Cross-species Anti-latent TGF-β 1 antibodies and methods of use

Abstract

To provide cross-species anti-latent TGF-β 1 antibodies which inhibit a protease mediated activation of latent TGF-β 1 without inhibiting integrin mediated activation of latent TGF-β 1.SOLUTION: To obtain the anti-latent TGF-β 1 antibodies of the present invention, anti-latent TGF-β 1 antibodies which inhibit a protease mediated activation of latent TGF-β 1 without inhibiting integrin mediated activation of latent TGF-β 1 are screened, and then humanized, and further optimized. The invention also provides combination therapies comprising an anti-latent TGF-β 1 antibody and one or more immune checkpoint inhibitors, preferably PD-1 axis binding antagonists.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anti-latent TGF-β1 antibodies and methods of using the same. [Background technology]

[0002] Transforming growth factor β (transforming growth factor beta; TGF-β) is a member of the TGF-β superfamily of cytokines, which consists of the TGF-β isoforms activin, inhibin, Nodal, bone morphogenetic proteins (BMPs), anti-Müllerian hormone (AMH), and growth differentiation factors (GDFs). Members of this superfamily are dimeric proteins with conserved structures and have pleiotropic functions in vitro and in vivo (Non-Patent Documents 1 and 2). TGF-β isoforms are involved in many cellular processes, including growth inhibition, cell migration, invasion, epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) remodeling, and immunosuppression (Non-Patent Document 3). However, despite being dynamically regulated and involved in tissue homeostasis, TGF-β isoforms are often chronically overexpressed in disease states, including cancer, fibrosis, and inflammation. Overproduction of TGF-β promotes disease progression by altering cell proliferation, migration, or phenotype.

[0003] Three distinct TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) have been identified in mammals, sharing 70–82% homology at the amino acid level (Non-Patent Document 4). All three TGF-β isoforms bind to TGF-β receptor type 2 (TGFR2) as homodimers (their active forms); TGFR2 then recruits and activates TGF-β receptor type 1 (TGFR1), activating receptor signaling (Non-Patent Document 5). However, the expression levels of the three isoforms vary depending on the tissue (Non-Patent Document 6), and their functions differ, as demonstrated by the phenotypes of knockout mice (Non-Patent Documents 7–11).

[0004] Like other members of the TGF-β superfamily, TGF-β is synthesized as a precursor protein that forms a homodimer with its latency-associated peptide (LAP) and latent TGF-β binding protein (LTBP) to form a large complex called the large latent complex (LLC). The TGF-β gene encodes a preproprotein sequence consisting of a signal peptide, a propeptide terminating in a proprotein convertase (PPC) cleavage site, and the mature TGF-β sequence. Furin hydrolyzes the PPC cleavage site, generating separate homodimers derived from TGF-β and the propeptide. These two homodimers remain noncovalently associated and are secreted. This latent complex maintains TGF-β in an inactive form that cannot bind to its receptor (Non-Patent Documents 12, 13). The TGF-β activation process involves the release of LLC from the ECM, followed by further proteolysis of LAP to release active TGF-β to its receptor (Non-Patent Document 3). Latent TGF-β is cleaved by a wide range of proteases, including plasmin (PLN), plasma kallikrein (PLK), matrix metalloproteinase (MMP) 2, and MMP-9 (Non-Patent Document 14), as well as by thrombospondin 1 (TSP-1) (Non-Patent Document 15), to release active TGF-β. While not wishing to be bound by any theory, MMP-2 and MMP-9 proteolytically cleave latent TGF-β1, releasing mature TGF-β1 from the latent form. Both MMP-2 and MMP-9 are synthesized as inactive pro-MMPs. Pro-MMP-2 is activated by a complex of membrane-type MMP-1 (MT1-MMP / MMP-14) and tissue inhibitor of metalloproteinase 2 (TIMP-2). Pro-MMP-9 is activated through an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin generates active MMP-3 from its zymogen, which cleaves the propeptide from the 92-kDa pro-MMP-9, yielding the 82-kDa enzymatically active enzyme.Although the cleavage site of MMPs has not been specifically determined, it has been reported that MMP3 specifically cleaves the site between 79 Ala and 80 Leu of latent TGF-β to activate TGF-β (WO2005 / 023870). Alternatively, mechanical stretching may activate TGF-β by binding to the RGD motif present in LAP, inducing the release of mature TGF-β from its latent complex (Non-Patent Documents 16 and 17).

[0005] After activation, dimeric TGF-β ligands bind to the extracellular domains of type I and type II receptors, inducing their proximity and positioning the receptor's intracellular serine / threonine kinase domain in a configuration that promotes phosphorylation and subsequent activation of the type I receptor. This activation of the type I receptor leads to amplification of signal transduction via at least two seemingly independent pathways: the SMAD-dependent canonical pathway and the SMAD-independent or noncanonical pathway. In the SMAD-dependent pathway, activation of TGFR1 (also known as ALK5) leads to the phosphorylation of SMAD proteins. SMAD2 and SMAD3 are substrates of TGFR1. Upon receptor phosphorylation, SMADs translocate to the nucleus along with a common mediator, SMAD4, where they interact with other transcription factors to regulate transcriptional responses (Non-Patent Document 18). In the non-canonical pathway, the activated TGF-β receptor complex transmits signals through other factors, such as tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4), TRAF6, TGF-β-activated kinase 1 (TAK1, also known as MAP3K7), p38 mitogen-activated protein kinase (p38 MAPK), RHO, phosphoinositide 3-kinase (PI3K), AKT (also known as protein kinase B), extracellular signal-regulated kinase (ERK), JUN N-terminal kinase (JNK), or nuclear factor kappa B (NF-κB). Thus, the cellular response to TGF-β signaling is brought about by the dynamic cooperation of canonical and non-canonical signaling cascades.

[0006] Fibrosis, or the accumulation of ECM molecules that forms scar tissue, is a common feature of chronic tissue injury. Pulmonary fibrosis, renal fibrosis, and liver cirrhosis are particularly common fibrotic diseases, and there are significant unmet medical needs for these diseases. TGF-β strongly promotes extracellular matrix production by mesenchymal cells while simultaneously suppressing epithelial cell proliferation, contributing to the pathogenesis of sclerosing diseases. Overexpression of the active form of TGF-β1 in the liver of transgenic mice is sufficient to induce fibrosis in multiple organs (Non-Patent Document 19). Meanwhile, TGF-β also plays an important role in maintaining our health. For example, TGF-β suppresses the excessive production of proteases in the lungs and inhibits the destruction of lung tissue that leads to emphysema. Furthermore, mice lacking TGF-β1 are either prenatally lethal (approximately 50% at 10.5 days postcoitum) or die prematurely after birth, exhibiting severe inflammatory lesions in many organs, including the lungs (vasculitis, perivascular cell infiltration, and interstitial pneumonia) and heart (endocarditis and myocarditis), suggesting that TGF-β1 plays a crucial role in maintaining immune homeostasis (Non-Patent Document 7).

[0007] Research using neutralizing antibodies against TGF-β and animal models has revealed that inhibiting the action of TGF-β can prevent or cure sclerosing diseases. Because TGF-β is produced as a precursor protein, several approaches have been reported to prevent its activation from its latent form. Another method for preventing activation from its latent form is to use inhibitors or antibodies that bind to latent TGF-β to block cleavage by proteases, such as PLK and PLN. Various antibodies using this method to inhibit TGF-β activation have been reported to prevent or treat liver fibrosis / cirrhosis (Patent Document 1). In addition, several publications mention anti-LAP antibodies for treating cancer (Patent Document 2) and TGF-β1-binding immunoglobulins for treating TGF-β1-related disorders (Patent Document 3). [Prior art documents] [Patent documents]

[0008] [License 1] WO 2011102483 [License 2] WO 2016115345 [License 3] WO 2017156500 [Non-licensed literature]

[0009] [Non-licensed Document 1] McCartney-Francis, NL et al. Int. Rev. Immunol. 16, 553-580 (1998) [Non-licensed Document 2] Massague, J. Annu. Rev. Biochem. 67, 753-791 (1998) [Non-licensed Document 3] Derynck, R. & Miyazono, K. Cold Spring Harbor Press (2008) [Non-licensed Document 4] Yu, L. et al. Kidney Int. 64, 844-856 (2003). [Non-licensed Document 5] Xu, P., Liu, J. & Derynck, R. et al. FEBS Lett. 586, 1871-1884 (2012). [Non-licensed Document 6] Millan, FA et al. Development 111, 131-143 (1991). [Non-licensed Document 7] Kulkarni, AB et al. Proc. Natl Acad. Sci. USA 90, 770-774 (1993). [Non-licensed Document 8] Shull, MM et al. Nature 359, 693-699 (1992). [Non-licensed Document 9] Dickson, M. C. et al. Development 121, 1845-1854 (1995).

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Summary of the Invention

[0010] One object of the present invention is to provide cross-species humanized and optimized anti-latent TGF-β1 antibodies that inhibit protease-mediated activation of latent TGF-β1 without inhibiting integrin-mediated activation of latent TGF-β1. The present invention also provides combination therapies comprising an anti-latent TGF-β1 antibody and one or more immune checkpoint inhibitors. [Means for solving the problem]

[0011] As a result of intensive research under the above circumstances, the present inventors have created a species-cross-reactive, humanized, and optimized anti-latent TGF-β1 antibody that inhibits protease-mediated activation of TGF-β1 without inhibiting integrin-mediated activation of latent TGF-β1. Furthermore, this anti-latent TGF-β1 antibody showed anti-tumor effects when administered in combination with one or more immune checkpoint inhibitors.

[0012] The present invention provides: A1. Anti-latent TGF-β1 antibodies, including: (a) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 20, 21, and 22, respectively; (b) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively; (c) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively; or (d) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 38, 39, and 40, respectively. A2. The anti-latent TGF-β1 antibody of A1, further comprising: (a) HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively; (b) HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively; (c) HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 36, and 37, respectively; and (d) HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 42, and 43, respectively. A3. Anti-latent TGF-β1 antibodies, including: (a) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 20, 21, and 22, respectively, and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively; (b) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively, and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively; (c) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively, and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 36, and 37, respectively; or (d) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 38, 39, and 40, respectively, and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 42, and 43, respectively. A4. The anti-latent TGF-β1 antibody according to any one of A1 to A3, comprising: (a) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13, or (iii) the VH sequence of (i) and the VL sequence of (ii); (b) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15, or (iii) the VH sequence of (i) and the VL sequence of (ii); (c) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, or (iii) the VH sequence of (i) and the VL sequence of (ii); or (d) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 18, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19, or (iii) the VH sequence of (i) and the VL sequence of (ii). A5. An anti-latent TGF-β1 antibody according to A4, comprising a VH sequence of SEQ ID NO: 12, 14, 16, or 18. A6. An anti-latent TGF-β1 antibody according to A4 or A5, comprising a VL sequence of SEQ ID NO: 13, 15, 17, or 19. A7. The anti-latent TGF-β1 antibody according to any one of A4 to A6, comprising: (a) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 13; (b) the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 15; (c) the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 17; or (d) The VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:19. A8. Anti-latent TGF-β1 antibodies, including: (a) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 13; (b) the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 15; (c) the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 17; or (d) The VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:19. A9. An anti-latent TGF-β1 antibody according to any one of A1 to A8, which is a human antibody, a humanized antibody, or a chimeric antibody. A10. The anti-latent TGF-β1 antibody according to any one of A1 to A9, which is a full-length IgG antibody, preferably a full-length IgG1 antibody. A11. An anti-latent TGF-β1 antibody according to any one of A1 to A9, which is a bispecific antibody. A12. The anti-latent TGF-β1 antibody according to any one of A1 to A11, which comprises a modified IgG1 Fc region having a reduced effector function compared to the wild-type IgG1 Fc region. A13. The anti-latent TGF-β1 antibody according to A12, wherein the modified IgG1 Fc region comprises an amino acid substitution at EU235 and / or EU236 according to the EU index. A14. The anti-latent TGF-β1 antibody according to A12 or A13, wherein the modified IgG1 Fc region comprises amino acid substitutions of L235R and G236R according to the EU index. A15. The anti-latent TGF-β1 antibody according to any one of A12 to A14, wherein the modified IgG1 Fc region further has enhanced FcRn-binding activity compared to the wild-type IgG1 Fc region. A16. The anti-latent TGF-β1 antibody according to A15, wherein the modified IgG1 Fc region comprises one or more amino acid substitutions at positions selected from the group consisting of EU428, EU434, EU438, and EU440 according to the EU index. A17. The anti-latent TGF-β1 antibody according to A15 or A16, wherein the modified IgG1 Fc region comprises the following amino acid substitutions: M428L, N434A, Q438R, and S440E. A18. The anti-latent TGF-β1 antibody according to any one of A1 to A11, which comprises a modified IgG1 Fc region containing the amino acid substitutions K214R, L235R, and G236R. A19. The anti-latent TGF-β1 antibody according to any one of A1 to A11, comprising a modified IgG1 Fc region containing the amino acid substitutions K214R, L235R, G236R, M428L, N434A, Q438R, and S440E. A20. The anti-latent TGF-β1 antibody according to any one of A1 to A11, which is an antibody fragment. A21. Anti-latent TGF-β1 antibodies, including: (a) the full-length heavy chain sequence of SEQ ID NO:47 and the full-length light chain sequence of SEQ ID NO:60; (b) the full-length heavy chain sequence of SEQ ID NO:48 and the full-length light chain sequence of SEQ ID NO:61; (c) the full-length heavy chain sequence of SEQ ID NO:49 and the full-length light chain sequence of SEQ ID NO:62; (d) the full-length heavy chain sequence of SEQ ID NO:50 and the full-length light chain sequence of SEQ ID NO:63; (e) the full-length heavy chain sequence of SEQ ID NO: 51 and the full-length light chain sequence of SEQ ID NO: 64; (f) the full-length heavy chain sequence of SEQ ID NO: 52 and the full-length light chain sequence of SEQ ID NO: 65; (g) the full-length heavy chain sequence of SEQ ID NO: 53 and the full-length light chain sequence of SEQ ID NO: 66; or (h) The full-length heavy chain sequence of SEQ ID NO:54 and the full-length light chain sequence of SEQ ID NO:67. A22. The anti-latent TGF-β1 antibody according to any one of A1 to A21, wherein the latent TGF-β1 is human latent TGF-β1, mouse latent TGF-β1, or cynomolgus monkey latent TGF-β1. A23. An anti-latent TGF-β1 antibody according to any one of A1 to A22, which binds to human latent TGF-β1, mouse latent TGF-β1, and cynomolgus monkey latent TGF-β1. A24. An anti-latent TGF-β1 antibody according to any one of A1 to A23, which binds to the latency associated peptide (LAP) region of latent TGF-β1. A25. An immunoconjugate comprising the anti-latent TGF-β1 antibody according to any one of A1 to A24 and a cytotoxic agent. A26. An isolated nucleic acid encoding the anti-latent TGF-β1 antibody according to any one of A1 to A24. A27. A vector containing the nucleic acid according to A26. A28. A host cell comprising the nucleic acid according to A26 or the vector according to A27. A29. A method for producing an anti-latent TGF-β1 antibody, comprising culturing the host cell according to A28 so that the antibody is produced. A30. The method according to A29, further comprising the step of recovering the antibody from the host cells. B1. An anti-latent TGF-β1 antibody according to any one of A1 to A24 or an immunoconjugate according to A25 for use as a pharmaceutical. B2. An anti-latent TGF-β1 antibody according to any one of A1 to A24 or an immunoconjugate according to A25 for use in the treatment of fibrosis or cancer. B3. Use of an anti-latent TGF-β1 antibody according to any one of A1 to A24 or an immunoconjugate according to A25 in the manufacture of a medicament for the treatment of fibrosis or cancer. B4. An anti-latent TGF-β1 antibody according to any one of A1 to A24 or an immunoconjugate according to A25, for use in combination with an additional therapeutic agent, preferably an immune checkpoint inhibitor, for the treatment of cancer. B5. An anti-latent TGF-β1 antibody or immunoconjugate according to B4, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody. B6. The anti-latent TGF-β1 antibody or immunoconjugate of B4, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. B7. An anti-latent TGF-β1 antibody or immunoconjugate according to any one of B4 to B6, wherein the immune checkpoint inhibitor is administered simultaneously with the anti-latent TGF-β1 antibody or immunoconjugate. B8. An anti-latent TGF-β1 antibody or immunoconjugate according to any one of B4 to B6, wherein the immune checkpoint inhibitor is administered before or after administration of the anti-latent TGF-β1 antibody or immunoconjugate. C1. A pharmaceutical preparation comprising an anti-latent TGF-β1 antibody according to any one of A1 to A24 or an immunoconjugate according to A25, and a pharmaceutically acceptable carrier. C2. The pharmaceutical formulation of C1, further comprising an additional therapeutic agent, preferably an immune checkpoint inhibitor. C3. The pharmaceutical formulation of C2, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody. C4. The pharmaceutical formulation of C2, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. C5. A pharmaceutical formulation according to any one of C1 to C4 for use in the treatment of fibrosis or cancer. C6. A pharmaceutical formulation according to any one of C1 to C4 for use in combination with an additional therapeutic agent, preferably an immune checkpoint inhibitor, for the treatment of cancer. C7. The pharmaceutical formulation of C6, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody. C8. The pharmaceutical formulation of C6, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. C9. The pharmaceutical formulation described in any one of C6 to C8, wherein the immune checkpoint inhibitor is administered simultaneously with the pharmaceutical formulation. C10. A pharmaceutical formulation according to any one of C6 to C8, wherein the immune checkpoint inhibitor is administered before or after administration of the pharmaceutical formulation. D1. A pharmaceutical formulation comprising an immune checkpoint inhibitor and a pharmaceutically acceptable carrier, for use in combination with an anti-latent TGF-β1 antibody of any one of claims A1 to A24 or an immunoconjugate of claim A25 for the treatment of cancer. D2. The pharmaceutical formulation of D1, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody. D3. The pharmaceutical formulation of D1, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. D4. The pharmaceutical formulation according to any one of D1 to D3, wherein the anti-latent TGF-β1 antibody or immunoconjugate is administered simultaneously with the pharmaceutical formulation. D5. The pharmaceutical formulation according to any one of D1 to D3, wherein the anti-latent TGF-β1 antibody or immunoconjugate is administered before or after administration of the pharmaceutical formulation. E1. A method for treating an individual with fibrosis or cancer, comprising administering to the individual an effective amount of an anti-latent TGF-β1 antibody described in any one of A1 to A24 or an immunoconjugate described in A25. E2. The method of E1, further comprising administering to the individual an additional therapeutic agent, preferably an immune checkpoint inhibitor. E3. The method of E1 or E2, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody. E4. The method of E3, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. E5. The method of any one of E1-E4, wherein the immune checkpoint inhibitor is administered simultaneously with an anti-latent TGF-β1 antibody or immunoconjugate. E6. The method of any one of E1-E4, wherein the immune checkpoint inhibitor is administered before or after administration of the anti-latent TGF-β1 antibody or immunoconjugate. [Brief explanation of the drawings]

