Anti-latent tgf-beta1 antibodies and methods of use

Abstract

It is an object of the present invention to provide anti-latent TGF-beta 1 antibodies and methods of using such antibodies. The present invention provides an anti-latent TGF-[beta] 1 antibody which inhibits protease-mediated activation of latent TGF-[beta] 1 without inhibiting integrin-mediated activation of latent TGF-[beta] 1 or which partially inhibits activation of integrin-mediated latent TGF-[beta] 1, or the antibody inhibits protease-mediated latent TGF-beta 1 activation but has a small effect on inhibition of integrin-mediated latent TGF-beta 1 activation. The present invention also provides optimized anti-latent TGF-beta 1 antibodies wherein the pH dependent binding properties are improved. The anti-latent TGF-beta 1 antibody can be used to treat fibrosis or cancer. For example, the anti-latent TGF-beta1 antibodies may be used to treat renal fibrosis, hepatic fibrosis, and pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to anti-latent TGF-β1 antibodies and methods of use thereof. BACKGROUND

[0002] Transforming growth factor-β (transforming growth factor beta; TGF-β) is a member of the TGF-β superfamily of cytokines, which consists of TGF-β isoforms, activins, inhibins, Nodal, bone morphogenetic proteins (BMPs), anti-Mullerian hormone (AMH), and growth and differentiation factors (GDFs). Members of this superfamily are dimeric proteins with conserved structure and have pleiotropic functions in vitro and in vivo (NPLs 1, 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 (NPL 3). However, while TGF-β isoforms are typically dynamically regulated and involved in maintaining tissue homeostasis, TGF-β isoforms are often chronically overexpressed in disease states, including cancer, fibrosis, and inflammation, and this overproduction of TGF-β drives disease progression by modulating cell growth, migration, or phenotype.

[0003] Three separate TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) have been identified in mammals and share 70-82% homology at the amino acid level (NPL 4). All three TGF-β isoforms bind to TGF-β receptor type 2 (TGFR2) as a homodimer, which is the active form; TGFR2 then recruits and activates TGF-β receptor type 1 (TGFR1) to activate receptor signaling (NPL 5). However, the expression levels of the three isoforms vary according to tissue (NPL 6), and their functions are distinct, as evidenced by the phenotypes of knockout mice (NPLs 7-11).

[0004] Like other members of the TGF-β superfamily, TGF-β is synthesized as a precursor protein that forms a homodimer in interaction with its latency-associated peptide (LAP) and latency TGF-β binding protein (LTBP) to form a larger complex called large latency complex (LLC). The TGF-β gene encodes a prepro sequence consisting of a signal peptide, a propeptide ending with a proprotein convertase (PPC) cleavage site, and a mature TGF-β sequence. The PPC cleavage site is hydrolyzed by furin protease, yielding the individual TGF-β and propeptide-derived homodimer. These two homodimers remain non-covalently associated and are secreted. This latent complex keeps TGF-β in an inactive form that cannot bind its receptor (NPLs 12, 13). The TGF-β activation process involves the release of LLC from ECM, followed by further proteolysis of LAP to release active TGF-β to its receptor (NPL 3). Latent TGF-β is cleaved by a wide range of proteases including plasmin (PLN), plasma kallikrein (PLK), matrix metalloproteinases (MMPs) 2 and 9 (NPL 14) and by thrombospondin 1 (TSP-1) (NPL 15) to release active TGF-β. Without wishing to be bound by any theory, MMP2 as well as MMP9 proteolytically cleave latent TGF-β1 and release mature TGF-β1 from the latent form. Both MMP2 and MMP9 are synthesized as inactive MMP precursors. The MMP2 precursor is activated by a complex of membrane type 1 MMP (MT1-MMP / MMP14) and metalloproteinase tissue inhibitor 2 (TIMP-2). The MMP9 precursor is activated by a protease cascade involving interactions of plasmin and stromelysin 1 (MMP-3). Plasmin is produced from its zymogen to active MMP-3. Active MMP-3 cleaves the propeptide from the 92-kDa MMP-9 precursor, generating the 82-kDa enzymatically active enzyme. The cleavage site of MMP has not been specifically determined; however, it was reported that MMP3 specifically cleaves the site between 79Ala and 80Leu of latent TGF-β, thereby activating TGF-β (WO2005 / 023870). Alternatively, integrins can activate TGF-β by binding to the RGD motif present in LAP upon mechanical stretch to induce release of mature TGF-β from its latent complex (NPLs 16, 17).

[0005] Upon activation, the dimeric TGF-β ligand binds to the extracellular domains of both type I and type II receptors and induces close proximity, placing the intracellular serine / threonine kinase domains of the receptors in a conformation that promotes phosphorylation and subsequent activation of the type I receptor. This activation of the type I receptor leads to signaling through at least two seemingly independent pathways: the SMAD-dependent canonical pathway and the SMAD-independent or non-canonical pathway. In the SMAD-dependent pathway, activation of TGFR1 (also known as ALK5) leads to phosphorylation of SMAD proteins. SMAD2 and SMAD3 are substrates of TGFR1. Upon receptor phosphorylation, SMADs translocate to the nucleus, where they interact with other transcription factors to modulate the transcriptional response (NPL 18). In the non-canonical pathway, the activated TGF-β receptor complex 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-κB (NF-κB). Thus, the cellular response to TGF-β signaling results from a dynamic combination of canonical and non-canonical signaling cascades.

[0006] Fibrosis, or accumulation of ECM molecules that make up scar tissue, is a common feature of chronic tissue injury. Renal fibrosis, liver fibrosis, and lung / pulmonary fibrosis are more common fibrotic diseases that collectively represent a huge unmet clinical need. TGF-β strongly promotes the production of extracellular matrix by mesenchymal cells, while it inhibits the growth of epithelial cells, which contributes to the pathogenesis of sclerotic diseases. Overexpression of the active form of TGF-β1 in the liver of transgenic mice was sufficient to induce fibrotic diseases in multiple organs (NPL 19). On the other hand, TGF-β also plays an important role in maintaining our health. For example, TGF-β suppresses the overproduction of proteases in the lung and prevents destruction of lung tissue leading to emphysema. In addition, TGF-β1 -deficient mice exhibit prenatal lethality (about 50% at 10.5 days post-coitus) or death of their offspring shortly after birth, with massive inflammatory lesions observed in many organs, including the lung (vasculitis, perivascular cuffing, and interstitial pneumonitis) and heart (endocarditis and myocarditis), suggesting that TGF-β1 plays a crucial role in maintaining immune homeostasis (NPL 7).

