VH <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Thr Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 17 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> LCR1901_VH_1G10 <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asp Tyr Ser Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 18 <211> 120 <212> PRT <213> Artificial sequence <220> <223> LCR1901_VH_1G10m <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asp Tyr Ser Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> LCR1901_VH_1G10m_Glv1_03(K) <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asp Tyr Ser Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Mus musculus <400> 20 Ser Phe Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Asn Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 21 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> LCR1901_Glv1 <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> LCR1901_VK_Glv1_03(F) <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> LCR1901_VK_Glv1_05(H) <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ile Asn Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 24 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> CH IgG4(S228P) <400> 24 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 25 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CK IGKC*01 <400> 25 Arg Thr Ala Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 26 <211> 6 <212> PRT <213> Mus musculus <400> 26 Ser Ser Ser Trp Ile His 1 5 <210> 27 <211> 16 <212> PRT <213> Mus musculus <400> 27 Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Thr Gly Lys Phe Lys 1 5 10 15 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 Humanized Antibody <400> 28 Gly Tyr Thr Phe Ser Ser Ser Trp Ile His 1 5 10 <210> 29 <211> 19 <212> PRT <213> Synthetic Sequence <220> <223> VH CDR2 Humanized <400> 29 Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asp Tyr Ser Glu 1 5 10 15 Lys Phe Gln <210> 30 <211> 11 <212> PRT <213> Synthetic Sequence <220> <223> VH CDR3 Humanized <400> 30 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Leu 1 5 10 <210> 31 <211> 13 <212> PRT <213> Synthetic Sequence <220> <223> VL CDR1 Humanized <400> 31 Lys Ala Ser Gln Ser Val Ile Asn Ala Val Ala Trp Tyr 1 5 10 <210> 32 <211> 11 <212> PRT <213> Synthetic Sequence <220> <223> VL CDR2 Humanized <400> 32 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr 1 5 10 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VL CDR3 humanized <400> 33 Gln Gln Asp Tyr Ser Ser Pro Tyr 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 variant humanized <400> 34 Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asp Tyr Ser Glu Lys Phe Gln 1 5 10 15 <210> 35 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 variant humanized <400> 35 Arg Met Ile Thr Thr Gln Ala Ala Leu Asp Tyr 1 5 10 <210> 36 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH LCR1901_VH_1G10m_02(J) <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asp Tyr Ser Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gln Ala Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 Humanized <400> 37 Arg Met Ile Thr Thr Gln Ala Ala Leu 1 5 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 38 ctgaaaggaa ggatatagaa tgtgc 25 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 39 gtcgtctttc cagacgttag 20 <210> 40 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 40 gagggtggtt ctggcgagtc caatgcsgcs gca 33 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 41 gagggtggtt ctggcgagtc caatgccryg gca 33 <210> 42 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 42 gagggtggtt ctggcgagtc caatgccatg gca 33 <210> 43 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 43 atggaccctt ggtcgacgct gaggagacgg tgaccagggt tcc 43 <210> 44 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 44 atggaccctt ggtcgacgct gaggagacgg tgaccgtggt ccc 43 <210> 45 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 45 atggaccctt ggtcgacgct gaggagacrg tgaccagggt scc 43 <210> 46 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 46 atggaccctt ggtcgacgct gaagagacgg tgaccattgt ccc 43 <210> 47 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 47 ctggctcttg gcgcggctag ccctgcsats gct 33 <210> 48 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 48 ctggctcttg gcgcggctag ccctgcgmkc gct 33 <210> 49 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 49 ctggctcttg gcgcggctag ccctgtgcgc gct 33 <210> 50 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 50 accaccagat ggtgcggccg cagttcgttt gatytccacc ttgg 44 <210> 51 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 51 accaccagat ggtgcggccg cagttcgttt gatctccagc ttgg 44 <210> 52 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 52 accaccagat ggtgcggccg cagttcgttt gatatccact ttgg 44 <210> 53 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 53 accaccagat ggtgcggccg cagttcgttt aatctccagt cgtg 44 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 54 gaggatttga tattcacctg g 21 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 55 gaattcgatc aggactgaac agag 24

Claims

1. An isolated antibody or antigen-binding fragment thereof that recognizes human and murine transforming growth factor β3 (TGF-β3) and neutralizes the activity of TGF-β3, wherein the isolated antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the CDR1 sequence GYTFSSSWIH (SEQ ID NO:28), the CDR2 sequence WIGRIYPGDGDTDYSEKFQ (SEQ ID NO:29), and the CDR3 sequence ARRMITTQAAL (SEQ ID NO:30), and the light chain variable region comprising the CDR1 sequence KASQSVINAVAWY (SEQ ID NO:31), the CDR2 sequence LLIYYASNRYT (SEQ ID NO:32), and the CDR3 sequence QQDYSSPY (SEQ ID NO:33).