[0013] [Figure 1A] Figure 1A shows the results of antibody binding to latent TGF-β1 on the cell surface of BaF3 cells. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 1B]Figure 1B shows the results of antibody binding to cell surface latent TGF-β1 on FreeStyle™ 293-F cells. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 2A] Figure 2A shows the results of antibody binding to mouse mature TGF-β1. GC1008-F1332m represents an anti-mature TGF-β antibody as a positive control. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 2B] Figure 2B shows the results of antibody binding to human mature TGF-β1. GC1008-F1332m represents an anti-mature TGF-β antibody as a positive control. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 2C] Figure 2C shows the results of antibody binding to mouse LAP. GC1008-F1332m represents an anti-mature TGF-β antibody. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 3A] Figure 3A shows the results of antibody activity against spontaneous mouse latent TGF-β1 activation. mSLC represents recombinant mouse latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 3B] Figure 3B shows the results of antibody activity against spontaneous human latent TGF-β1 activation. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 4A] Figure 4A shows the results of antibody activity against plasmin (PLN)-mediated mouse latent TGF-β1 activation. mSLC represents recombinant mouse latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 4B] Figure 4B shows the results of antibody activity against plasmin (PLN)-mediated human latent TGF-β1 activation. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 5A] Figure 5A shows the results of antibody activity against kallikrein (PLK)-mediated mouse latent TGF-β1 activation. mSLC represents recombinant mouse latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 5B]Figure 5B shows the results of antibody activity against kallikrein (PLK)-mediated human latent TGF-β1 activation. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 6A] Figure 6A shows the results of antibody activity against matrix metalloproteinase (MMP) 2-mediated human latent TGF-β1 activation. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 6B] Figure 6B shows the results of antibody activity against matrix metalloproteinase (MMP) 9-mediated human latent TGF-β1 activation. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 7A] Figure 7A shows the results of antibody activity against plasmin (PLN)-mediated cleavage of mouse latent TGF-β1. Cam represents camostat, a protease inhibitor used as a control. AE04 (hT0947AE04-SG191), AE07 (hT0947AE07-SG191), AE08 (hT0947AE08-SG191), and AE09 (hT0947AE09-SG191) represent anti-latent TGF-β1 antibodies. [Figure 7B]Figure 7B shows the results of antibody activity against plasmin (PLN)-mediated cleavage of human latent TGF-β1. Cam represents camostat, a protease inhibitor used as a control. AE04 (hT0947AE04-SG191), AE07 (hT0947AE07-SG191), AE08 (hT0947AE08-SG191), and AE09 (hT0947AE09-SG191) represent anti-latent TGF-β1 antibodies. [Figure 8] Figure 8 shows the results of antibody activity against integrin-mediated mouse TGF-β1 activation in mouse PBMCs. IC17-hIgG1 represents an anti-KLH antibody as a negative control. GC1008-F1332m represents an anti-mature TGF-β antibody as a positive control. RGE represents the RGE peptide. RGD represents the RGD peptide as a positive control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies. [Figure 9] Figure 9 shows tumor growth curves for the isotype control treatment group (●), anti-PD-L1 treatment group (■), hT0947AE04-mF18 treatment group (▲), and hT0947AE04-mF18 + anti-PD-L1 treatment group (×). Each point represents the mean tumor volume for each group (N=10). [Figure 10] Figure 10 shows the tumor growth curves for the isotype control treatment group (●), anti-PD-L1 treatment group (■), hT0947AE04-mF18 treatment group (▲), and hT0947AE04-mF18 + anti-PD-L1 treatment group (×). Each point represents the mean tumor volume for each group (N=10). [Figure 11] Figure 11 shows the tumor growth curves for the vehicle-treated group (●), anti-PD-L1-treated group (■), hT0947AE04-mF18 + anti-PD-L1-treated group (×), hT0947AE07-SG181 + anti-PD-L1-treated group (▲), and hT0947AE08-SG181 + anti-PD-L1-treated group (○). Each point represents the mean tumor volume for each group (N=10). [Figure 12] Figure 12 shows the results of hydroxyproline content in the kidney. Monoclonal antibodies were evaluated in a unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis model. The sham-operated group represents a non-disease-induced control. IC17dk-SG181 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191 represent anti-latent TGF-β1 antibodies. [Figure 13] Figure 13 shows the tumor growth curves for the vehicle-treated group (●), anti-mouse PD-L1 antibody-treated group (■), hT0947AE07-SG191 (10 mg / kg) + anti-mouse PD-L1 antibody-treated group (▲), and hT0947AE07-SG191 (30 mg / kg) + anti-mouse PD-L1 antibody-treated group (○). Each point represents the mean tumor volume for the corresponding group (N=10 in each group). DETAILED DESCRIPTION OF THE INVENTION

[0014] Description of Aspects I. Definition An "acceptor human framework," for purposes of this specification, is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0015] The term "binding activity" refers to the strength of the total noncovalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when a binding pair reflects a monovalent 1:1 interaction, the binding activity is specifically referred to as intrinsic binding affinity (affinity). When one member of a binding pair is capable of both monovalent and polyvalent binding, the binding activity is the sum of the respective binding strengths. The binding activity of molecule X to its partner Y can generally be expressed by the dissociation constant (KD) or the "amount of analyte bound per unit amount of ligand" (hereinafter sometimes referred to as "binding amount"). Those skilled in the art will generally understand that a lower dissociation constant indicates higher binding activity, and a higher "amount of analyte bound per unit amount of ligand" or "binding amount" indicates higher binding activity. Avidity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring avidity are described below.

[0016] An "avidity matured," "affinity matured," "avidity increased (enhanced)," or "affinity increased (enhanced)" antigen-binding molecule or antibody refers to an antibody that has one or more alterations (e.g., substitutions) in one or more hypervariable regions (HVRs), which improve the antigen-binding activity of the antigen-binding molecule or antibody against its antigen, compared to a parent antigen-binding molecule or antibody that does not have such alterations.

[0017] The terms "anti-latent TGF-β1 antibody" or "antibody capable of binding to latent TGF-β1" refer to an antibody capable of binding to latent TGF-β1 with sufficient binding activity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeted to latent TGF-β1. In one embodiment, an "antibody capable of binding to latent TGF-β1" is an antibody that specifically binds to latent TGF-β1. In one embodiment, the degree of binding activity of an anti-latent TGF-β1 antibody to an unrelated, non-latent TGF-β1 protein is less than about 10% of the binding activity of the antibody to latent TGF-β1, as measured (e.g., by radioimmunoassay (RIA)). In certain embodiments, an antibody capable of binding to TGF-β1 has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, the anti-latent TGF-β1 antibody binds to an epitope of latent TGF-β1 that is conserved among latent TGF-β1 from different species.

[0018] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. The term "antibody" also encompasses any antigen-binding molecule comprising an immunoglobulin variable heavy chain and / or variable light chain structure.

[0019] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0020] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen by 50% or more in a competition assay, or conversely, a reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0021] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer. In one example, the cancer is resistant to and / or shows limited response to immune checkpoint inhibitors.

[0022] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0023] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0024] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents, as disclosed below.

[0025] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0026] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0027] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0028] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0029] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0030] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0031] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0032] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0033] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0034] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0035] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0036] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0037] An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0038] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0039] An "isolated nucleic acid encoding an anti-latent TGF-β1 antibody" or a "nucleic acid encoding an anti-latent TGF-β1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including nucleic acid molecules carried on a single vector or separate vectors, and including nucleic acid molecules present in one or more locations in a host cell.

[0040] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0041] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0042] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0043] The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0044] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0045] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0046] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0047] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0048] The term "TGF-β1," as used herein, unless otherwise indicated, refers to any native TGF-β1 from any vertebrate source, including mammals, e.g., primates (e.g., humans), and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed TGF-β1 and any form of TGF-β1 resulting from intracellular processing. The term also encompasses naturally occurring variants of TGF-β1, such as splice variants or allelic variants. The amino acid sequence of an exemplary human TGF-β1 preproprotein is set forth in SEQ ID NO: 68 (NCBI RefSeq: NP_000651.3), and the nucleic acid sequence encoding an exemplary human TGF-β1 is set forth in SEQ ID NO: 69 (NCBI RefSeq: NM_000660.6). The amino acid sequence of an exemplary mouse TGF-β1 preproprotein is set forth in SEQ ID NO: 70 (NCBI RefSeq: NP_035707.1), and the nucleic acid sequence encoding an exemplary mouse TGF-β1 is set forth in SEQ ID NO: 71 (NCBI RefSeq: NM_011577.2). The amino acid sequence of an exemplary cynomolgus monkey TGF-β1 preproprotein is set forth in SEQ ID NO: 72 (NCBI RefSeq: XP_005589396.1), and the nucleic acid sequence encoding an exemplary cynomolgus monkey TGF-β1 is set forth in SEQ ID NO: 73 (NCBI RefSeq: XM_005589339.2). The term "TGF-β1" encompasses both latent TGF-β1 and mature TGF-β1.

[0049] The term "latent TGF-β1," as used herein, refers to any TGF-β1 that is unable to form a latent TGF-β1 complex ("cell surface latent TGF-β1," LLC, or SLC (see below)) and / or bind to its receptor. Transforming growth factor β1 (TGF-β1) is a member of the TGF-β superfamily. Like other members of the TGF-β superfamily, TGF-β is synthesized as a precursor protein that forms a homodimer that interacts with its latency-associated peptide (LAP) and latent TGF-β binding protein (LTBP) to form a larger complex called the large latent complex (LLC). The amino acid sequence of an exemplary latent human TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30-390 of SEQ ID NO:68. The amino acid sequence of an exemplary mouse latent TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30-390 of SEQ ID NO:70. An exemplary amino acid sequence of latent cynomolgus TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30 to 390 of SEQ ID NO:72.