[0007] The results of studies using neutralizing antibodies against TGF-β and animal models have shown that it is possible to prevent or cure sclerotic diseases by inhibiting the action of TGF-β. Since TGF-β is produced as a precursor protein, there are several reported methods to prevent activation from the latent form. Another method to prevent activation from the latent form is to block the cleavage of proteases such as PLK and PLN using an inhibitor or antibody that binds to latent TGF-β. Several antibodies using this method of inhibiting TGF-β activation have been reported to prevent or treat liver cirrhosis (PTL 1). In addition, there have been some literature mentioning anti-LAP antibodies for treating cancer (PTL 2) and TGFβ1 binding immunoglobulins for treating TGFβ1 related disorders (PTL 3).

[0008] Some anti-latent TGF-β1 antibodies have been described (PTL 4, PTL 5, and PTL 6).

[0009] List of Citations

[0010] Patent Literature

[0011] [PTL 1] WO 2011102483

[0012] [PTL 2] WO 2016115345

[0013] [PTL 3] WO 2017156500

[0014] [PTL 4] WO 2018 / 043734

[0015] [PTL 5] WO 2019 / 163927

[0016] [PTL 6] WO 2021 / 039945

[0017] Non-Patent Literature

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[0020] [NPL 3] Derynck, R. & Miyazono, K. Cold Spring Harbor Press (2008)

[0021] [NPL 4] Yu, L. et al. Kidney Int. 64, 844-856 (2003).

[0022] [NPL 5] Xu, P., Liu, J. and Derynck, R. et al. FEBS Lett. 586, 1871-1884 (2012).

[0023] [NPL 6] Millan, F. A. et al. Development 111, 131-143 (1991).

[0024] [NPL 7] Kulkarni, A. B. et al. Proc. Natl Acad. Sci. USA 90, 770-774 (1993).

[0025] [NPL 8] Shull, M. M. et al. Nature 359, 693-699 (1992).

[0026] [NPL 9] Dickson, M. C. et al. Development 121, 1845-1854 (1995).

[0027] [NPL 10] Sanford, L. P. et al. Development 124, 2659-2670 (1997).

[0028] [NPL 11] Proetzel, G. et al. Nature Genet. 11, 409-414 (1995).

[0029] [NPL 12] Dubois, C. M. et al. J. Biol. Chem. 270, 10618-10624 (1995)

[0030] [NPL 13] Nunes, I. et al. J. Am. Optom. Assoc. 69, 643-648 (1998)

[0031] [NPL 14] Annes, J. et al. J. Cell Sci. 116, 217-224 (2003).

[0032] [NPL 15] Schultz-Cherry, S. et al. J. Biol. Chem. 269, 26775-26782 (1994).

[0033] [NPL 16] Munger, J. S. et al. Cell 96, 319-328 (1999).

[0034] [NPL 17] Shi, M. et al. Nature 474, 343-349 (2011).

[0035] [NPL 18] Shi, Y. and Massague, et al. Cell 113, 685-700 (2003).

[0036] [NPL 19] Sanderson, N. et al. Proc. Natl Acad. Sci. USA 92, 2572-2576 (1995). SUMMARY

[0037] TECHNICAL PROBLEM

[0038] An object of the present application is to provide a cross-species, humanized and optimized anti-latent TGF-β1 antibody which inhibits activation of latent TGF-β1 mediated by a protease without inhibiting or partially inhibiting activation of latent TGF-β1 mediated by integrin, or which inhibits activation of latent TGF-β1 mediated by a protease but has a less influence on inhibiting activation of latent TGF-β1 mediated by integrin. The present application also provides an anti-TGF-β1 antibody having a pH-dependent binding affinity to latent TGF-β1.

[0039] SOLUTION TO THE PROBLEM

[0040] The present inventors have intensively studied in the above-described circumstances, and thus have produced a cross-species, humanized and optimized anti-latent TGF-β1 antibody which inhibits activation of latent TGF-β1 mediated by a protease without inhibiting or partially inhibiting activation of latent TGF-β1 mediated by integrin, or which inhibits activation of latent TGF-β1 mediated by a protease but has a less influence on inhibiting activation of latent TGF-β1 mediated by integrin. In addition, the present inventors have produced an anti-TGF-β1 antibody having a pH-dependent binding affinity to latent TGF-β1.

[0041] More specifically, the present application provides:

[0042] A1. An anti-latent TGF-β1 antibody comprising:

[0043] (a) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 36, respectively;

[0044] (b) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 28, 32 and 37, respectively;

[0045] (c) HVR-H1, HVR-H2, and HVR-H3, respectively, comprise the amino acid sequences of SEQ ID NOs: 27, 30, and 39; or

[0046] (d) HVR-H1, HVR-H2, and HVR-H3, respectively, comprise the amino acid sequences of SEQ ID NOs: 27, 33, and 40.

[0047] A1-1. An anti-latent TGF-β1 antibody comprising:

[0048] (a) HVR-H1, HVR-H2, and HVR-H3, respectively, consist of the amino acid sequences of SEQ ID NOs: 29, 30, and 36;

[0049] (b) HVR-H1, HVR-H2, and HVR-H3, respectively, consist of the amino acid sequences of SEQ ID NOs: 28, 32, and 37;

[0050] (c) HVR-H1, HVR-H2, and HVR-H3, respectively, consist of the amino acid sequences of SEQ ID NOs: 27, 30, and 39; or

[0051] (d) HVR-H1, HVR-H2, and HVR-H3, respectively, consist of the amino acid sequences of SEQ ID NOs: 27, 33, and 40.

[0052] A2. The anti-latent TGF-β1 antibody of A1, further comprising:

[0053] (a) HVR-L1, HVR-L2, and HVR-L3, respectively, comprise the amino acid sequences of SEQ ID NOs: 49, 52, and 56;

[0054] (b) HVR-L1, HVR-L2, and HVR-L3, respectively, comprise the amino acid sequences of SEQ ID NOs: 50, 52, and 56;

[0055] (c) HVR-L1, HVR-L2, and HVR-L3, respectively, comprise the amino acid sequences of SEQ ID NOs: 48, 53, and 56; and

[0056] (d) HVR-L1, HVR-L2, and HVR-L3, respectively, comprise the amino acid sequences of SEQ ID NOs: 51, 52, and 56.