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof does not react with TGF-β1 or TGF-β2.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region that is a sequence having at least 90% amino acid identity with SEQ ID NO:18, wherein the variant retains TGF-β3 reactivity and neutralization.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region that is a sequence having at least 90% amino acid identity with SEQ ID NO:19, wherein the variant retains TGF-β3 reactivity and neutralization.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region that is a sequence having at least 90% amino acid identity with SEQ ID NO:36, wherein the variant retains TGF-β3 reactivity and neutralization.

6. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a light chain variable region that is a sequence having at least 90% amino acid identity with SEQ ID NO:22, wherein the variant retains TGF-β3 reactivity and neutralization.

7. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a light chain variable region that is a sequence having at least 90% amino acid identity with SEQ ID NO:23, wherein the variant retains TGF-β3 reactivity and neutralization.

8. The isolated antibody according to claim 1, wherein the isolated antibody is selected from: (a) Antibody 1901-1C, wherein the antibody 1901-1C comprises a heavy chain sequence SEQ ID NO:18 and a light chain sequence SEQ ID NO:22; (b) Antibody 1901-1A, wherein the antibody 1901-1A comprises a heavy chain sequence SEQ ID NO:18 and a light chain sequence SEQ ID NO:23; (c) Antibody 1901-1D, wherein the antibody 1901-1D comprises a heavy chain sequence SEQ ID NO:19 and a light chain sequence SEQ ID NO:22; and (d) Antibody 1901-1B, wherein the antibody 1901-1B comprises a heavy chain sequence SEQ ID NO:19 and a light chain sequence SEQ ID NO:

23.

9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 3 to 8, wherein the isolated antibody or antigen-binding fragment thereof does not react with TGF-β1 or TGF-β2.

10. An isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof recognizes human and murine TGF-β3 and does not recognize or bind to human or murine TGF-β1 or TGF-β2, and the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region that is a sequence having at least 90% amino acid identity with SEQ ID NO:18 or SEQ ID NO:19 or SEQ ID NO:36, wherein the variant retains TGF-β3 reactivity and neutralizing activity and lacks TGF-β1 and TGF-β2 reactivity.

11. An isolated antibody or antigen-binding fragment thereof according to claim 10, wherein the isolated antibody or antigen-binding fragment thereof further comprises a light chain variable region that is a sequence having at least 90% amino acid identity with SEQ ID NO:22 or SEQ ID NO:23, wherein the variant retains TGF-β3 reactivity and neutralizing activity and lacks TGF-β1 and TGF-β2 reactivity.

12. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1-8, 10-11, wherein the isolated antibody or antigen-binding fragment thereof is humanized.

13. An isolated antibody or antigen-binding fragment thereof according to claim 12, wherein the isolated antibody or antigen-binding fragment thereof is chimeric.

14. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1-8, 10-11, wherein the isolated antibody or antigen-binding fragment thereof is in the form of an F(ab’)2, scFv fragment or minibody.

15. An antibody conjugate, wherein in the antibody conjugate, a radiolabel is attached to the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-8, 10-11.

16. An isolated nucleic acid comprising a sequence encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-8, 10-11.

17. A method for preparing an antibody or an antigen-binding fragment thereof as defined in any one of claims 1-8, 10-11, the method comprising expressing the nucleic acid according to claim 16 under conditions that cause the expression of the antibody or an antigen-binding fragment thereof, and recovering the antibody or an antigen-binding fragment thereof.

18. Use of an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 14 in the preparation of a medicament for treating cancer or reducing the recurrence or metastasis of cancer in a mammal, wherein the cancer or the tumor in the cancer expresses TGF-β3, and the cancer is selected from glioma, fibrosarcoma, colon cancer, breast cancer, melanoma, prostate cancer and lung cancer.

19. The use according to claim 18, wherein the medicament is prepared for intratumoral injection.

20. A kit for diagnosing or prognosticating a cancer in which the TGF-β3 antigen is expressed, the kit comprising an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 14, optionally having a buffer, and / or a stabilizer, and / or instructions for use.

21. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 14 and a pharmaceutically acceptable vehicle, carrier or diluent.

22. A lymphoid cell genetically engineered to express and secrete an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 14.

23. The lymphoid cell according to claim 22, wherein the cell is further engineered to express a receptor.

24. The lymphoid cell according to claim 23, wherein the receptor is a chimeric antigen receptor (CAR).

25. The lymphoid cell according to claim 23, wherein the receptor is a T cell receptor.

26. The lymphoid cell according to claims 22 to 25, wherein the cell is further engineered to express and secrete one or more soluble proteins.

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