[0050] The complex formed by the TGF-β homodimer and its LAP is called the small latent complex (SLC). This latent complex maintains TGF-β in an inactive form that cannot bind to its receptor. SLC can be covalently linked to an additional protein, latent TGF-β binding protein (LTBP), to form the large latent complex (LLC). Four different LTBP isoforms are known: LTBP-1, LTBP-2, LTBP-3, and LTBP-4. LTBP-1, LTBP-3, and LTBP-4 have been reported to bind to SLC (see, for example, Rifkin et al., J. Biol. Chem. 2005 Mar. 4;280(9):7409-12). SLC can also be covalently linked to other additional proteins, such as repeat-dominant glycoprotein A (GARP) or leucine-rich repeat-containing protein 33 (LRRC33). GARP and LRRC have transmembrane domains and bind to LAP on the cell surface (see, e.g., Wang et al., Mol Biol Cell. 2012 Mar;23(6):1129-39). Regarding LLC, it has been reported that LLC covalently binds to the extracellular matrix (ECM) through the N-terminus of LTBP (see, e.g., Saharinen et al., Cytokine Growth Factor Rev. 1999 Jun;10(2):99-117). In some embodiments, latent TGF-β1 bound to the ECM on the cell surface is referred to as "cell surface latent TGF-β1."

[0051] The terms "active TGF-β1," "mature TGF-β1," or "active mature TGF-β1," as used herein, refer to any TGF-β1 homodimer that does not form a latent TGF-β1 complex (LLC or SLC) and can bind to its receptor. The TGF-β1 activation process involves the release of LLC from the ECM, followed by further proteolysis of LAP, which releases active TGF-β to its receptor. A wide range of proteases, including plasmin (PLN), prekallikrein (PLK), matrix metalloproteinase (MMP) 2, MMP 9, MMP 13, MMP 14, thrombin, tryptase, and calpain, are known to cleave latent TGF-β and release active TGF-β. In the context of the present invention, these proteases may be collectively referred to as "(latent) TGF-β-cleaving proteases" or "(latent) TGF-β1-cleaving proteases." In addition to proteases, thrombospondin-1 (TSP-1), neuropilin-1 (Nrp1), ADAMSTS1, and F-spondin activate latent TGF-β. Alternatively, mechanical stretching can induce integrins (preferably integrin αVβ8 and / or αVβ6) to activate TGF-β by binding to the RGD motif present in LAP, inducing the release of mature TGF-β from its latent complex form.

[0052] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0053] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0054] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0055] II. Compositions and Methods In one aspect, the present invention is based, in part, on anti-latent TGF-β1 antibodies and their uses. In certain embodiments, antibodies that bind to TGF-β1 are provided. The antibodies of the present invention are useful, for example, in the diagnosis or treatment of fibrosis, preferably myocardial fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, skin fibrosis, ocular fibrosis, and myelofibrosis. The antibodies of the present invention are also useful, for example, in the diagnosis or treatment of cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0056] A. Exemplary Anti-Latent TGF-β1 Antibodies In one aspect, the present invention provides an isolated antibody that binds to latent TGF-β1. In a further embodiment, the anti-latent TGF-β1 antibody binds to the latency associated protein (LAP) region of latent TGF-β1. An example of the LAP region includes amino acids 30-278 of human TGF-β1 preproprotein (SEQ ID NO: 1). LAP, as described above, is a component of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody binds to the latent TGF-β1 region of latent TGF-β1. -8 nM or less, 10 -9 nM or less, or 10 -10 It binds to latent TGF-β1 with a dissociation constant (KD) of less than nM.

[0057] In one aspect, the anti-latent TGF-β1 antibody binds to latent TGF-β1 that forms LLC and / or latent TGF-β1 that forms a complex with GARP or LRRC33. In a specific embodiment, the anti-latent TGF-β1 antibody binds to cell surface latent TGF-β1, which is latent TGF-β1 bound to the extracellular matrix (ECM) on the cell surface. In another aspect, the anti-latent TGF-β1 antibody binds to latent TGF-β1, where the LAP region of latent TGF-β1 is not linked to LTBP and forms a small latent complex (SLC). In a specific embodiment, the SLC is present in a soluble form. In some embodiments, the anti-latent TGF-β1 antibody binds to latent TGF-β1 in a concentration of 10 -8 nM or less, 10 -9 nM or less, or 10 -10 It binds to latent TGF-β1 (cell surface latent TGF-β1, LLC or SLC) with a dissociation constant (KD) of less than nM.

[0058] In one aspect, an anti-latent TGF-β1 antibody inhibits the activation of latent TGF-β1. As used herein, the term "activation" of latent TGF-β1 refers to any process in which mature TGF-β1 is released from LAP, a component of latent TGF-β1. Activation of latent TGF-β1 can be detected, for example, by measuring mature TGF-β1 and / or mature TGF-β1 activity using various techniques known in the art or described herein. In some embodiments, an anti-latent TGF-β1 antibody inhibits the release of mature TGF-β1 from latent TGF-β1. As described above, it has been reported that mature TGF-β1 is released from latent TGF-β1 by activators such as proteases, integrins, and other non-protease activators. Non-limiting examples of proteases that activate latent TGF-β1 include plasmin (PLN), prekallikrein (PLK), matrix metalloproteinase (MMP) 2, and MMP9. In some embodiments, anti-latent TGF-β1 antibodies inhibit protease-mediated and / or integrin-mediated release of mature TGF-β1 from latent TGF-β1. As described above, proteases cleave the LAP region of latent TGF-β1, releasing mature TGF-β1. In some embodiments, the cleavage site by PLN and / or PLK is located within a fragment consisting of amino acids 56 to 59 of the LAP polypeptide.

[0059] In one aspect, an anti-latent TGF-β1 antibody inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP portion of latent TGF-β1. In some embodiments, an anti-latent TGF-β1 antibody inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1, allowing proteases to cleave the LAP region of latent TGF-β1 while the anti-latent TGF-β1 antibody is bound to the LAP region. In some embodiments, an anti-latent TGF-β1 antibody does not block protease access to latent TGF-β1, particularly to the cleavage sites by PLN and / or PLK. In other embodiments, an anti-latent TGF-β1 antibody does not bind to the protease cleavage sites, particularly the cleavage sites by PLN and / or PLK, of the LAP portion of latent TGF-β1.

[0060] In some embodiments, an anti-latent TGF-β1 antibody that inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1 is an antibody that (i) inhibits cleavage of the LAP region mediated by one or more proteases, but (ii) does not inhibit cleavage of the LAP region mediated by other proteases. For example, an anti-latent TGF-β1 antibody (1-i) inhibits MMP2- and / or MMP9-mediated release of mature TGF-β1 by inhibiting MMP2- and / or MMP9-mediated cleavage of the LAP portion of latent TGF-β1, or (1-ii) inhibits PLN- and / or PLK-mediated release of mature TGF-β1 without inhibiting PLN- and / or PLK-mediated cleavage of the LAP portion of latent TGF-β1. Alternatively, the anti-latent TGF-β1 antibody (2-i) inhibits PLN- and / or PLK-mediated release of mature TGF-β1 by inhibiting PLN- and / or PLK-mediated cleavage of the LAP portion of latent TGF-β1, and (2-ii) inhibits MMP2- and / or MMP9-mediated release of mature TGF-β1 without inhibiting MMP2- and / or MMP9-mediated cleavage of the LAP portion of latent TGF-β1. Alternatively, the anti-latent TGF-β1 antibody (3-i) inhibits PLN- and / or PLK-mediated release of mature TGF-β1 without inhibiting PLN- and / or PLK-mediated cleavage of the LAP portion of latent TGF-β1, and (3-ii) inhibits MMP2- and / or MMP9-mediated release of mature TGF-β1 without inhibiting MMP2- and / or MMP9-mediated cleavage of the LAP portion of latent TGF-β1.

[0061] In some embodiments, antibodies that "inhibit activation of latent TGF-β1" include antibodies that result in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more decrease in activation of TGF-β1. In other embodiments, antibodies that "inhibit protease-mediated release of mature TGF-β1 from latent TGF-β1" include antibodies that result in at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more decrease in protease-mediated release of mature TGF-β1 from latent TGF-β1. In a further embodiment, antibodies that inhibit protease-mediated release of mature TGF-β1 from latent TGF-β1 "without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1" include antibodies that result in a reduction of 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of protease-mediated cleavage of the LAP region of latent TGF-β1.

[0062] In some embodiments, an anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1. As used herein, when an anti-latent TGF-β1 antibody "stabilizes" the structure of the LAP region, the LAP region bound by the anti-latent TGF-β1 antibody is maintained in a specific structure that cannot release mature TGF-β1. In further embodiments, latent TGF-β1 stabilized by an anti-latent TGF-β1 antibody can be activated by integrins (preferably integrin αVβ8 and / or integrin αVβ6). In certain embodiments, the LAP region stabilized by an anti-latent TGF-β1 antibody is either cleaved or not cleaved by a protease. In some embodiments, the anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1, allowing proteases to cleave the LAP region while the anti-latent TGF-β1 antibody is bound to the LAP region of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1 without blocking protease access to latent TGF-β1, particularly to the cleavage sites by PLN and / or PLK. In other embodiments, the anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1 without blocking protease access to latent TGF-β1, particularly to the cleavage sites by MMP2 and / or MMP9.

[0063] In one aspect, an anti-latent TGF-β1 antibody does not bind to mature TGF-β1. In some embodiments, an anti-latent TGF-β1 antibody binds to latent TGF-β1 with a higher binding avidity than to mature TGF-β1. In certain embodiments, an antibody of the present invention binds to latent TGF-β1 with a binding avidity that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times higher than to mature TGF-β1.

[0064] In one aspect, the anti-latent TGF-β1 antibody does not inhibit or does not significantly inhibit integrin-mediated TGF-β1 activation, i.e., integrin-mediated release of mature TGF-β1 from latent TGF-β1. Preferably, the integrin herein is integrin αVβ8 and / or integrin αVβ6. In some embodiments, antibodies that "do not inhibit or do not significantly inhibit integrin-mediated TGF-β1 activation" include antibodies that result in a reduction of integrin-mediated TGF-β1 activation, i.e., integrin-mediated release of mature TGF-β1 from latent TGF-β1, by 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one aspect, anti-latent TGF-β1 antibodies produce reduced or fewer toxicity and / or side effects associated with anti-TGF-β antagonists. In some embodiments, anti-latent TGF-β1 antibodies such as those described herein have a superior safety-efficacy profile compared to agents that are active against mature TGF-β1 or agents that are active against latent TGF-β1 but inhibit both protease-mediated and integrin-mediated activation of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibodies of the present disclosure have reduced cardiotoxicity while having superior or comparable efficacy to anti-mature TGF-β1 antibodies. Without being bound by any theory, the anti-latent TGF-β1 antibodies of the present disclosure do not inhibit or do not significantly inhibit integrin-mediated TGF-β1 activation, thereby reducing or minimizing toxicity and / or side effects resulting from (i) integrin-mediated TGF-β1 activation or (ii) inhibition of TGF-β1 signaling at sites where TGF-β1 is activated by integrins. Therefore, the anti-latent TGF-β1 antibodies of the present disclosure can be administered to a subject in need thereof in therapeutically effective amounts without causing side effects, particularly cardiac toxicity. Such an approach would thus expand the dosage range that can achieve both efficacy and safety / tolerability in patients. Accordingly, the present invention provides a method for treating a disease associated with TGF-β1 signaling by administering to a subject an effective amount of an anti-latent TGF-β1 antibody that does not inhibit or does not significantly inhibit integrin-mediated TGF-β1 activation. The present invention encompasses the use of anti-latent TGF-β1 antibodies to reduce the toxicity and / or side effects associated with TGF-β1 inhibition in subjects.In some embodiments, toxicity and / or side effects may include cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, bone / cartilage toxicity, reproductive toxicity, and nephrotoxicity.In some embodiments, cardiovascular toxicity includes, but is not limited to, cardiac valve lesions, such as bleeding, inflammation, degeneration and proliferation of valvular interstitial cells.In some embodiments, toxicity and / or side effects may include bleeding.In some embodiments, the toxicity and / or side effects may include skin lesions or tumors, hi some embodiments, the toxicity and / or side effects may include tumor progression.

[0065] In some embodiments, the anti-latent TGF-β1 antibody of the invention: binds to latent TGF-β1; binds to latent TGF-β1, forming SLC; binds to latent TGF-β1, forming LLC; binds to latent TGF-β1 in complex with GARP or LRRC33; binds to cell surface latent TGF-β1; Binds to the LAP region of latent TGF-β1; binds to LAP; 10 -8 nM or less, 10 -9 nM or less, or 10 -10 Binds to latent TGF-β1 with a sub-nM dissociation constant (KD); inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1; does not inhibit protease-mediated cleavage of the LAP region of latent TGF-β1; does not inhibit or does not significantly inhibit integrin-mediated release of mature TGF-β1 from latent TGF-β1; and / or Anti-TGF-β1 antagonists, such as anti-mature TGF-β antibodies, result in reduced or fewer toxicity and / or side effects. In a further embodiment, the anti-latent TGF-β1 antibody of the invention: Monoclonal antibodies; human, humanized, or chimeric antibodies; full-length IgG antibodies; and / or antibody fragments is.

[0066] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25.

[0067] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31.

[0068] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37.

[0069] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0070] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25.

[0071] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31.

[0072] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37.

[0073] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0074] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2, and HVR-H3 of the VH sequence set forth in SEQ ID NO: 12, and HVR-L1, HVR-L2, and HVR-L3 of the VL sequence set forth in SEQ ID NO: 13, wherein the HVRs are defined by (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b) and / or (c).

[0075] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2, and HVR-H3 of the VH sequence set forth in SEQ ID NO: 14, and HVR-L1, HVR-L2, and HVR-L3 of the VL sequence set forth in SEQ ID NO: 15, wherein the HVRs are defined by (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b) and / or (c).

[0076] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2, and HVR-H3 of the VH sequence set forth in SEQ ID NO: 16, and HVR-L1, HVR-L2, and HVR-L3 of the VL sequence set forth in SEQ ID NO: 17, wherein the HVRs are defined by (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b) and / or (c).

[0077] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2, and HVR-H3 of the VH sequence set forth in SEQ ID NO: 18, and HVR-L1, HVR-L2, and HVR-L3 of the VL sequence set forth in SEQ ID NO: 19, wherein the HVRs are defined by (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b) and / or (c).