[0057] A2-1. The anti-latent TGF-β1 antibody of A1-1, further comprising:

[0058] (a) HVR-L1, HVR-L2, and HVR-L3 consist of the amino acid sequences of SEQ ID NOs: 49, 52, and 56, respectively;

[0059] (b) HVR-L1, HVR-L2, and HVR-L3 consist of the amino acid sequences of SEQ ID NOs: 50, 52, and 56, respectively;

[0060] (c) HVR-L1, HVR-L2, and HVR-L3 consist of the amino acid sequences of SEQ ID NOs: 48, 53, and 56, respectively; and

[0061] (d) HVR-L1, HVR-L2, and HVR-L3 consist of the amino acid sequences of SEQ ID NOs: 51, 52, and 56, respectively.

[0062] A3. An anti-latent TGF-β1 antibody comprising:

[0063] (a) HVR-H1, HVR-H2, and HVR-H3 comprise the amino acid sequences of SEQ ID NOs: 29, 30, and 36, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprise the amino acid sequences of SEQ ID NOs: 49, 52, and 56, respectively;

[0064] (b) HVR-H1, HVR-H2, and HVR-H3 comprise the amino acid sequences of SEQ ID NOs: 28, 32, and 37, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprise the amino acid sequences of SEQ ID NOs: 50, 52, and 56, respectively;

[0065] (c) HVR-H1, HVR-H2, and HVR-H3 comprise the amino acid sequences of SEQ ID NOs: 27, 30, and 39, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprise the amino acid sequences of SEQ ID NOs: 48, 53, and 56, respectively; or

[0066] (d) HVR-H1, HVR-H2, and HVR-H3 comprise the amino acid sequences of SEQ ID NOs: 27, 33, and 40, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprise the amino acid sequences of SEQ ID NOs: 51, 52, and 56, respectively.

[0067] A3-1. An anti-latent TGF-β1 antibody comprising:

[0068] (a) HVR-H1, HVR-H2, and HVR-H3 consisting of the amino acid sequences of SEQ ID NOs: 29, 30, and 36, respectively; and HVR-L1, HVR-L2, and HVR-L3 consisting of the amino acid sequences of SEQ ID NOs: 49, 52, and 56, respectively;

[0069] (b) HVR-H1, HVR-H2, and HVR-H3 consisting of the amino acid sequences of SEQ ID NOs: 28, 32, and 37, respectively; and HVR-L1, HVR-L2, and HVR-L3 consisting of the amino acid sequences of SEQ ID NOs: 50, 52, and 56, respectively;

[0070] (c) HVR-H1, HVR-H2, and HVR-H3 consisting of the amino acid sequences of SEQ ID NOs: 27, 30, and 39, respectively; and HVR-L1, HVR-L2, and HVR-L3 consisting of the amino acid sequences of SEQ ID NOs: 48, 53, and 56, respectively; or

[0071] (d) HVR-H1, HVR-H2, and HVR-H3 consisting of the amino acid sequences of SEQ ID NOs: 27, 33, and 40, respectively; and HVR-L1, HVR-L2, and HVR-L3 consisting of the amino acid sequences of SEQ ID NOs: 51, 52, and 56, respectively.

[0072] A4. The anti-LiV-TGF-βl antibody of any one of A1 to A3-1, comprising:

[0073] (a) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, or (iii) a VH sequence as in (i) and a VL sequence as in (ii);

[0074] (b) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 21, or (iii) a VH sequence as in (i) and a VL sequence as in (ii);

[0075] (c) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, or (iii) a VH sequence as in (i) and a VL sequence as in (ii); or

[0076] (d) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10, and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0077] A4-1. The anti-LTGF-βl antibody of any one of A1 to A4, which retains the binding activity of the antibody to human latent TGF-βl.

[0078] A5. The anti-LTGF-βl antibody of A4 or A4-1, which comprises a VH sequence of SEQ ID NO: 4, 9, 11, or 10.

[0079] A6. The anti-LTGF-βl antibody of any one of A4 to A5, which comprises a VL sequence of SEQ ID NO: 16, 21, 17, or 20.

[0080] A7. The anti-LTGF-βl antibody of any one of A4 to A6, which comprises:

[0081] (a) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 16;

[0082] (b) a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 21;

[0083] (c) a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 17; or

[0084] (d) a VH sequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO: 20.

[0085] A8. An anti-LTGF-βl antibody, which comprises:

[0086] (a) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 16;

[0087] (b) a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 21;

[0088] (c) a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 17; or

[0089] (d) a VH sequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO: 20.

[0090] A9. The anti-latent TGF-betal antibody of any one of A1 to A8, which is a human, humanized, or chimeric antibody.

[0091] A9-1. The anti-latent TGF-betal antibody of A9, which is a human antibody.

[0092] A9-2. The anti-latent TGF-betal antibody of A9, which is a humanized antibody.

[0093] A9-3. The anti-latent TGF-betal antibody of A9, which is a chimeric antibody.

[0094] A10. The anti-latent TGF-betal antibody of any one of A1 to A9-3, which is an IgG antibody.

[0095] A10-1. The anti-latent TGF-betal antibody of any one of A1 to A10, which is an IgGl antibody.

[0096] A11. The anti-latent TGF-betal antibody of any one of A1 to A9-3, which is a bispecific antibody.

[0097] A12. The anti-latent TGF-betal antibody of any one of A1 to A11, wherein the anti-latent TGF-betal antibody comprises a modified IgGl Fc region having reduced effector function compared to a wild-type IgGl Fc region.

[0098] A13. The anti-latent TGF-betal antibody of A12, wherein the modified IgGl Fc region comprises an amino acid substitution at position 235 and / or 236 according to the EU index.

[0099] A14. The anti-latent TGF-betal antibody of A12 or A13, wherein in the modified IgGl Fc region, the amino acid residues at positions 235 and 236 according to the EU index are changed to arginine (R).

[0100] A14-1. The anti-latent TGF-betal antibody of any one of A12 to A14, wherein the modified IgGl Fc region comprises amino acid substitutions of L235R and G236R according to the EU index.

[0101] A15. The anti-latent TGF-βl antibody of any one of A12 to A14-1, wherein the modified IgGl Fc region further has enhanced binding activity to FcRn compared to a wild-type IgGl Fc region.

[0102] A16. The anti-latent TGF-βl antibody of A15, wherein the modified IgGl Fc region comprises one or more amino acid substitutions at positions selected from the group consisting of 428, 434, 438, and 440 according to EU index.

[0103] A17. The anti-latent TGF-βl antibody of A15 or A16, wherein in the modified IgGl Fc region, the amino acid residues at positions 428, 434, 438, and 440 are changed to leucine (L), alanine (A), arginine (R), and glutamic acid (E), respectively.

[0104] A17-1. The anti-latent TGF-βl antibody of any one of A15 to A17, wherein the modified IgGl Fc region comprises amino acid substitutions of M428L, N434A, Q438R, and S440E.

[0105] A18. The anti-latent TGF-βl antibody of any one of A1 to A11, wherein the anti-latent TGF-βl antibody comprises a modified IgGl Fc region, wherein in the modified IgGl Fc region, the amino acid residues at positions 214, 235, and 236 are each changed to arginine (R).

[0106] A18-1. The anti-latent TGF-βl antibody of any one of A1 to A11, wherein the anti-latent TGF-βl antibody comprises a modified IgGl Fc region, wherein the modified IgGl Fc region comprises amino acid substitutions of K214R, L235R, and G236R.

[0107] A19. The anti-latent TGF-βl antibody of any one of A1 to A11, wherein the anti-latent TGF-βl antibody comprises a modified IgGl Fc region, wherein in the modified IgGl Fc region, the amino acid residues at positions 214, 235, 236, 428, 434, 438, and 440 are changed to arginine (R), arginine (R), arginine (R), leucine (L), alanine (A), arginine (R), and glutamic acid (E), respectively.

[0108] A19-1. The anti-latent TGF-β1 antibody of any one of A1 to A11, wherein the anti-latent TGF-β1 antibody comprises a modified IgG1 Fc region, wherein the modified IgG1 Fc region comprises amino acid substitutions of K214R, L235R, G236R, M428L, N434A, Q438R, and S440E.

[0109] A20-1. The anti-latent TGF-β1 antibody of any one of A1 to A19-1, wherein the anti-latent TGF-β1 binds to latent TGF-β1 in a pH-dependent manner.

[0110] A20-2. The anti-latent TGF-β1 antibody of A20-1, wherein the anti-latent TGF-β1 antibody binds to latent TGF-β1 with a higher antigen-binding affinity under neutral pH conditions than under acidic pH conditions.

[0111] A20-3. The anti-latent TGF-β1 antibody of A20-2, wherein the anti-latent TGF-β1 antibody binds to latent TGF-β1 with a higher antigen-binding affinity at pH 7.4 than at pH 5.8.

[0112] A20-4. The anti-latent TGF-β1 antibody of A20-3, wherein the anti-latent TGF-β1 antibody has a dissociation constant ratio between pH 5.8 and pH 7.4 for latent TGF-β1 ([KD(pH 5.8) / KD(pH 7.4)]) of 5 or greater.

[0113] A20-5. The anti-latent TGF-β1 antibody of A20-4, wherein the dissociation constant ratio is 50 or greater.

[0114] A20-6. The anti-latent TGF-β1 antibody of A20-4, wherein the dissociation constant ratio is 100 or greater.

[0115] A20-7. The anti-latent TGF-β1 antibody of A20-4, wherein the dissociation constant ratio is 120 or greater.

[0116] A20-8. The anti-latent TGF-β1 antibody of A20-4, wherein the dissociation constant ratio is 130 or greater.

[0117] A20-9. The anti-latent TGF-β1 antibody of A20-4, wherein the dissociation constant ratio is 160 or greater.

[0118] A20-10. The anti-LTGF-βl antibody of any one of A20-4 to A20-9, wherein the dissociation constant ratio is measured using a surface plasmon resonance method.

[0119] A21. An anti-LTGF-βl antibody comprising:

[0120] (a) a heavy chain sequence of SEQ ID NO: 82 and a light chain sequence of SEQ ID NO: 83;

[0121] (b) a heavy chain sequence of SEQ ID NO: 84 and a light chain sequence of SEQ ID NO: 85;

[0122] (c) a heavy chain sequence of SEQ ID NO: 86 and a light chain sequence of SEQ ID NO: 87;

[0123] (d) a heavy chain sequence of SEQ ID NO: 88 and a light chain sequence of SEQ ID NO: 89; (e) a heavy chain sequence of SEQ ID NO: 90 and a light chain sequence of SEQ ID NO: 83;

[0124] (f) a heavy chain sequence of SEQ ID NO: 91 and a light chain sequence of SEQ ID NO: 85;

[0125] (g) a heavy chain sequence of SEQ ID NO: 92 and a light chain sequence of SEQ ID NO: 87; or

[0126] (h) a heavy chain sequence of SEQ ID NO: 93 and a light chain sequence of SEQ ID NO: 89.

[0127] A22. The anti-LTGF-βl antibody of any one of A1 to A21, wherein the LTGF-βl is human LTGF-βl, mouse LTGF-βl, or cynomolgus monkey LTGF-βl.

[0128] A23. The anti-LTGF-βl antibody of any one of A1 to A22, wherein the anti-LTGF-βl antibody binds to human LTGF-βl, mouse LTGF-βl, and cynomolgus monkey LTGF-βl.

[0129] A24. The anti-LTGF-βl antibody of any one of A1 to A23, wherein the anti-LTGF-βl antibody binds to a latency associated peptide (LAP) region of LTGF-βl.

[0130] A24-1. The anti-latent TGF-β1 antibody of any one of A1 to A24, wherein the anti-latent TGF-β1 antibody:

[0131] i) inhibits activation of latent TGF-β1 ;

[0132] ii) inhibits release of mature TGF-β1 from latent TGF-β1 ;

[0133] iii) inhibits protease-mediated and / or integrin-mediated release of mature TGF-β1 from latent TGF-β1 ;

[0134] iv) 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 ;

[0135] v) inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1 and allows protease cleavage of the LAP region while the anti-latent TGF-β1 antibody is bound to the LAP region of the latent TGF-β1 ;

[0136] vi) does not block protease access to latent TGF-β1 ;

[0137] vii) does not bind to the protease cleavage site of the LAP portion of latent TGF-β1 ;

[0138] viii) inhibits protease-mediated activation of latent TGF-β1 without inhibiting or partially inhibiting integrin-mediated activation of latent TGF-β1 ; and / or

[0139] ix) inhibits protease-mediated activation of latent TGF-β1 with less impact on inhibiting integrin-mediated activation of latent TGF-β1.