[0078] In any of the above embodiments, the anti-latent TGF-β1 antibody is humanized. In one embodiment, the anti-latent TGF-β1 antibody comprises the HVR of any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0079] In another aspect, an anti-latent TGF-β1 antibody comprises a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, 14, 16, or 18. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-latent TGF-β1 antibody comprising such a sequence retains the ability to bind to latent TGF-β1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 12, 14, 16, or 18. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-latent TGF-β1 antibody comprises a VH sequence in SEQ ID NO: 12, 14, 16, or 18, including post-translational modifications of said sequences. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, 26, 32, or 38, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21, 27, 33, or 39, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22, 28, 34, or 40. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0080] In another aspect, an anti-latent TGF-β1 antibody is provided, comprising a light chain variable region (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13, 15, 17, or 19. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-latent TGF-β1 antibody comprising such a sequence retains the ability to bind to latent TGF-β1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 13, 15, 17, or 19. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-latent TGF-β1 antibody comprises a VL sequence in SEQ ID NO: 13, 15, 17, or 19, including post-translational modifications of said sequences. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23, 29, 35, or 41, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, 30, 36, or 42, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25, 31, 37, or 43. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0081] In another aspect, an anti-latent TGF-β1 antibody is provided, comprising the VH of any of the above embodiments and the VL of any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0082] In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 15, respectively, including post-translational modifications of said sequences, including, but not limited to, modification of the N-terminal glutamine or glutamic acid at the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0083] In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17, respectively, including post-translational modifications of said sequences, including, but not limited to, modification of the N-terminal glutamine or glutamic acid at the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0084] In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 19, respectively, including post-translational modifications of said sequences, including, but not limited to, modification of the N-terminal glutamine or glutamic acid at the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0085] In a further aspect, the present invention provides antibodies that bind to the same epitope as the anti-latent TGF-β1 antibodies provided herein. For example, in certain embodiments, (1) An anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (2) an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31; (3) an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37; or (4) An anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43. Antibodies that bind to the same epitope as

[0086] In a further aspect, the present invention provides antibodies that bind to human, monkey, mouse, and / or rat latent TGF-β1. In certain embodiments, the present invention provides antibodies that bind to human, monkey, and mouse latent TGF-β1. In certain embodiments, the present invention provides antibodies that bind to human, monkey, and mouse latent TGF-β1 that forms SLC. In certain embodiments, the present invention provides antibodies that bind to human, monkey, and mouse latent TGF-β1 that forms LLC. In certain embodiments, the present invention provides antibodies that bind to human, monkey, and mouse latent TGF-β1 that forms LLC. In certain embodiments, the present invention provides antibodies that bind to human, monkey, and mouse latent TGF-β1 that forms a complex with GARP or LRRC33. In certain embodiments, the present invention provides antibodies that bind to human, monkey, and mouse cell surface latent TGF-β1.

[0087] In a further aspect, the present invention provides antibodies that bind to the same epitope as any one of the anti-latent TGF-β1 antibodies provided herein. The epitope may be present on human, monkey, mouse, and / or rat TGF-β1. For example, in certain embodiments, the present invention provides antibodies that bind to the same epitope as a reference antibody, wherein the reference antibody is (1) An anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (2) an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31; (3) an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37; or (4) An anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43. is.

[0088] In a further aspect, the present invention provides antibodies that compete with the anti-latent TGF-β1 antibodies provided herein for binding to human, monkey, mouse, and / or rat TGF-β1. For example, in certain embodiments, the antibodies compete with the anti-latent TGF-β1 antibodies provided herein for binding to human, monkey, mouse, and / or rat TGF-β1: (1) An anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (2) an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31; (3) an anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37; or (4) An anti-latent TGF-β1 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43. Antibodies that compete with

[0089] In a further aspect of the present invention, the anti-latent TGF-β1 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-latent TGF-β1 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1, IgG2, IgG3, or IgG4 antibody, or other antibody classes or isotypes defined herein. In a further aspect, the anti-latent TGF-β1 antibody also includes any antigen-binding molecule comprising the variable heavy and / or variable light chain structure of an immunoglobulin.

[0090] In further aspects, an anti-latent TGF-β1 antibody according to any of the above embodiments may incorporate, alone or in combination, any of the features described in items 1-7 below.

[0091] 1. Antibody binding activity In certain embodiments, the antibodies provided herein have a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (KD) of 1 M.

[0092] In one embodiment, the binding activity of an antibody is measured by a radiolabeled antigen binding assay (RIA) and expressed as KD. In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding activity of the Fab to the antigen is measured at the lowest concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0093] In one embodiment, antibody binding activity is measured using a ligand capture method, for example, using a BIACORE® T200 or BIACORE® 4000 (GE Healthcare, Uppsala, Sweden), which relies on surface plasmon resonance analysis as its measurement principle. The instrument is operated using BIACORE® Control Software. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the manufacturer's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate for an appropriate injection time. Binding activity is measured using a buffer containing 0.05% polysorbate 20 (also known as Tween®-20) as the measurement buffer, at a flow rate of 10 to 30 μL / min, preferably at a measurement temperature of 25°C or 37°C. In the case of measurements in which an antibody is used as a ligand to be captured by a ligand capture molecule, the antibody is injected and the target amount of antibody is captured, followed by the injection of a serial dilution of antigen and / or Fc receptor (analyte) prepared using the measurement buffer. In the case of measurements in which an antigen and / or Fc receptor is used as a ligand to be captured by a ligand capture molecule, the antigen and / or Fc receptor is injected and the target amount is captured, followed by the injection of a serial dilution of antibody (analyte) prepared using the measurement buffer.

[0094] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. Kinetic parameters can be calculated by simultaneously fitting the binding and dissociation sensorgrams with a 1:1 binding model, and the binding rate constant (k or ka), dissociation rate constant (k or k), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, especially when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (KD) can be calculated using a steady state model. As an additional parameter related to binding activity, the "amount of analyte bound per unit amount of ligand" can be calculated by dividing the amount of analyte bound at a specific concentration (resonance unit: RU) by the amount of captured ligand.

[0095] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0096] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0097] A single-domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0098] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0099] The present invention also relates to antigen-binding molecules that bind to TGF-β1, including, but not limited to, minibodies (low-molecular-weight antibodies) and scaffold proteins. In the present invention, any scaffold protein can be used as long as it is a peptide that has a stable three-dimensional structure and can at least bind to an antigen. Such peptides include, for example, fragments of antibody variable regions, fibronectin, protein A domains, LDL receptor A domains, lipocalins, and other molecules described in Nygren et al. (Current Opinion in Structural Biology, (1997) 7:463-469; Journal of Immunol Methods, (2004) 290:3-28), Binz et al. (Nature Biotech. (2005) 23:1257-1266), and Hosse et al. (Protein Science, (2006) 15:14-27). In the context of this specification, when referring to such antibodies, for example, "anti-latent TGF-β1 antibody" should be replaced with "anti-latent TGF-β1 antigen-binding molecule".

[0100] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0101] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and binding activity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or binding activity of the antibody.

[0102] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Further described in Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0103] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see Baca et al., J. Biol. Chem. 272:10678-10684 (1997)). and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0104] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0105] Human antibodies may be prepared by administering an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0106] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0107] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0108] 5. Library-derived Antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0109] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-avidity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0110] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0111] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for TGF-β1 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of TGF-β1. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing TGF-β1. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0112] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be constructed by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1993)). (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0113] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0114] As used herein, the antibody or fragment also includes a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to TGF-β1 and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0115] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding activity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0116] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0117] [Table 1]

[0118] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0119] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property compared to the parent antibody (e.g., increased binding activity, decreased immunogenicity) and / or will substantially retain a particular biological property of the parent antibody. An exemplary substitutional variant is an avidity-matured antibody, which can be conveniently generated using, for example, phage-display-based avidity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a particular biological activity (e.g., binding activity).

[0120] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve the binding activity of antibodies. Such modifications can be made in "hot spots" of HVRs, i.e., residues encoded by codons that frequently mutate during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigens, and the resulting mutant VH or VL can be tested for binding activity. Avidity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of avidity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired binding activity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0121] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, as long as such modifications do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may, for example, be outside the antigen-contacting residues of the HVRs. In certain embodiments of the above-described mutant VH and VL sequences, each HVR is unaltered or contains only one, two, or three amino acid substitutions.

[0122] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or can be excluded from the list of substitution candidates. Mutants can be screened to determine whether they contain desired properties.

[0123] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0124] b) Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0125] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, usually attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention may be performed to create antibody variants with specific improved properties.

[0126] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0127] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0128] c) Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions. In another embodiment, the human Fc variant may comprise a chimeric human Fc region sequence (e.g., a human IgG1 / 4 or human IgG2 / 4 Fc region), or a chimeric human Fc region sequence further comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0129] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated by the present invention, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0130] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0131] Certain antibody variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0132] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0133] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0134] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0135] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0136] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0137] e) Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3 dioxolane, poly-1,3,6 trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0138] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0139] B. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods or constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-latent TGF-β1 antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one aspect, a method for producing an anti-latent TGF-β1 antibody is provided, comprising culturing a host cell containing nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-latent TGF-β1 antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0140] For recombinant production of an anti-latent TGF-β1 antibody, nucleic acid encoding the antibody (e.g., such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0141] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0142] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0143] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0144] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0145] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include DHFR cells; MRC5 cells; and FS4 cells. -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0146] C. Assay The anti-latent TGF-β1 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0147] 1. Binding and Other Assays In one aspect, antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blot, surface plasmon resonance (e.g., BIACORE® or similar techniques (e.g., KinExa or OCTET®)), etc.

[0148] In another aspect, a competition assay can be used to identify antibodies that compete with any of the anti-latent TGF-β1 antibodies described herein, preferably hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191, for binding to latent TGF-β1. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as that bound by any of the anti-latent TGF-β1 antibodies described herein, preferably hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191. Details of exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). Methods for mapping epitopes include, but are not limited to, X-ray crystallography and alanine scanning mutagenesis.

[0149] In certain embodiments, when such a competing antibody is present in excess, it blocks (reduces) the binding of the reference antibody to TGF-β1 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95% or more. In certain embodiments, such a competing antibody binds to the same epitope (e.g., linear or conformational epitope) as that bound by the anti-latent TGF-β1 antibody described herein. In a further aspect, the reference antibody is hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191.

[0150] In an exemplary competitive assay, immobilized latent TGF-β1 is incubated in a solution containing a first labeled antibody (reference antibody) that binds to latent TGF-β1 (e.g., hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to latent TGF-β1. The second antibody may be present in a hybridoma supernatant. As a control, immobilized latent TGF-β1 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to TGF-β1, excess unbound antibody is removed, and the amount of label bound to the immobilized latent TGF-β1 is measured. If the amount of label bound to immobilized latent TGF-β1 in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to latent TGF-β1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0151] In certain embodiments, binding of an anti-latent TGF-β1 antibody to cell surface latent TGF-β1 can be tested by known methods, such as ELISA, Western blot, BIAcore, flow cytometry, etc. For example, cells expressing latent TGF-β1 can be contacted with either an anti-latent TGF-β1 antibody directly conjugated to PE or APC, or an unconjugated anti-latent TGF-β1 antibody followed by a PE- or APC-conjugated secondary antibody, and staining of cell surface latent TGF-β1 can be detected. See, for example, Oida et al., PLoS One. 2010 Nov 24;5(11):e15523; Su et al., Hum Mol Genet. 2015 Jul 15;24(14):4024-36.

[0152] 2. Activity Assay In one aspect, an assay for identifying biologically active anti-latent TGF-β1 antibodies is provided. Biological activities include, for example, inhibiting activation of latent TGF-β1, inhibiting release of mature TGF-β1 from latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 without blocking access of proteases to latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 while allowing proteases to cleave the LAP region of latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibiting or partially inhibiting integrin-mediated TGF-β1 activation, and the like. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0153] In certain embodiments, antibodies of the invention are tested for such biological activities.

[0154] In some embodiments, whether a test antibody inhibits the activation of latent TGF-β1, i.e., inhibits the release of mature TGF-β1 from latent TGF-β1, is determined by contacting an activator of latent TGF-β1 (e.g., a protease, an integrin, another non-protease activator, etc.) with latent TGF-β1 in the presence or absence of the test antibody, and then detecting mature TGF-β1 using a method known in the art, such as electrophoresis, chromatography, immunoblot analysis, enzyme-linked immunosorbent assay (ELISA), or mass spectrometry. In one example, the activator can be isolated (e.g., an isolated protease or integrin) and / or non-isolated (e.g., mouse, monkey, or human PBMCs containing integrins). It is also known that activation of latent TGF-β1, i.e., the release of mature TGF-β1 from latent TGF-β1, occurs even in the absence of an activator (spontaneous activation of latent TGF-β1). In some embodiments, whether a test antibody inhibits spontaneous activation of latent TGF-β1 is determined by incubating latent TGF-β1 with or without the test antibody and then detecting mature TGF-β1 using the methods described above. In some embodiments, if a decrease in the amount of mature TGF-β1 is detected in the presence of the test antibody (after contact with the test antibody) compared to the amount detected in the absence of the test antibody, the test antibody is identified as an antibody capable of inhibiting the activation of latent TGF-β1. In one example, the amount of mature TGF-β1, whether decreased or increased, can be measured in terms of the concentration of mature TGF-β1 (e.g., g / ml, mg / ml, microgram / ml, ng / ml, pg / ml, etc.). In another example, the amount of mature TGF-β, whether decreased or increased, can be measured in terms of the optical density (OD) (e.g., mm or nm wavelength, etc.) of a label directly or indirectly bound to mature TGF-β.

[0155] In certain embodiments, inhibition of TGF-β1 activation comprises at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more decrease in the amount of mature TGF-β1 in the assay compared to a negative control under similar conditions. In some embodiments, it represents at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more inhibition of TGF-β1 activation, i.e., inhibition of release of mature TGF-β1.

[0156] In some embodiments, whether a test antibody inhibits the activation of latent TGF-β1, i.e., inhibits the release of mature TGF-β1 from latent TGF-β1, is also determined by detecting mature TGF-β1 activity, such as binding to a TGF-β1 receptor or mediating signal transduction in cells expressing the TGF-β1 receptor. In some embodiments, binding of mature TGF-β1 to a TGF-β1 receptor can be detected using a receptor binding assay. In some embodiments, activity mediating TGF-β1 signal transduction can be determined by detecting activation of the TGF-β1 / Smad pathway. Cells useful in such assays can be those that express endogenous TGF-β1 receptor or those generated by transfection of cells with a TGF-β1 receptor gene. For example, HEK-Blue™ TGF-β cells used in the examples described herein or those transiently or stably genetically modified to express a transgene encoding a TGF-β1 receptor can be used. TGF-β1-mediated signaling can be detected at any level in the signaling pathway, for example, by examining the phosphorylation of Smad polypeptides, by examining the expression of genes regulated by TGF-β1, including receptor genes, or by measuring the proliferation of TGF-β1-dependent cells.