[0140] A24-1a. The anti-latent TGF-β1 antibody of A24-1, wherein the protease is selected from the group consisting of plasmin (PLN), plasma kallikrein (PLK), matrix metalloproteinase 2 (MMP2), and matrix metalloproteinase 9 (MMP9).

[0141] A24-2. An antibody fragment of the anti-latent TGF-β1 antibody of any one of A1 to A24-1a.

[0142] A25. An immunoconjugate comprising the anti-latent TGF-β1 antibody of any one of A1 to A24-1a or the antibody fragment of A24-2 and a cytotoxic agent.

[0143] A26. An isolated nucleic acid encoding the anti-Latent TGFp-1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2.

[0144] A27. A vector comprising the nucleic acid according to A26.

[0145] A28. A host cell comprising the nucleic acid according to A26 or the vector according to A27.

[0146] A29. A method of producing an anti-Latent TGFp-1 antibody, the method comprising culturing the host cell according to A28, thereby producing the antibody.

[0147] A29-2. A method of producing an anti-Latent TGFp-1 antibody, the method comprising:

[0148] (a) contacting a biological sample comprising Latent TGF-β1 and a protease with a test antibody;

[0149] (b) detecting (i) whether the test antibody inhibits cleavage of the LAP region of Latent TGF-β1 and (ii) whether the test antibody inhibits activation of Latent TGF-β1;

[0150] (c) selecting the test antibody that inhibits activation of Latent TGF-β1 without inhibiting cleavage of the LAP portion of Latent TGF-β1 by the protease.

[0151] (d) obtaining amino acid sequence information of the anti-Latent TGF-β1 antibody selected in step (c); and

[0152] (e) introducing a gene encoding the anti-Latent TGF-β1 antibody into a host cell; and

[0153] (f) culturing the host cell, thereby producing the antibody.

[0154] A30. The method according to A29 or A29-2, further comprising recovering the antibody from the host cell.

[0155] B1. The anti-Latent TGFp-1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2 or the immunoconjugate according to A25 for use as a medicament.

[0156] B1-1. The anti-Latent TGFp-1 antibody according to B1 for use as a medicament for subcutaneous administration.

[0157] B2. The anti-LTGFβ-1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2 or the immunoconjugate according to A25 for use in the treatment of fibrosis.

[0158] B2a. The anti-LTGFβ-1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2 or the immunoconjugate according to A25 for use in the treatment of cancer.

[0159] B2-1. The anti-LTGFβ-1 antibody according to B2 or B2a for use in the subcutaneous administration of the antibody.

[0160] B2-2. The anti-LTGFβ-1 antibody according to B2 or B2-1, wherein the fibrosis is selected from the group consisting of renal fibrosis, liver fibrosis and lung fibrosis.

[0161] B2-3. The anti-LTGFβ-1 antibody according to B2-2, wherein the fibrosis is renal fibrosis.

[0162] B2-4. The anti-LTGFβ-1 antibody according to B2-2, wherein the fibrosis is liver fibrosis.

[0163] B2-5. The anti-LTGFβ-1 antibody according to B2-2, wherein the fibrosis is lung fibrosis.

[0164] B3. Use of an anti-LTGFβ-1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2 or the immunoconjugate according to A25 for the manufacture of a medicament for the treatment of fibrosis.

[0165] B3a. Use of an anti-LTGFβ-1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2 or the immunoconjugate according to A25 for the manufacture of a medicament for the treatment of cancer.

[0166] B3-1. The use according to B3 or B3a, the medicament being for subcutaneous administration.

[0167] B3-2. The use according to B3 or B3-1, wherein the fibrosis is selected from the group consisting of renal fibrosis, liver fibrosis and lung fibrosis.

[0168] B3-3. The anti-LTGFβ-1 antibody according to B3-2, wherein the fibrosis is renal fibrosis.

[0169] B3-4. The anti-LTBP1 antibody according to B3-2, wherein the fibrosis is liver fibrosis.

[0170] B3-5. The anti-LTBP1 antibody according to B3-2, wherein the fibrosis is lung fibrosis.

[0171] C1. A pharmaceutical preparation comprising the anti-LTBP1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2 or the immunoconjugate according to A25, and a pharmaceutically acceptable carrier.

[0172] C2. The pharmaceutical preparation according to C1, wherein the viscosity of the pharmaceutical preparation is less than 70 mPa-s.

[0173] C3. The pharmaceutical preparation according to C1 or C2, wherein the concentration of the antibody in the pharmaceutical preparation is 50 to 350 mg / ml.

[0174] C4. The pharmaceutical preparation according to any one of C1 to C3, which is administered subcutaneously.

[0175] C5. The pharmaceutical preparation according to any one of C1 to C4, for use in the treatment of fibrosis.

[0176] C5a. The pharmaceutical preparation according to any one of C1 to C4, for use in the treatment of cancer.

[0177] C6. The pharmaceutical preparation according to C5, wherein the fibrosis is selected from the group consisting of kidney fibrosis, liver fibrosis, and lung fibrosis.

[0178] C7. The anti-LTBP1 antibody according to C6, wherein the fibrosis is kidney fibrosis.

[0179] C8. The anti-LTBP1 antibody according to C6, wherein the fibrosis is liver fibrosis.

[0180] C9. The anti-LTBP1 antibody according to C6, wherein the fibrosis is lung fibrosis.

[0181] E1. A method of treating an individual having fibrosis, the method comprising administering to the individual an effective amount of the anti-LTBP1 antibody according to any one of A1 to A24-1a or the antibody fragment according to A24-2 or the immunoconjugate according to A25.

[0182] E1a. A method of treating an individual having cancer, the method comprising administering to the individual an effective amount of an anti-latent TGFp-1 antibody according to any one of A1 to A24-1a or an antibody fragment according to A24-2 or an immunoconjugate according to A25.

[0183] E2. The method of E1 or E1a, comprising subcutaneously administering to the individual an effective amount of the anti-latent TGFp-1 antibody.

[0184] E3. The method of E1 or E2, wherein the fibrosis is selected from the group consisting of kidney fibrosis, liver fibrosis, and lung fibrosis.

[0185] E4. The anti-latent TGFp-1 antibody of E3, wherein the fibrosis is kidney fibrosis.

[0186] E5. The anti-latent TGFp-1 antibody of E3, wherein the fibrosis is liver fibrosis.

[0187] E6. The anti-latent TGFp-1 antibody of E3, wherein the fibrosis is lung fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0188] [Figure 1] Figure 1 depicts an assessment of the neutralization activity of human latent TGF-β1 on PLNs.

[0189] [Figure 2] Figure 2 depicts an assessment of the antibody kinetics against human latent TGF-β1.

[0190] [Figure 3] Figure 3 depicts an assessment of the KD for human latent TGF-β1 at pH 7.4 and the koff for human latent TGF-β1 at pH 5.8 for antibody variants generated during antibody optimization.

[0191] [Figure 4] Figure 4 depicts an assessment of human CD8 - CD25 低 IL-2 secretion by PBMC following antibody incubation for immunogenicity risk assessment.

[0192] [Figure 5] Figure 5 shows ka, kd, and KD values for anti-latent TGF-β1 antibody binding to recombinant human, cynomolgus monkey (“cyno”), and mouse latent TGF-β1 at pH 7.4.

[0193] [Figure 6] Figure 6 shows ka, kd, and KD values for anti-latent TGF-β1 antibody binding to recombinant human, cynomolgus monkey, and mouse latent TGF-β1 at pH 5.8.

[0194] [Figure 7] Figure 7 shows koff and KD ratios (pH 5.8 / pH 7.4) for anti-latent TGF-β1 antibodies based on Figures 5 and 6.

[0195] [Figure 8] Figure 8 depicts the results of cell surface latent TGF-β1 binding on 293-F cells and BaF3 cells for anti-latent TGF-β1 antibodies. TM 293-F cells and BaF3 cells.

[0196] [Figure 9] Figure 9 depicts the results of anti-latent TGF-β1 antibody activity against spontaneous latent TGF-β1 activation.

[0197] [Figure 10] Figure 10 depicts the results of anti-latent TGF-β1 antibody activity against plasmin (PLN)-mediated latent TGF-β1 activation.

[0198] [Figure 11] Figure 11 depicts the results of anti-latent TGF-β1 antibody activity against plasma kallikrein, matrix metalloproteinase (MMP) 2, and MMP9-mediated human latent TGF-β1 activation.

[0199] [Figure 12] Figure 12 depicts the results of anti-latent TGF-β1 antibody activity against integrin-mediated mouse TGF-β1 activation in mouse PBMCs.

[0200] [Figure 13] Figure 13 depicts the results of anti-latent TGF-β1 antibody activity against integrin avb6-mediated human TGF-β1 activation in Detroit562.

[0201] [Figure 14-1] Figure 14-1 depicts a pharmacokinetic (PK) study of anti-human latent TGF-β1 antibodies in cynomolgus monkeys.

[0202] [Figure 14-2] Figure 14-2 is a continuation of Figure 14-1.

[0203] [Figure 14-3] Figure 14-3 is a continuation of Figure 14-2.

[0204] [Figure 14-4] Figure 14-4 is a continuation of Figure 14-3.

[0205] [Figure 14-5] Figure 14-5 is a continuation of Figure 14-4.

[0206] [Figure 14-6] Figure 14-6 is a continuation of Figure 14-5.

[0207] [Figure 14-7] Figure 14-7 depicts a PK study of anti-human latent TGF-β1 antibodies in mice.

[0208] [Figure 14-8] Figure 14-8 is a continuation of Figure 14-7.

[0209] [Figure 14-9] Figure 14-9 is a continuation of Figure 14-8.

[0210] [Figure 14-10] Figure 14-10 is a continuation of Figure 14-9.

[0211] [Figure 15] Figure 15 depicts results for hydroxyproline content in the kidney. Efficacy of the antibody was evaluated in a mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO). The sham group represents a non-disease induced control.

[0212] [Figure 16] Figure 16 depicts results for glomerulosclerosis in the kidney. Efficacy of the antibody was evaluated in a mouse model of kidney inflammation induced by anti-GBM serum (aGBM model). The normal mouse group represents a non-disease induced control.

[0213] [Figure 17] Figure 17 depicts results for plasma creatinine. Efficacy of the antibody was evaluated in a COL4A3 KO mouse (Alport mouse model). The normal mouse group represents a non-disease induced control.

[0214] [Figure 18] Figure 18 shows viscosity of the anti-latent TGF-βl antibody at 150 mg / mL antibody concentration in formulation buffer at pH 6.0.

[0215] [Figure 19] Figure 19 shows SEC analysis of the anti-latent TGF-βl antibody at 40 degrees Celsius for 2 weeks and 4 weeks.

[0216] [Figure 20] Figure 20 depicts results for collagen type 1 alpha 1 (Col1a1) mRNA in the liver. Efficacy of the antibody was evaluated in a mouse model of NASH / liver fibrosis induced by choline deficient, L-amino acid defined high fat diet (CDAHFD). The normal diet group represents a non-disease control.

[0217] [Figure 21] Figure 21 depicts results for hydroxyproline content, collagen type 1 alpha 1 (Col1a1) mRNA, and serpinase 1 mRNA in the lung. Efficacy of the antibody was evaluated in a bleomycin induced model of lung fibrosis. The saline group represents a non-disease control.

[0218] [Figure 22] Figure 22 depicts wild-type Fc region sequences for IgGl, IgG2, IgG3, and IgG4. DETAILED DESCRIPTION

[0219] Those of ordinary skill in the art are generally familiar with and commonly use routine methods to use the techniques and procedures described or referenced herein, such as, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rded. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M. Ausubel et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I. Freshney ed. (1987)); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (H. A. Erlich, ed., 1992); and Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strobe eds., 1991); and the research literature.Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B. Lippincott Company, 1993).

[0220] I. Definitions

[0221] A "recipient human framework" for purposes herein is a framework comprising an 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. A recipient human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence as that human immunoglobulin framework or human consensus framework, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, a VL recipient human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0222] The term "binding activity" refers to the strength of the sum total of noncovalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In this context, "binding activity" is not strictly limited to 1 : 1 interactions between members of a binding pair (e.g., an antibody and an antigen). For example, when members of a binding pair exhibit 1 : 1 monovalent interactions, the binding activity is specifically referred to as intrinsic binding affinity (affinity). When members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of each of the binding strengths. The binding activity of a molecule X to its partner Y can be expressed in terms of a dissociation constant (KD) or "amount of analyte bound per amount of ligand" (hereinafter can be referred to as "amount bound"). Those of ordinary skill in the art will generally understand that lower values of dissociation constant (KD) indicate higher binding activity; and higher values of "amount of analyte bound per amount of ligand" or "amount bound" indicate higher binding activity. Binding affinity can be measured by routine methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

[0223] "Binding activity matured," "affinity matured," antigen binding molecule or antibody, "increased (enhanced) binding activity" or "increased (enhanced) affinity" antigen binding molecule or antibody refers to an antibody having one or more alterations (e.g., substitutions) in one or more hypervariable regions (HVRs) that result in improvement in the binding activity of the antigen binding molecule or antibody for an antigen, as compared to a parent antigen binding molecule or a parent antibody that does not carry such alterations.