[0157] In some embodiments, activity mediating TGF-β1 signaling can also be determined by detecting activation of the TGF-β1 / Smad pathway by examining phosphorylation of Smad polypeptides (see, e.g., Fukasawa et al., Kidney International. 65(1):63-74 (2004) and Ganapathy et al., Molecular Cancer 26;9:122 (2010)). In other embodiments, activity in mediating TGF-β1 signaling can be determined by testing the ability of TGF-β to inhibit cell migration in "wounded" monolayer cultures of BAE cells, testing the ability of TGF-β to inhibit cell proliferation, testing the ability of TGF-β to suppress plasminogen activator (PA) activity, testing the ability of TGF-β to upregulate plasminogen activator inhibitor 1 (PAI-1), etc. (see Mazzieri et al., Methods in Molecular Biology 142:13-27 (2000)).

[0158] Inhibition of TGF-β1 activation can also be detected and / or measured using methods described and exemplified in the Examples. Using these or other suitable types of assays, test antibodies can be screened for their ability to inhibit TGF-β1 activation. In certain embodiments, inhibition of TGF-β1 activation comprises at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or greater decrease in TGF-β1 activation in the assay compared to a negative control under similar conditions. In some embodiments, it represents at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater inhibition of TGF-β1 activation. In certain embodiments, inhibition of TGF-β1 activation comprises at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more decrease in the amount of mature TGF-β1 detected in the assay compared to a negative control under similar conditions, hi some embodiments, it represents at least a 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more decrease in the amount of mature TGF-β1.

[0159] In some embodiments, whether a test antibody inhibits the cleavage of the LAP portion of latent TGF-β1 is determined by contacting latent TGF-β1 with a protease in the presence or absence of the test antibody, and then detecting the cleavage product of latent TGF-β1 and / or uncleaved latent TGF-β1 using various methods known in the art, such as electrophoresis, chromatography, immunoblot analysis, enzyme-linked immunosorbent assay (ELISA) or mass spectrometry. For example, if a protein tag (e.g., a FLAG tag, etc.) is added to the N-terminus of the LAP region of latent TGF-β1, the portion to which the protein tag is added will be cut off when protease-mediated cleavage occurs. Therefore, the cleavage product of latent TGF-β1 can be detected by detecting latent TGF-β1 (or the LAP region of latent TGF-β1) without the protein tag, and / or uncleaved latent TGF-β1 can be detected by detecting latent TGF-β1 with the protein tag.

[0160] As another example, if a protein tag (e.g., a FLAG tag) is added to the N-terminus of the LAP region of latent TGF-β1 and the location of the protease cleavage site is not near the N-terminus of the LAP region of latent TGF-β1, the LAP region bearing the protein tag will be shortened upon protease-mediated cleavage. Thus, the cleavage product of latent TGF-β1 can be detected by detecting latent TGF-β1 bearing the shortened LAP region bearing the protein tag (or the shortened LAP region of latent TGF-β1).

[0161] In some embodiments, if a decrease in the amount of cleavage products of latent TGF-β1 is detected in the presence of the test antibody (or after contact with the test antibody) compared to the amount detected in the absence of the test antibody, the test antibody is identified as an antibody capable of inhibiting the cleavage of latent TGF-β1. Conversely, if the amount of cleavage products of latent TGF-β1 is not significantly decreased in the presence of the test antibody (or after contact with the test antibody) compared to the amount detected in the absence of the test antibody, the test antibody is identified as an antibody that does not inhibit the cleavage of latent TGF-β1. In some embodiments, if an increase in the amount of uncleaved latent TGF-β1 is detected in the presence of the test antibody (or after contact with the test antibody) compared to the amount detected in the absence of the test antibody, the test antibody is identified as an antibody that can inhibit the cleavage of latent TGF-β1. Conversely, if the amount of uncleaved latent TGF-β1 is not significantly increased in the presence of the test antibody (or after contact with the test antibody) compared to the amount detected in the absence of the test antibody, the test antibody is identified as an antibody that does not inhibit the cleavage of latent TGF-β1. In certain embodiments, whether the test antibody blocks protease access to latent TGF-β1 is determined by a method for detecting protein interaction between a protease and latent TGF-β1, such as ELISA or surface plasmon resonance (e.g., BIACORE® or similar technology (e.g., KinExa or OCTET®)). If a decrease in the interaction between a protease and latent TGF-β1 is detected in the presence of the test antibody (or after contact with the test antibody) compared to the interaction detected in the absence of the test antibody, the test antibody is identified as an antibody that can block protease access to latent TGF-β1.

[0162] In certain embodiments, non-inhibition of cleavage of latent TGF-β1 comprises at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more increase in the amount of cleavage product of latent TGF-β1 in the assay compared to a negative control under similar conditions. In some embodiments, non-inhibition of cleavage of latent TGF-β1 comprises at least a 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less increase in the amount of uncleaved latent TGF-β1 in the assay compared to a negative control under similar conditions.

[0163] In some embodiments, the anti-latent TGF-β1 antibody may be subjected to other biological activity assays, e.g., to assess its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the target antigen and intended use of the antibody. For example, the biological effects of TGF-β1 blockade with anti-latent TGF-β1 antibodies can be evaluated in a unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis model (e.g., as described in Chevalier RL et al., Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy. Kidney Int. 2009 Jun;75(11):1145-1152), a choline-deficient L-amino acid-limited high-fat diet (CDAHFD)-induced NASH / liver fibrosis mouse model, a bleomycin (BLM)-induced pulmonary fibrosis mouse model, and / or a syngeneic tumor model (e.g., as described in Mariathasan S et al., TGF-beta attenuates tumor response to PD-L1 blockade by contributing to exclusion of T cells. Nature. 2018 Feb 22;554(7693):544-548). In further embodiments, anti-latent TGF-β1 antibodies may be subjected to biological activity assays described herein.

[0164] 3. Screening Method In one aspect, methods for screening antibodies of the present invention include various methods described herein and known in the art. For example, a method for screening anti-latent TGF-β1 antibodies includes the following steps: (a) contacting a biological sample containing latent TGF-β1 and a protease with a test antibody; (b) (i) detecting whether the test antibody inhibits cleavage of the LAP region of latent TGF-β1, and (ii) detecting whether the test antibody inhibits activation of latent TGF-β1; and (c) selecting test antibodies that inhibit the activation of latent TGF-β1 without inhibiting the protease-mediated cleavage of the LAP portion of latent TGF-β1;

[0165] Alternatively, instead of the above steps (b) and (c), the method for screening an anti-latent TGF-β1 antibody may include, for example, the following steps (b) and (c): (b) measuring (i) the amount of uncleaved latent TGF-β1 and (ii) the amount of mature TGF-β1; and (c) selecting a test antibody that inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1, if the amount of uncleaved latent TGF-β1 does not significantly increase and the amount of mature TGF-β1 decreases compared to when the test antibody is not present. Alternatively, instead of the above steps (b) and (c), the method for screening an anti-latent TGF-β1 antibody may include, for example, the following steps (b) and (c): (b) measuring (i) the amount of cleavage products of latent TGF-β1 and (ii) the level of mature TGF-β1 activity; and (c) selecting a test antibody that inhibits protease-mediated activation of latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1 if the amount of cleavage product is not significantly reduced and the level of mature TGF-β1 activity is reduced compared to when the test antibody is not present. Furthermore, the present invention provides a method for producing an anti-latent TGF-β1 antibody, which method comprises, for example, the following steps (d) and (e) in addition to the above steps (a) to (c): (d) obtaining amino acid sequence information of the anti-latent TGF-β1 antibody selected in step (c); and (e) introducing a gene encoding the anti-latent TGF-β1 antibody into a host cell.

[0166] In this context, the term "not significantly increased / decreased" in the phrases "the amount of uncleaved latent TGF-β1 is not significantly increased" and "the amount of cleavage products (of latent TGF-β1) is not significantly decreased" means that the level / degree of increase / decrease may be zero, or may not be zero but may be near zero, or may be technically negligible or sufficiently low to be considered as realistic / substantially zero by those skilled in the art. For example, in an immunoblot analysis, if a researcher cannot detect or observe any significant signal / band (or a relatively high or strong signal) for uncleaved latent TGF-β1, the amount of uncleaved latent TGF-β1 is considered to be "not significantly increased" or the amount of cleavage products (of latent TGF-β1) is considered to be "not significantly decreased." In addition, the term "not significantly increased / decreased" is used interchangeably with the term "not substantially increased / decreased."

[0167] In some embodiments, whether a test antibody inhibits cleavage of the LAP region of latent TGF-β1 and whether a test antibody inhibits activation of latent TGF-β1 can be determined by various assays described herein and known in the art.

[0168] D. Immunoconjugates The present invention also provides immunoconjugates comprising the anti-latent TGF-β1 antibodies herein conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragment thereof), or a radioactive isotope).

[0169] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to: maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342). (1993); and see Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0170] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0171] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioactive isotopes include Pb and Lu. When radioactive conjugates are used for detection, they are used in combination with radioactive atoms for scintigraphic examinations (e.g., Tc-99m or 123 I), or spin labels (again, e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0172] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein linking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0173] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0174] E. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-latent TGF-β1 antibodies provided herein is useful for detecting the presence of TGF-β1, e.g., latent TGF-β1, in a biological sample. The term "detection," as used herein, encompasses quantitative or qualitative detection / measurement. In certain embodiments, the biological sample comprises a cell or tissue, such as serum, whole blood, plasma, a biopsy sample, a tissue sample, a cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, breast milk, colostrum, mammary gland secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, peritoneal fluid, ocular fluid, and mucus.

[0175] In one embodiment, an anti-latent TGF-β1 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of TGF-β1, e.g., latent TGF-β1, in a biological sample is provided. For example, the method for detecting the presence of latent TGF-β1 includes: (a) contacting a biological sample with an anti-latent TGF-β1 antibody of the invention described herein under conditions that allow the anti-latent TGF-β1 antibody to bind to latent TGF-β1; and (b) detecting whether a complex is formed between the anti-latent TGF-β1 antibody and latent TGF-β1; Includes.

[0176] Such a method can be an in vitro or in vivo method. In one embodiment, the anti-latent TGF-β1 antibody is used to select subjects suitable for treatment with the anti-latent TGF-β1 antibody, for example, when TGF-β1, for example, latent TGF-β1, is a biomarker for patient selection. That is, the anti-latent TGF-β1 antibody is useful as a diagnostic agent for targeting TGF-β1.

[0177] More specifically, the anti-latent TGF-β1 antibody is useful for diagnosing fibrosis, preferably myocardial fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, skin fibrosis, ocular muscle fibrosis, and bone marrow fibrosis. The anti-latent TGF-β1 antibody of the present invention is also useful for diagnosing cancer.

[0178] In some aspects, the present invention provides a method for inhibiting the release of mature TGF-β1 from latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1 in a biological sample, the method comprising contacting a biological sample containing latent TGF-β1 with an anti-TGF-β1 antibody of the present invention under conditions that allow the antibody to bind to latent TGF-β1.

[0179] In certain embodiments, labeled anti-latent TGF-β1 antibodies are provided, e.g., for detection / diagnostic purposes. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and indirectly detectable moieties (e.g., enzymes or ligands), e.g., through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C. 125 I, 3 H and 131Fluorophores such as I, rare earth chelates or those linked to fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0180] F. Pharmaceutical Formulations Pharmaceutical formulations of the anti-latent TGF-β1 antibodies described herein are prepared in the form of a lyophilized formulation or aqueous solution by mixing the antibody having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.; small (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of use thereof (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0181] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0182] In one aspect, the present invention provides a pharmaceutical formulation comprising an anti-latent TGF-β1 antibody for the treatment of fibrosis, preferably for the treatment of myocardial fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, skin fibrosis, ocular muscle fibrosis, and bone marrow fibrosis. The present invention also provides a pharmaceutical formulation comprising an anti-latent TGF-β1 antibody for the treatment of cancer.

[0183] The combinations herein may also contain more than one active ingredient as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to additionally provide an immune checkpoint inhibitor, as described below under "III. Combination Therapies."

[0184] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0185] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0186] Preparations to be used for in vivo administration are generally sterile, and sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0187] G. Therapeutic Methods and Compositions Any of the anti-latent TGF-β1 antibodies provided herein can be used in therapeutic methods. In one aspect, an anti-latent TGF-β1 antibody for use as a pharmaceutical is provided. In a further aspect, an anti-latent TGF-β1 antibody for use in treating cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc. is provided. In a specific embodiment, an anti-latent TGF-β1 antibody for use in a therapeutic method is provided. In a specific embodiment, the present invention provides an anti-latent TGF-β1 antibody for use in a method of treating an individual with cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc., comprising the step of administering to the individual an effective amount of an anti-latent TGF-β1 antibody. In one such embodiment, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the present invention provides an anti-latent TGF-β1 antibody for use in inhibiting protease-mediated activation of latent TGF-β1. In certain embodiments, the present invention provides an anti-latent TGF-β1 antibody for use in a method of inhibiting protease-mediated activation of latent TGF-β1 in an individual, comprising administering to the individual an effective amount of the anti-latent TGF-β1 antibody to inhibit protease-mediated activation of latent TGF-β1. The "individual" in any of the above embodiments is preferably a human.

[0188] In a further aspect, the present invention provides use of an anti-latent TGF-β1 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc. In a further embodiment, the medicament is for use in a method for treating cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc., comprising the step of administering an effective amount of the medicament to an individual having cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc. In one such embodiment, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the medicament is for inhibiting protease-mediated activation of latent TGF-β1. In a further embodiment, the medicament is for use in a method for inhibiting protease-mediated activation of latent TGF-β1 in an individual, comprising the step of administering to the individual an effective amount of the medicament to inhibit protease-mediated activation of latent TGF-β1. The "individual" in any of the above embodiments is preferably a human.

[0189] In a further aspect, the present invention provides a method for treating cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc. In one embodiment, the method comprises administering to an individual having such cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc., an effective amount of an anti-TGF-β1 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In some embodiments, the antibody and the agent are administered simultaneously. The "individual" in any of the above embodiments may be a human.

[0190] In a further aspect, the present invention provides a method for inhibiting protease-mediated activation of latent TGF-β1 in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-latent TGF-β1 antibody to inhibit protease-mediated activation of latent TGF-β1. In one embodiment, the "individual" is a human.

[0191] In a further aspect, the present invention provides pharmaceutical formulations (e.g., for use in any of the therapeutic methods described above) comprising any of the anti-latent TGF-β1 antibodies provided herein. In one embodiment, the pharmaceutical formulation comprises any of the anti-latent TGF-β1 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-latent TGF-β1 antibodies provided herein and at least one additional therapeutic agent (e.g., as described below).