[0224] The terms "anti-latent TGF-β1 antibody" and "antibody that can bind to latent TGF-β1" refer to an antibody that is capable of binding to latent TGF-β1 with sufficient binding activity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting latent TGF-β1. In one embodiment, an "antibody that can bind to latent TGF-β1" is an antibody that specifically binds to latent TGF-β1. In one embodiment, the extent 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 that can bind to TGF-β1 has a dissociation constant (KD) of 1 micromolar or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10-9 M to 10 -13 M) of the anti-latent TGF-β1 antibody. In certain embodiments, the anti-latent TGF-β1 antibody binds to an epitope of latent TGF-β1 that is conserved in latent TGF-β1 from different species.

[0225] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) so long as they exhibit the desired antigen-binding activity. An "antibody" can be an antigen-binding molecule comprising a variable heavy chain and / or a variable light chain structure of an immunoglobulin.

[0226] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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. An "antibody fragment" can be an antigen-binding molecule comprising a variable heavy chain and / or a variable light chain structure of an immunoglobulin.

[0227] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of that antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

[0228] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include but are not limited to a malignant tumor, a lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), a blastoma, a sarcoma, and a leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the nervous system, cancer of the male or female reproductive system, a combination of one or more of the same, or other cancer. In one example, the cancer is resistant to and / or shows a limited response to an immune checkpoint inhibitor.

[0229] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular species or originating from a different species.

[0230] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0231] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At, 211 At、 131 I、 125 I、 90 Y、 186 Re、 188 Re、 153 Sm、 212 Bi、 32 P、 212 Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), duanorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes, such as nucleolytic enzymes; antibodies; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and the various antitumor or anticancer agents disclosed below.

[0232] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody's isotype. Examples of antibody effector functions include: Clq 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 receptor); and B cell activation.

[0233] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0234] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The 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 carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region can or can not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called 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).

[0235] An Fc region of an IgG that occurs in nature, also referred to as a "wild-type Fc region," means an Fc region comprising an amino acid sequence identical to that of an Fc region derived from an IgG found in nature. An Fc region of an IgG that occurs in nature, also referred to as a "wild-type IgGl Fc region," is shown in Figure 22 (SEQ ID NOs: 105-108). For example, the figure refers to an Fc region derived from a naturally-occurring human IgGl (also referred to as a "wild-type human IgGl Fc region"), an Fc region derived from a naturally-occurring human IgG2 (also referred to as a "wild-type human IgG2 Fc region"), an Fc region derived from a naturally-occurring human IgG3 (also referred to as a "wild-type human IgG3 Fc region"), and an Fc region derived from a naturally-occurring human IgG4 (also referred to as a "wild-type human IgG4 Fc region"). An Fc region of an IgG that occurs in nature can also include mutants that arise spontaneously therefrom.

[0236] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR residues are typically conserved within a species and often only modestly modifiable without adversely affecting the specificity of an antibody. The variable domain of a native antibody comprises four FR regions (FR1, FR2, FR3, and FR4) that are interposed among three CDR regions (CDR1, CDR2, and CDR3).

[0237] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody with a structure substantially similar to a native antibody structure or with heavy chains that have an Fc region as defined herein.

[0238] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny can not be completely identical to the parent cell in nucleic acid content, for example, mutations that occur during replication. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0239] A "human antibody" is one which possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or derived from an antibody produced by a human or a human cell using somatic hypermutation techniques. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.

[0240] A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in the human immunoglobulin VL or VH framework sequences. Generally, the selection of amino acid residues in the human consensus framework is based on the analysis of a large number of human VL or VH sequences. Generally, the selection of amino acid residues in the human consensus framework is based on the analysis of a large number of human VL or VH sequences. Generally, the subgroup of VL or VH sequences is selected from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as 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 the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0241] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can 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.

[0242] The term "hypervariable region" or "HVR", as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen-contact residues ("antigenic contact points"). Generally, antibodies comprise six HVRs: three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

[0243] (a) the hypervariable loops that occur at amino acid residues 26-32 (LI), 50- 52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0244] (b) the CDRs that occur at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (HI), 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));

[0245] (c) the antigenic contact points that occur at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (HI), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

[0246] (d) combinations of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (HI), 26-35b (HI), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0247] Unless otherwise indicated, HVR residues and other residues in a variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0248] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0249] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and 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 a human.

[0250] An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. See, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007) for a review of methods for assessing antibody purity.

[0251] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from that of normal nucleic acid.

[0252] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from that of normal nucleic acid.

[0253] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the monoclonal antibody, such variants typically being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and use of transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0254] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody can be present in a pharmaceutical formulation.

[0255] A "native antibody" refers to a naturally occurring immunoglobulin molecule with varying structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 Da that is composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a constant light (CL) domain. The light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of their constant domains.

[0256] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0257] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0258] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been made available to the public at U.S. Copyright Office, Washington D.C., 20559, where it was registered under U.S. Copyright Registration TXU510087, along with user documentation. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are unchanged. In the case of amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A has, or comprises, a certain % amino acid sequence identity to a given amino acid sequence B) is calculated as follows:

[0259] 100 times the fraction X / Y

[0260] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where 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 both lengths are zero. Except as otherwise specifically noted, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0261] The term "pharmaceutical formulation" refers to a preparation which allows a biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation will be administered.

[0262] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than an active ingredient, which is nontoxic to a subject to which it is administered in doses necessary to achieve the desired therapeutic effect. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizing agents, or preservatives.