[0192] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or repeated doses over various time periods, bolus administration, and pulse infusion.

[0193] The antibodies of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of agent delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will typically be used in the same dosages and by any route of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0194] The appropriate dose of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered prophylactically or therapeutically, previous medical history, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0195] It is understood that any of the products described herein may contain an immunoconjugate of the invention in place of, or in addition to, an anti-latent TGF-β1 antibody.

[0196] III. Combination Therapy

[0197] The anti-latent TGF-β1 antibodies of the present invention can be used alone or in combination with other agents in therapy, preferably for the treatment of cancer or fibrosis, more preferably for the treatment of cancer. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the antibody and the agent are administered simultaneously. In certain embodiments, the additional therapeutic agent is one or more immune checkpoint inhibitors, such as inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, CD160, CD57, CD244, LAG-3, CD272, KLRG1, CD26, CD39, CD73, CD305, TIGIT, TIM-3, and / or VISTA. In some embodiments, the immune checkpoint inhibitor is, for example, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CD160 antibody, an anti-CD57 antibody, an anti-CD244 antibody, an anti-LAG-3 antibody, an anti-CD272 antibody, an anti-KLRG1 antibody, an anti-CD26 antibody, an anti-CD39 antibody, an anti-CD73 antibody, an anti-CD305 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, and / or an anti-VISTA antibody. Preferably, the immune checkpoint inhibitor is a PD-1 axis binding antagonist. More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, or cemiplimab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab, preferably atezolizumab. Preferably, the combination therapy comprises an anti-latent TGF-β1 antibody of the present invention and atezolizumab. In some embodiments, the combination therapy comprising an anti-latent TGF-β1 antibody of the present invention and one or more immune checkpoint inhibitors has an additive or synergistic effect, e.g., an additive, combined, or synergistic anti-tumor effect, compared to monotherapy with an anti-TGFβ antibody or monotherapy with an immune checkpoint inhibitor.

[0198] In one aspect, the combination therapy of the present invention is for the treatment of cancer or fibrosis, preferably cancer. In one embodiment, the cancer is resistant to immune checkpoint inhibitors and / or shows limited response to immune checkpoint inhibitors. Without being bound by any theory, the lack of response of some immune checkpoint-resistant cancers and / or cancers that show limited response to immune checkpoint inhibitors is associated with the signature of TGF-β signaling in fibroblasts, particularly in patients in whom CD8+ T cells are excluded from the tumor parenchyma and instead found in the fibroblast- and collagen-rich peritumoral stroma. Therefore, an anti-latent TGF-β1 antibody combined with an immune checkpoint inhibitor can reduce TGF-β signaling in stromal cells, promoting T cell infiltration into the tumor core and exerting enhanced antitumor activity.

[0199] Programmed cell death protein 1 (PD-1; also known as CD274 or B7-H1) is a type I membrane protein that belongs to the CD28 / CTLA-4 family of T cell regulators. PD-1 has two ligands, PD-L1 and PD-L2, which belong to the B7 family. PD-1 and its ligands are thought to negatively regulate immune responses, including T cell responses. PD-L1 and PD-1 are highly expressed in several types of cancer and are thought to be involved in cancer immune evasion. Inhibitors that block the interaction between PD-1 and PD-L1, such as "immune checkpoint inhibitors," can enhance T cell responses and increase antitumor activity.

[0200] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, resulting in the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing) so as to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis. As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0201] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes, which are mediated by signaling through PD-1, so as to improve the function of dysfunctional T cells (e.g., enhance effector responses to antigen recognition). In a specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab), or AMP-224 or AMP-514 (MEDI0680). In another specific aspect, the PD-1 binding antagonist is selected from the group consisting of PDR001, REGN2810, BGB A317, and SHR-1210.

[0202] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L1 with its binding partners, such as PD-1 and B7-1. In one embodiment, the PD-L1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes, which are mediated by signaling through PD-L1, so as to improve the function of dysfunctional T cells (e.g., enhance effector responses to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In specific aspects, the anti-PD-L1 antibody is YW243.55.S70 (atezolizumab), MDX-1105, avelumab, MPDL3280A, or MEDI4736 (durvalumab).

[0203] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, the PD-L2 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that are mediated by signaling through PD-L2, so as to improve dysfunctional T cell function (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0204] Such combination therapy as described above encompasses combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration (where the antibody of the present invention can be administered before, simultaneously with, or after administration of one or more additional therapeutic agents). In one embodiment, administration of the anti-latent TGF-β1 antibody and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The antibody of the present invention can also be used in combination with radiation therapy.

[0205] In one aspect, when the above-mentioned combination therapy involves combined administration and two or more therapeutic agents are contained in the same pharmaceutical formulation, the pharmaceutical formulation herein comprises, for example, an anti-latent TGF-β1 antibody of the present invention and one or more of the above-mentioned immune checkpoint inhibitors. Preferably, the pharmaceutical formulation herein comprises an anti-latent TGF-β1 antibody of the present invention, a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab), and a pharmaceutically acceptable carrier.

[0206] In one aspect, the present invention provides an anti-latent TGF-β1 antibody for use in combination with an additional therapeutic agent for the treatment of one or more diseases. In another aspect, the present invention provides a pharmaceutical formulation comprising an anti-latent TGF-β1 antibody for use in combination with an additional therapeutic agent for the treatment of one or more diseases. In one embodiment, the one or more diseases are cancer and / or fibrosis, preferably cancer. In one embodiment, the additional therapeutic agent is one or more immune checkpoint inhibitors as described above. Preferably, the present invention provides an anti-latent TGF-β1 antibody for use in combination with a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab) for the treatment of cancer.

[0207] In one aspect, the present invention provides a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab) for use in combination with an anti-latent TGF-β1 antibody for the treatment of one or more diseases. In another aspect, the present invention provides a pharmaceutical formulation comprising a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab) for use in combination with an anti-latent TGF-β1 antibody for the treatment of one or more diseases. In one embodiment, the one or more diseases is cancer and / or fibrosis, preferably cancer.

[0208] IV. Products, Kits

[0209] A.Product In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders (e.g., fibrosis and cancer) is provided. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition (e.g., fibrosis and cancer), and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an antibody or immunoconjugate of the present invention. The label or package insert indicates that the composition is used for treating the selected condition (e.g., fibrosis and cancer). The article of manufacture may further comprise: (a) a first container with a composition containing an antibody / immunoconjugate of the invention contained therein; and (b) a second container with a composition containing an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture of this aspect of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition (e.g., fibrosis and cancer). Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0210] It will be appreciated that any of the above-described products may contain an immunoconjugate of the present invention instead of or in addition to an anti-latent TGF-β1 antibody.

[0211] B. Kit The present disclosure provides kits for use in methods for treating, preventing, and / or diagnosing disorders described herein, particularly for treating individuals with fibrosis or cancer. The kits include an anti-latent TGF-β1 antibody of the present disclosure, an immunoconjugate comprising the anti-latent TGF-β1 antibody, an isolated nucleic acid encoding the anti-latent TGF-β1 antibody, or a vector comprising the nucleic acid, all of which are disclosed herein or produced by a method of the present disclosure. The kits may further include any therapeutic agent, such as an immune checkpoint inhibitor, including an anti-PD-L1 antibody, as exemplified in "III. Combination Therapies" herein. The kits may be packaged with additional pharmaceutically acceptable carriers or vehicles disclosed herein or instructions for use of the kit. Similar to the products described herein, the kits may include materials useful for treating fibrosis or cancer; a container and a label on or package insert associated with the container; the composition alone or in combination with another composition effective in treating fibrosis or cancer; a sterile access port, etc. The kit may further comprise a label or package insert indicating that the composition can be used to treat fibrosis or cancer. Alternatively, or in addition, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further comprise other buffers, diluents, filters, needles, and syringes, and other materials desirable from a commercial or user standpoint. [Example]

[0212] The following are examples of the methods and compositions of the present invention. In light of the above general description, it will be understood that various other embodiments may be practiced.

[0213] Example 1: Antigen expression and purification (1-1) Expression and purification of latent TGF-β1 The sequences used for expression and purification are as follows: FLAG-tagged human latent TGF-β1 (SEQ ID NOs: 1 and 2), FLAG-tagged mouse latent TGF-β1 (SEQ ID NOs: 3 and 4), and FLAG-tagged cynomolgus monkey latent TGF-β1 (SEQ ID NOs: 5 and 6). Each of these FLAG-tagged latent TGF-β1s contains, from the N-terminus to the C-terminus, a signal sequence derived from rat serum albumin (SEQ ID NO: 7), a FLAG tag, and the sequence of latent TGF-β1. These FLAG-tagged latent TGF-β1s each have a substitution of Ser at the 30th Cys residue, which corresponds to the "C33S mutation" (see, for example, Yoshinaga K, et al. Perturbation of transforming growth factor (TGF)-β1 association with latent TGF-beta binding protein yields inflammation and tumors. Proc Natl Acad Sci U S A. 2008;105(48):18758-18763).

[0214] FLAG-tagged human latent TGF-β1 (hereafter referred to as "human latent TGF-β1 (SLC)" or "human latent TGF-β1"), FLAG-tagged mouse latent TGF-β1 (hereafter referred to as "mouse latent TGF-β1 (SLC)" or "mouse latent TGF-β1"), or FLAG-tagged cynomolgus monkey latent TGF-β1 (hereafter referred to as "monkey latent TGF-β1 (SLC)" or "monkey latent TGF-β1") was transiently expressed in FreeStyle 293-F or Expi293F cell lines (Thermo Fisher Scientific). Conditioned media expressing human, mouse, or monkey latent TGF-β1 (SLC) were applied to a column packed with anti-FLAG M2 affinity resin (Sigma), and latent TGF-β1 (SLC) was eluted with FLAG peptide (Sigma). Fractions containing human, mouse, or monkey latent TGF-β1 (SLC) were collected and then applied to a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1x PBS, after which the fractions containing human, mouse, or monkey latent TGF-β1 (SLC) were pooled and stored at -80°C.

[0215] (1-2) Expression and purification of mouse latency-associated peptide (LAP) The sequences used for expression and purification are as follows: FLAG-tagged mouse LAP (SEQ ID NO: 8, 9) having, from the N-terminus to the C-terminus, a rat serum albumin-derived signal sequence (SEQ ID NO: 7), a FLAG tag, and the sequence of latency-associated protein (LAP). The 30th Cys residue of FLAG-tagged LAP was substituted with Ser, which corresponds to a "C33S mutation." The expression and purification of FLAG-tagged mouse LAP (SEQ ID NO: 8, 9) (hereinafter referred to as "recombinant mouse latency-associated protein (LAP)") were carried out in exactly the same manner as described in Example (1-1).

[0216] Example 2: Humanization and optimization of anti-TGF-β1 antibodies (2-1) Humanization The chimeric parent antibody, anti-TGF-β antibody TBA0947, was humanized as follows. First, the heavy and light chain variable regions of the humanized antibody were designed using the variable region of TBA0947 and human germline frameworks. Next, the polynucleotides of the designed heavy and light chain variable regions were cloned into expression vectors containing the heavy chain constant region SG181 sequence (SEQ ID NO: 10) and the light chain constant region SK1 sequence (SEQ ID NO: 11), respectively. The humanized antibody was transiently expressed in FreeStyle 293-F cells (Thermo Fisher Scientific) and subjected to Biacore analysis. Humanized antibodies that showed Biacore binding activity at least similar to that of the parent antibody were selected.

[0217] (2-2) Optimization The humanized antibodies obtained in Example (2-1) were optimized to produce hT0947AE04, hT0947AE07, hT0947AE08, and hT0947AE09, which have improved binding activity to latent TGF-β1 (SLC). Briefly, comprehensive mutagenesis was performed on all residues in the complementarity-determining regions (CDRs) of both the heavy and light chains. Each amino acid was substituted with the original amino acid and 18 other natural amino acids, excluding cysteine. The variants were transiently expressed in FreeStyle 293-F cells (Thermo Fisher Scientific) and purified from the culture supernatant for Biacore analysis. Target variants with improved binding activity to both human and mouse latent TGF-β1 (SLC) were selected. Subsequently, antibodies with combinations of these mutations in the CDRs were constructed.

[0218] (2-3) Optimized antibody amino acid sequence The amino acid sequences of the variable regions of hT0947AE04, hT0947AE07, hT0947AE08, and hT0947AE09 were determined as follows: The heavy chain variable region of hT0947AE04 (hT0947AE04H) comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region of hT0947AE04 (hT0947AE04L) comprises the amino acid sequence of SEQ ID NO: 13. The heavy chain variable region of hT0947AE07 (hT0947AE07H) comprises the amino acid sequence of SEQ ID NO: 14, and the light chain variable region of hT0947AE07 (hT0947AE07L) comprises the amino acid sequence of SEQ ID NO: 15. The heavy chain variable region of hT0947AE08 (hT0947AE08H) comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region of hT0947AE08 (hT0947AE08L) comprises the amino acid sequence of SEQ ID NO: 17. The heavy chain variable region of hT0947AE09 (hT0947AE09H) comprises the amino acid sequence of SEQ ID NO: 18, and the light chain variable region of hT0947AE09 (hT0947AE09L) comprises the amino acid sequence of SEQ ID NO: 19.

[0219] The amino acid sequences of the CDRs (HVRs) of hT0947AE04, hT0947AE07, hT0947AE08, and hT0947AE09 were identified according to Kabat as follows: hT0947AE04 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 23, 24 and 25, respectively. hT0947AE07 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 31, respectively. hT0947AE08 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 33 and 34, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 35, 36 and 37, respectively. hT0947AE09 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively.

[0220] (2-4) Preparation of full-length heavy and light chains Multiple amino acid substitutions were introduced into the heavy chain constant region SG1 (SEQ ID NO: 44). SG1 is a wild-type human IgG1 heavy chain constant region lacking the last two C-terminal amino acids, Gly-Lys (GK). As a result, SG181 (SEQ ID NO: 10) and SG191 (SEQ ID NO: 45) were generated. SG181 contains the amino acid substitutions L235R / G236R (amino acid substitutions that reduce effector function) and K214R, as indicated by the EU index. SG191 contains the amino acid substitutions L235R / G236R (amino acid substitutions that reduce effector function), M428L / N434A (amino acid substitutions that improve binding to FcRn), Q438R / S440E (amino acid substitutions that reduce rheumatoid factor binding), and K214R, as indicated by the EU index. Furthermore, a mouse IgG heavy chain constant region mF18 (SEQ ID NO: 46) containing P235K / S239K (amino acid substitutions that reduce effector function) was constructed.

[0221] Each of the heavy chain variable regions was combined with the heavy chain constant region SG181 (SEQ ID NO: 10), SG191 (SEQ ID NO: 45), or mF18 (SEQ ID NO: 46), thus generating full-length heavy chain sequences with the following amino acid sequences: (a1) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 47, which includes hT0947AE04H (heavy chain variable region) and SG181 (heavy chain constant region). (a2) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 48, including hT0947AE07H (heavy chain variable region) and SG181 (heavy chain constant region). (a3) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 49, including hT0947AE08H (heavy chain variable region) and SG181 (heavy chain constant region). (a4) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 50, including hT0947AE09H (heavy chain variable region) and SG181 (heavy chain constant region). (b1) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 51, including hT0947AE04H (heavy chain variable region) and SG191 (heavy chain constant region). (b2) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 52, including hT0947AE07H (heavy chain variable region) and SG191 (heavy chain constant region) (b3) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 53, including hT0947AE08H (heavy chain variable region) and SG191 (heavy chain constant region). (b4) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 54, including hT0947AE09H (heavy chain variable region) and SG191 (heavy chain constant region). (c1) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 55, including hT0947AE04H (heavy chain variable region) and mF18 (heavy chain constant region) (c2) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 56, including hT0947AE07H (heavy chain variable region) and mF18 (heavy chain constant region); (c3) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 57, including hT0947AE08H (heavy chain variable region) and mF18 (heavy chain constant region) (c4) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 58, including hT0947AE09H (heavy chain variable region) and mF18 (heavy chain constant region).

[0222] Each of the light chain variable regions was combined with either the human IgG light chain constant region (kappa) SK1 (SEQ ID NO: 11) or the mouse IgG light chain constant region (kappa) mk1 (SEQ ID NO: 59), thus generating a full-length light chain sequence with the following amino acid sequence: (d1) A full-length light chain comprising the amino acid sequence of SEQ ID NO: 60, including hT0947AE04L (light chain variable region) and SK1 (light chain constant region). (d2) a full-length light chain comprising the amino acid sequence of SEQ ID NO: 61, including hT0947AE07L (light chain variable region) and SK1 (light chain constant region) (d3) a full-length light chain comprising the amino acid sequence of SEQ ID NO: 62, including hT0947AE08L (light chain variable region) and SK1 (light chain constant region) (d4) a full-length light chain comprising the amino acid sequence of SEQ ID NO: 63, including hT0947AE09L (light chain variable region) and SK1 (light chain constant region) (e1) A full-length light chain comprising the amino acid sequence of SEQ ID NO: 64, including hT0947AE04L (light chain variable region) and mk1 (light chain constant region). (e2) a full-length light chain comprising the amino acid sequence of SEQ ID NO: 65, including hT0947AE07L (light chain variable region) and mk1 (light chain constant region) (e3) A full-length light chain comprising the amino acid sequence of SEQ ID NO: 66, including hT0947AE08L (light chain variable region) and mk1 (light chain constant region). (e4) A full-length light chain comprising the amino acid sequence of SEQ ID NO: 67, including hT0947AE09L (light chain variable region) and mk1 (light chain constant region).

[0223] Next, each full-length heavy chain and light chain was combined to prepare the antibodies shown in Table 2. The prepared antibodies were named as shown in Table 2 and will be referred to herein by their respective names.

[0224] [Table 2]

[0225] Example 3: Biacore analysis for evaluating the binding activity of anti-latent TGF-β1 antibodies The binding activity of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191) to human, cynomolgus monkey, or mouse latent TGF-β1 (SLC) was measured using a Biacore 8k instrument (GE Healthcare). Mouse anti-human Ig kappa light chain antibody (BD Pharmingen) was immobilized on all flow cells of a CM5 sensor chip using an amine coupling kit (GE Healthcare). After capturing the antibody on the anti-kappa sensor surface at a capture level of approximately 20 RU (resonance units), human, cynomolgus monkey, or mouse latent TGF-β1 (SLC) prepared in Example (1-1) was added to the flow cell. All antibodies and analytes were prepared in ACES (pH 7.4) containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The assay temperature was set at 37°C. The sensor surface was regenerated with 10 mM Glycine-HCl (pH 2.1) after each cycle. Data were processed using Biacore Insight software, version 1.1.1.7442 (GE Healthcare), and binding activity was determined by fitting to a 1:1 binding model. The binding activity (ka, kd, and KD) of the anti-latent TGF-β1 antibodies to human, cynomolgus monkey, or mouse latent TGF-β1 is shown in Table 3.

[0226] [Table 3]

[0227] Example 4: Characterization of anti-latent TGF-β1 antibodies (4-1) Anti-latent TGF-β1 antibody bound to latent TGF-β1 on the cell surface The binding activity of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) to cell surface latent TGF-β1 was tested by FACS using Ba / F3 cells expressing mouse latent TGF-β1 or FreeStyle™ 293-F cells (ThermoFisher) expressing human latent TGF-β1. Anti-latent TGF-β1 antibodies (10 μg / mL each) were incubated with each cell line for 30 minutes at 4°C and then washed with FACS buffer (2% FBS, 2 mM EDTA in PBS). An anti-KLH antibody with a human IgG1 Fc region (IC17-hIgG1), which does not bind to either mouse or human latent TGF-β1, was used as a negative control. The cells were then incubated with Goat F(ab')2 anti-Human IgG, Mouse ad-PE (Southern Biotech, catalog 2043-09) at 4°C for 30 minutes and washed with FACS buffer. Data were acquired using FACSVerse (Becton Dickinson) and analyzed using FlowJo software (Tree Star) and GraphPad Prism software (GraphPad). As shown in Figure 1, all anti-latent TGF-β1 antibodies bound to mouse cell surface latent TGF-β1 expressed on Ba / F3 cells and human cell surface latent TGF-β1 expressed on FreeStyle™ 293-F cells.

[0228] (4-2) Anti-latent TGF-β1 antibody did not bind to mature TGF-β1 but bound to mouse LAP The binding activity of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) to mature TGF-β1 was tested by ELISA. A 384-well plate was coated with mouse or human mature TGF-β1 overnight at 4°C and then washed four times with PBS-T. After washing, the plate was blocked with blocking buffer (1x TBS / Tween-20 + 0.5% BSA + 1x Block Ace) at room temperature for at least 1 hour and then washed four times with PBS-T. After washing, the antibody solution was added to the plate and incubated for 2 hours at room temperature. The plate was then washed four times with PBS-T. After washing, diluted secondary antibody (goat anti-human IgG-HRP, Abcam, catalog ab98624) was added to the plate and incubated for 1 hour at room temperature. The plate was then washed four times with PBS-T. After washing, TMB solution was added to the plate and incubated for 15 minutes at room temperature. The reaction was then stopped by adding 1N sulfuric acid. Optical density (OD) was measured at 450 nm / 570 nm. An anti-KLH antibody (IC17-IgG1) was used as a negative control, and the anti-mature TGF-β antibody GC1008 (described in U.S. Patent No. 8,383,780) with a human IgG1 Fc region (GC1008-F1332m) was used as a positive control. As shown in Figures 2A and 2B, the anti-latent TGF-β1 antibody did not bind to either mouse mature TGF-β1 or human mature TGF-β1. Furthermore, the binding activity of the anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947A09-SG191) to mouse latency-associated protein (LAP) was tested by ELISA as described above. As shown in Figure 2C, the anti-latent TGF-β1 antibodies bound to mouse LAP.

[0229] (4-3) Anti-latent TGF-β1 antibody inhibited spontaneous activation of latent TGF-β1 Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were incubated for 1 hour at 37°C in the presence or absence of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). Anti-KLH antibody (IC17-IgG1) was used as a negative control. Spontaneous latent TGF-β1 activation and antibody-mediated inhibition of spontaneous latent TGF-β1 activation were analyzed by mature TGF-β1 ELISA (human TGF-β1 Quantikine ELISA Kit, R&D Systems) according to the manufacturer's protocol. As shown in Figure 3, the spontaneous activation of latent TGF-β1 was inhibited by anti-latent TGF-β1 antibody.

[0230] (4-4) Anti-latent TGF-β1 antibody inhibited plasmin (PLN)-mediated activation of latent TGF-β1 Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were incubated with human plasmin (Calbiochem) for 1 hour at 37°C in the presence or absence of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). The antibodies were preincubated with mouse or human latent TGF-β1 (SLC) for 30 minutes at room temperature before incubation with plasmin. An anti-KLH antibody (IC17-hIgG1) was used as a negative control. Plasmin-mediated activation of latent TGF-β1 and antibody-mediated inhibition were analyzed by mature TGF-β1 ELISA (Human TGF-β1 Quantikine ELISA Kit, R&D systems) according to the manufacturer's protocol. As shown in Figure 4, plasmin-mediated activation of latent TGF-β1 was inhibited by anti-latent TGF-β1 antibodies.

[0231] (4-5) Anti-latent TGF-β1 antibodies inhibited plasma kallikrein (PLK)-mediated activation of latent TGF-β1. Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were incubated with human kallikrein (Enzyme Research Laboratories) for 2 hours at 37°C in the presence or absence of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). The antibodies were preincubated with mouse or human latent TGF-β1 (SLC) for 30 minutes at room temperature before incubation with kallikrein. An anti-KLH antibody (IC17-hIgG1) was used as a negative control. Kallikrein-mediated activation of latent TGF-β1 and antibody-mediated inhibition were analyzed by mature TGF-β1 ELISA (Human TGF-β1 Quantikine ELISA Kit, R&D systems) according to the manufacturer's protocol. As shown in Figure 5, kallikrein-mediated activation of latent TGF-β1 was inhibited by anti-latent TGF-β1 antibodies.

[0232] (4-6) Anti-latent TGF-β1 antibodies inhibited MMP2- and MMP9-mediated human latent TGF-β1 activation Human latent TGF-β1 (SLC) prepared in Example (1-1) was incubated with activated metalloproteinase 2 (MMP2) or MMP9 (R&D Systems) for 2 hours at 37°C in the presence or absence of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). The antibodies were preincubated with human latent TGF-β1 (SLC) for 30 minutes at room temperature before incubation with MMP2 or MMP9. An anti-KLH antibody (IC17-hIgG1) was used as a negative control. MMP2- and MMP9-mediated human latent TGF-β1 activation and antibody-mediated inhibition were analyzed by mature TGF-β1 ELISA (Human TGF-β1 Quantikine ELISA Kit, R&D systems) according to the manufacturer's protocol. As shown in Figure 6, both MMP2-mediated and MMP9-mediated human latent TGF-β1 activation were inhibited by anti-latent TGF-β1 antibodies.

[0233] (4-7) Anti-latent TGF-β1 antibodies inhibited latent TGF-β1 activation without interfering with plasmin (PLN)-mediated cleavage of the latent TGF-β1 propeptide. Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were incubated with human plasmin (Calbiochem) for 1 hour at 37°C in the presence or absence of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). The antibodies were preincubated with mouse or human latent TGF-β1 (SLC) for 30 minutes at room temperature before incubation with plasmin. Camostat mesylate (TOCRIS), a serine protease inhibitor known to inhibit plasmin activity, was used as a control. The samples were mixed with 4x SDS-PAGE sample buffer (Wako), heated at 95°C for 5 minutes, and then loaded for SDS gel electrophoresis. Proteins were transferred to a membrane using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Latent TGF-β1 propeptide was detected using mouse anti-FLAG, M2-HRP antibody (Sigma-Aldrich). The membrane was incubated with ECL substrate, and images were acquired using an ImageQuant LAS 4000 (GE Healthcare). As shown in FIG. 7, cleavage of latent TGF-β1 propeptide by plasmin was not inhibited by anti-latent TGF-β1 antibody.

[0234] (4-8) Anti-latent TGF-β1 antibodies did not significantly inhibit integrin-mediated latent TGF-β1 activation in mouse PBMCs. To detect integrin-mediated latent TGF-β1 activation, we performed a mouse PBMC and HEK-Blue™ TGF-β cell coculture assay. Mouse PBMCs were isolated from mouse blood using Histopaque-1083 density gradient medium (Sigma-Aldrich). HEK-Blue™ TGF-β cells (Invivogen) expressing a Smad3 / 4-binding element (SBE)-inducible SEAP reporter gene can detect bioactive TGF-β1 (both mouse and human TGF-β1) by monitoring Smad3 / 4 activation. Active TGF-β1 stimulates the production and secretion of SEAP into the cell supernatant. The amount of secreted SEAP was assessed using QUANTI-Blue™ reagent (Invivogen).

[0235] HEK-Blue™ TGF-β cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 50 U / mL streptomycin, 50 μg / mL penicillin, 100 μg / mL normocin, 30 μg / mL blasticidin, 200 μg / mL HygroGold, and 100 μg / mL Zeocin. During the functional assay, the cell medium was replaced with assay medium (RPMI 1640 containing 10% FBS) and plated into a 96-well plate. Anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) and mouse PBMCs were then applied to the wells and incubated with the HEK-Blue™ TGF-β cells overnight. The cell supernatant was then mixed with QUANTI-Blue™, and the optical density (OD) at 620 nm was measured using a colorimetric plate reader. The RGD peptide (GRRGDLATIH, GenScript) is known to bind to integrins and function as a decoy integrin ligand to inhibit integrin-mediated TGF-β1 activation. Therefore, the RGD peptide was used as a positive control. Additionally, the RGE control peptide (GRRGELATIH, GenScript), which is known not to function as a decoy integrin ligand, was used as a negative control. An anti-KLH antibody (IC17-hIgG1) was used as a negative control. An anti-mature TGF-β1 antibody (GC1008-F1332m) was used as a positive control. F1332m is a human IgG1 heavy chain constant region containing amino acid substitutions that reduce effector function. As shown in Figure 8, anti-latent TGF-β1 antibodies did not significantly inhibit integrin-mediated TGF-β1 activation in mouse PBMCs.

[0236] Example 5: Antitumor activity of anti-latent TGF-β1 antibodies (1) The in vivo efficacy of the anti-latent TGF-β1 monoclonal antibody hT0947AE04-mF18, alone or in combination with an anti-PD-L1 antibody, was evaluated in a mouse allograft model using EMT6 mouse breast cancer cells and Balb / c mice, in which immune checkpoint inhibitor treatment alone has limited effect on tumor growth and survival (see Nature. 2018 Feb 22;554(7693):544-548).

[0237] (5-1) Establishment of a mouse allograft model The EMT6 mouse breast cancer cell line was obtained from the American Type Culture Collection (ATCC CRL-2755). Cells were cultured in RPMI-1640 medium (SIGMA) supplemented with 10% fetal bovine serum (FBS) (SIGMA) and 2 mM L-glutamine (SIGMA). Six-week-old specific pathogen-free Balb / c female mice were purchased from Charles River Japan and acclimated for two weeks before transplantation. EMT6 cells in the logarithmic growth phase were harvested, washed with Hank's balanced salt solution (HBSS) (SIGMA), and cultured at 1x10 6 The cells were resuspended in 50% HBSS and 50% Matrigel (Corning) at a concentration of 1 x 10 cells / mL. The left fifth mammary fat pad of each mouse was inoculated with 1 x 10 cells in 100 μL of HBSS:Matrigel (1:1). 5 EMT6 cells were transplanted. Mean tumor volume approximately 100-300 mm 3 After reaching a tumor mass of 1000 mg / kg (7 days after implantation), mice were randomly divided into groups based on tumor volume and body weight. Tumor volume was measured with a caliper and calculated as follows: Tumor volume (mm 3 ) = (1 / 2) x length (mm) x width (mm) 2

[0238] (5-2) Evaluation of antitumor activity After establishing the mouse model in Example (5-1), mice were treated with an isotype control antibody (a combination of mouse IgG1 antibody and rat IgG2b antibody, purchased from Bio X Cell), an anti-mouse PD-L1 antibody (rat IgG2b clone 10F.9G2, purchased from Bio X Cell), hT0947AE04-mF18, or a combination of hT0947AE04-mF18 and an anti-mouse PD-L1 antibody, as shown in Table 4. The antibodies were administered three times a week for three weeks. The first administration was intravenous, and the second and subsequent administrations were intraperitoneal.

[0239] [Table 4]

[0240] Tumor volumes were measured twice a week, and the results are shown in Figure 9.

[0241] Antitumor activity was also evaluated by tumor growth inhibition (TGI [%]). TGI [%] for a particular group on a particular day was calculated as follows: TGI[%] = {1-(T-T0) / (C-C0)} x 100 where "T" is the mean tumor volume of the group on the day of measurement, "T0" is the mean tumor volume of the group on the day of randomization, "C" is the mean tumor volume of Group 1 (isotype control) on the day of measurement, and "C0" is the mean tumor volume of Group 1 (isotype control) on the day of randomization. As a result, the TGI [%] values ​​14 days after the first administration were 51, 12, and 83, respectively, for the anti-mouse PD-L1 antibody (Group 2), hT0947AE04-mF18 (Group 3), and the combination of hT0947AE04-mF18 and anti-mouse PD-L1 antibody (Group 4). Therefore, a synergistic antitumor effect was observed between the anti-latent TGF-β1 (hT0947AE04-mF18) and the anti-PD-L1 antibody.

[0242] Survival curves were also plotted to assess the survival of each group. A "surviving" mouse was defined as follows: a mouse with a tumor volume of 1955 mm 3 As shown in Figure 10, combined treatment with hT0947AE04-mF18 and anti-mouse PD-L1 antibody (Group 4) significantly increased survival of mice compared to anti-mouse PD-L1 antibody-treated mice (Group 2) and hT0947AE04-mF18-treated mice (Group 3).

[0243] Example 6: Antitumor activity of anti-latent TGF-β1 antibodies (2) The in vivo efficacy of the anti-latent TGF-β1 monoclonal antibodies hT0947AE04-mF18, hT0947AE07-SG181, or hT0947AE08-SG181 in combination with an anti-PD-L1 antibody was evaluated in a mouse allograft model using EMT6 mouse breast cancer cells and Balb / c mice.

[0244] (6-1) Establishment of a mouse allograft model The EMT6 mouse breast cancer cell line was obtained from the American Type Culture Collection (ATCC CRL-2755). Cells were cultured in RPMI-1640 medium (SIGMA) supplemented with 10% fetal bovine serum (FBS; SIGMA) and 2 mM L-glutamine (SIGMA). Seven-week-old specific pathogen-free Balb / c female mice were purchased from Charles River Japan and acclimated for one week before transplantation. EMT6 cells in the logarithmic growth phase were harvested, washed with Hank's balanced salt solution (HBSS; SIGMA), and cultured at 1x10 6 The cells were resuspended in 50% HBSS and 50% Matrigel (Corning) at a concentration of 1 x 10 cells / mL. The left fifth mammary fat pad of a mouse was treated with 1 x 10 cells in 100 μL of HBSS:Matrigel (1:1). 5 EMT6 cells were transplanted. Mean tumor volume approximately 100-300 mm 3 After reaching a tumor mass of 1000 mg / kg (7 days after implantation), mice were randomly divided into groups based on tumor volume and body weight. Tumor volume was measured with a caliper and calculated as follows: Tumor volume (mm 3) = (1 / 2) x length (mm) x width (mm) 2

[0245] (6-2) Evaluation of antitumor activity After establishing the mouse model as described in Example (6-1), mice were treated with vehicle (150 mM NaCl / 20 mM His-HCl buffer, pH 6.0), anti-mouse PD-L1 antibody (rat IgG2b clone 10F.9G2, purchased from Bio X Cell), hT0947AE04-mF18 in combination with anti-mouse PD-L1 antibody, hT0947AE07-SG181 in combination with anti-mouse PD-L1 antibody, or hT0947AE08-SG181 in combination with anti-mouse PD-L1 antibody, as shown in Table 5. The antibodies were administered three times a week for three weeks. The first administration was intravenous, and subsequent administrations were intraperitoneal.

[0246] [Table 5]

[0247] Tumor volumes were measured twice a week, and the results are shown in Figure 11.

[0248] Antitumor activity was also evaluated by tumor growth inhibition (TGI [%]), which was calculated as {1-(T-T0) / (C-C0)} x 100, as in Example (5-2). The TGI [%] 14 days after the first administration in the anti-mouse PD-L1 antibody alone (Group 2), the combination of hT0947AE04-mF18 and anti-mouse PD-L1 antibody (Group 3), the combination of hT0947AE07-SG181 and anti-mouse PD-L1 antibody (Group 4), and the combination of hT0947AE08-SG181 and anti-mouse PD-L1 antibody (Group 5) were 64, 89, 86, and 76, respectively. Therefore, the anti-latent TGF-β1 antibody demonstrated efficacy in combination with an anti-PD-L1 antibody.

[0249] Example 7: In vivo efficacy of anti-latent TGF-β1 antibody in a UUO-induced mouse renal fibrosis model The in vivo efficacy of monoclonal antibodies hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191 was evaluated in a unilateral ureteral obstruction (UUO) mouse model, which is known to induce progressive renal fibrosis.

[0250] (7-1) Establishment of a UUO-induced mouse renal fibrosis model The in vivo efficacy of monoclonal antibodies hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191 was evaluated in a unilateral ureteral ligation (UUO) mouse model, which induces progressive renal fibrosis. Six-week-old specific pathogen-free C57BL / 6NTac male mice were purchased from Invivos Pte Ltd (Singapore) and acclimated for 1 week before treatment initiation. Animals were maintained at 20–26°C under a 12-hour light / 12-hour dark cycle with commercial standard chow (5P75; PMI Nutrition INT'L (LabDiet), MO, USA) and tap water ad libitum. Under isoflurane anesthesia, UUO surgery was performed. The left side of the abdomen was shaved, and a longitudinal incision was made in the skin. A second incision was made in the peritoneum, and the skin was retracted to expose the kidney. The kidney was then brought to the surface using forceps, and the left ureter was clamped in two places below the kidney with surgical silk. The ligated kidney was carefully returned to its correct anatomical position, after which the peritoneum and skin were sutured. Analgesics were administered to alleviate the animal's discomfort. In the sham-operated group, only the peritoneum and skin were incised and sutured.

[0251] (7-2) Evaluation of in vivo efficacy All monoclonal antibodies were administered intravenously at 15 mg / kg, three times a week, starting one day before surgery. In this study, anti-KLH antibody (IC17dk-SG181) was used as a negative control. A sham-operated group was administered anti-KLH antibody (IC17dk-SG181). Seven days after surgery, animals were weighed and sacrificed by exsanguination under isoflurane anesthesia. Blood samples were collected from the cardiac cavity or inferior vena cava and kept at -80°C until assay. Kidneys were immediately removed. For molecular analysis, portions of kidney tissue were flash-frozen in liquid nitrogen or dry ice. The kidney content of hydroxyproline, an amino acid found in collagen, was measured to assess extracellular matrix deposition in the tissue. Wet kidney tissue was dried at 95°C for 3 hours and weighed. 6N HCl (100 μL / 1 mg of dried tissue) was then added to the dried tissue and boiled overnight. The samples were washed using a filter, and 10 μL of each sample was plated onto a 96-well plate. The plates containing the samples were dried at 60°C, and hydroxyproline was measured using a hydroxyproline assay kit (BioVision). The results are shown in Figure 12. A significant increase in hydroxyproline content was observed in disease-induced kidneys, and all antibodies (hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191) inhibited renal fibrosis. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using Student's t-test. Differences were considered significant when the P value was <0.05.

[0252] Example 8: Toxicity evaluation of anti-latent TGF-β1 antibodies The potential toxicity of anti-latent TGF-β1 antibodies was evaluated in repeated-dose toxicity studies in normal mice and cynomolgus monkeys in comparison with the anti-mature TGF-β antibody GC1008-mF18 (anti-mature TGF-β antibody GC1008 with the mouse IgG Fc region mF18 (as described in U.S. Patent No. U.S. 8,383,780)). Because anti-latent TGF-β1 antibodies cross-react in mice and cynomolgus monkeys, mice and cynomolgus monkeys were selected as the animal species for evaluation in in vivo toxicity studies. See Table 6 for a summary of all toxicity studies.

[0253] [Table 6] IV = intravenous; Q2D = once every 2 days; Q2W = once every 2 weeks; NOAEL = no observed adverse effect level; a - Solvent, 150 mmol / L NaCl, 20 mmol / L histidine-HCl, pH 6.0 b - NOAEL is underlined c - solvent, 20 mmol / L histidine, 150 mmol / L arginine-aspartate, pH 6.0

[0254] In a 3-month mouse study (IV; 5 or 20 mg / kg Q2D, a total of 46 doses), anemia (20 mg / kg in the hT0947AE04-mF18 group; 5 and 20 mg / kg in the GC1008-mF18 group) and cardiac lesions (5 and 20 mg / kg in the GC1008-mF18 group; see Table 7) were observed. These findings were considered to be caused by on-target toxicity of TGF-β inhibition. Considering on-target toxicity in the 3-month mouse study, the NOAEL for hT0947AE04-mF18 was 5 mg / kg IV Q2D. Due to adverse effects in the 5 mg / kg group of GC1008-mF18, a NOAEL for GC1008-mF18 could not be determined under the conditions of the 3-month mouse study.

[0255] [Table 7] NE = not tested; ± = minimal; + = mild; 2+ = moderate. a - hT0947AE04-mF18-unrelated changes based on similar findings / degree in the vehicle control group (minor changes in 2 mice)

[0256] In a 6-week study in monkeys (IV; 10, 30, or 100 mg / kg Q2W, a total of 4 doses), no toxic changes were observed with IV administration of hT0947AE07-SG191, and the NOAEL was the highest dose of 100 mg / kg Q2W.

[0257] Example 9: Antitumor activity of anti-latent TGF-β1 antibodies (3) The in vivo efficacy of the anti-latent TGF-β1 monoclonal antibody hT0947AE07-SG191 in combination with an anti-mouse PD-L1 antibody was evaluated in an EMT6 murine breast cancer Balb / c mouse allograft model. The EMT6 mouse breast cancer cell line was obtained from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (SIGMA) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences Corporation). Six-week-old specific pathogen-free Balb / c female mice were purchased from Charles River Japan and acclimated for one week before transplantation. EMT6 cells in the logarithmic growth phase were harvested, washed with Hank's balanced salt solution (HBSS; SIGMA), and cultured at 1x10 6 The cells were resuspended in 50% HBSS and 50% Matrigel (Corning) at a concentration of 1 x 10 cells / mL. The left fifth mammary fat pad of each mouse was inoculated with 1 x 10 cells in 100 μL of HBSS:Matrigel (1:1). 5 EMT6 cells were transplanted. Mean tumor volume approximately 100-300 mm 3 After reaching 100 μm (7 days after implantation), the mice were randomly divided into groups based on tumor volume and body weight. Tumor volume was measured with a caliper and calculated as 1 / 2 × l × w 2(l = length, w = width) Mice were treated with vehicle (150 mM NaCl / 20 mM His-HCl buffer, pH 6.0), anti-mouse PD-L1 antibody (rat IgG2b clone 10F.9G2, purchased from Bio X cell, 10 mg / kg for the first dose, followed by 5 mg / kg thereafter), hT0947AE07-SG191 (10 mg / kg) in combination with anti-mouse PD-L1 antibody, or hT0947AE07-SG191 (30 mg / kg) in combination with anti-mouse PD-L1 antibody. The antibodies were administered intravenously for the first dose and intraperitoneally thereafter, three times weekly for two weeks. Tumor volumes were measured twice weekly. Antitumor activity was evaluated by tumor growth inhibition (TGI [%]), calculated as {1-(T-T0) / (C-C0)} × 100, where T is the mean tumor volume on the measurement day of the group and T is the mean tumor volume on the randomization day, and C and C are the mean tumor volumes of the vehicle control group. The results of this experiment are shown in FIG. The TGI [%] at 14 days after the first administration of the anti-mouse PD-L1 antibody alone, the combination of hT0947AE07-SG191 (10 mg / kg) with the anti-mouse PD-L1 antibody, and the combination of hT0947AE07-SG191 (30 mg / kg) with the anti-mouse PD-L1 antibody were 60, 77, and 80, respectively. hT0947AE07-SG191 demonstrated efficacy in combination with the anti-mouse PD-L1 antibody.

[0258] The present invention has been described in some detail herein by way of illustration and example for purposes of clarity of understanding, but the detailed descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety. [Industrial Applicability]

[0259] The present invention provides cross-species anti-latent TGF-β1 antibodies that inhibit protease-mediated activation of latent TGF-β1 without inhibiting integrin-mediated activation of latent TGF-β1. The present invention also provides combination therapies comprising anti-latent TGF-β1 antibodies and checkpoint inhibitors. The anti-latent TGF-β1 antibodies of the present invention, which can be administered in combination with checkpoint inhibitors, are expected to be useful in treating TGF-β1-related diseases such as fibrosis and cancer.

Claims

[Claim 1] The invention described herein.

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