[0263] The term "TGF-β1" as used herein, unless otherwise indicated, refers to any native TGF-β1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed TGF-β1 as well as any form of TGF-β1 that results from processing in the cell. The term also encompasses naturally occurring variants of TGF-β1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human TGF-β1 preproprotein is set forth in SEQ ID NO: 94 (NCBI RefSeq: NP_000651.3) and the nucleic acid sequence encoding an exemplary human TGF-β1 is set forth in SEQ ID NO: 95 (NCBI RefSeq: NM_000660.6). The amino acid sequence of an exemplary mouse TGF-β1 preproprotein is set forth in SEQ ID NO: 96 (NCBI RefSeq: NP_035707.1) and the nucleic acid sequence encoding an exemplary mouse TGF-β1 is set forth in SEQ ID NO: 97 (NCBI RefSeq: NM_011577.2). The amino acid sequence of an exemplary cynomolgus monkey TGF-β1 preproprotein is set forth in SEQ ID NO: 98 (NCBI RefSeq: XP_005589396.1) and the nucleic acid sequence encoding an exemplary cynomolgus monkey TGF-β1 is set forth in SEQ ID NO: 99 (NCBI RefSeq: XM_005589339.2). The term "TGF-β1" encompasses both latent TGF-β1 and mature TGF-β1.

[0264] The term“latent TGF-β1” as used herein refers to any TGF-β1 that forms a latent TGF-β1 complex (“cell surface latent TGF-β1”, LLC or SLC (see below)) and / or is unable to bind to its receptor. Transforming growth factor-beta 1 (TGF-β1) is a member of TGF-β, which 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 with its latency-associated peptide (LAP) and a latent TGF-β binding protein (LTBP) that interact to form a larger complex called a 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 to 390 of SEQ ID NO: 94. The amino acid sequence of an exemplary latent mouse TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30 to 390 of SEQ ID NO: 96. The amino acid sequence of an exemplary latent cynomolgus monkey TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30 to 390 of SEQ ID NO: 98.

[0265] The complex formed by the TGF-β homodimer and its LAP is called a small latent complex (SLC). This latent complex holds TGF-β in an inactive form that is unable to bind to its receptor. The SLC can be covalently linked to an additional protein, a latent TGF-β binding protein (LTBP), forming a large latent complex (LLC). There are four different LTBP subtypes known: LTBP-1, LTBP-2, LTBP-3, and LTBP-4. LTBP-1, LTBP-3, and LTBP-4 are reported to bind to the SLC (see, e.g., Rifkin et al., J Biol Chem. 2005 Mar 4;280(9):7409-12). The SLC can also be covalently linked to other additional proteins, such as Glycoprotein A Repetin Domain-Containing Protein (GARP) or Leucine-Rich Repeat Containing Protein 33 (LRRC33). GARP and LRRC have transmembrane domains and associate with LAP on the cell surface (see, e.g., Wang et al., Mol Biol Cell. 2012 Mar;23(6):1129-39). With respect to LLC, the LLC is reported to covalently associate with the extracellular matrix (ECM) via the N-terminus of the LTBP (see, e.g., Saharinen et al., Cytokine Growth Factor Rev. 1999 Jun;10(2):99-117.). In some embodiments, the latent TGF-β1 associated with the ECM on the cell surface is referred to as“cell surface latent TGF-β1”.

[0266] The term "active TGF-β1", "mature TGF-β1", or "active mature TGF-β1" as used herein refers to any TGF-β1 homodimer that does not form a latent TGF-β1 complex (LLC or SLC) and is capable of binding to its receptor. The TGF-β1 activation process involves release of LLC from ECM, followed by further proteolysis of LAP to release active TGF-β to its receptor. A broad range of proteases including plasmin (PLN), plasma kallikrein (PLK), matrix metalloproteinases (MMP) 2, MMP9, MMP13, MMP14, thrombin, trypsin-like and calpains are known to cleave latent TGF-β and release active TGF-β. In the context of the present invention, these proteases can 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, upon mechanical stretch, integrins (preferably integrin αVβ8 and / or integrin αVβ6) can activate TGF-β by binding to the RGD motif present in LAP and inducing release of mature TGF-β from its latent complex form.

[0267] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") means clinical intervention in an attempt to alter the natural progress of the individual being treated, and can be for prophylactic or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing rate of disease progression, ameliorating or palliating a disease state, and improving prognosis. In some embodiments, the antibodies of the present invention are used to delay development of a disease or to slow the progression of a disease.

[0268] 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 antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6thEd., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using only the VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0269] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0270] II. Compositions and Methods

[0271] In one aspect, the present application is based, in part, on anti-latent TGF-β1 antibodies and uses thereof. In certain embodiments, antibodies that bind to TGF-β1 are provided. The antibodies of the present application can be used, for example, in the diagnosis or treatment of fibrosis, preferably cardiac fibrosis, renal fibrosis, ocular fibrosis, bone marrow fibrosis, liver fibrosis, and lung / pulmonary fibrosis. The antibodies of the present application can also be used, for example, in the diagnosis or treatment of cancer. Examples of cancer include, but are not limited to, a malignant tumor, a lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), a blastoma, a sarcoma, and a leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, cancer of the vulva, thyroid cancer, hepatic carcinoma, leukemia and other lymphatic system cancers, and various types of head and neck cancer.

[0272] In the context of diagnosis and treatment, renal fibrosis includes, but is not limited to, the following: progressive kidney disease, IgA nephropathy, mesangial proliferative nephritis, mesangial proliferative glomerulonephritis, mesangial capillary glomerulonephritis, systemic lupus erythematosus, glomerulonephritis, renal interstitial fibrosis, renal failure, diabetic nephropathy, polycystic kidney disease, Alport syndrome, focal segmental glomerulosclerosis, or membranous nephropathy.

[0273] A. Exemplary Anti-Latent TGF-β1 Antibodies

[0274] In one aspect, the present application provides an isolated antibody that binds to latent TGF-β1. In further embodiments, the anti-latent TGF-β1 antibody binds to the latency associated protein (LAP) region of latent TGF-β1. Examples of the LAP region include amino acids 30 to 278 of human TGF-β1 proprotein (SEQ ID NO: 100). As described above, LAP is a component of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody binds to latent TGF-β1 with a dissociation constant (KD) of 10 -8 nM or less, 10 -9 nM or less, or 10 -10 nM or less.

[0275] In one aspect, the anti-latent TGF-β1 antibody binds to latent TGF-β1 to form an LLC, and / or binds to latent TGF-β1 in complex with GARP or LRRC33. In certain embodiments, the anti-latent TGF-β1 antibody binds to cell surface latent TGF-β1, which is latent TGF-β1 associated with extracellular matrix (ECM) on the surface of a cell. In another aspect, the anti-latent TGF-β1 antibody binds to latent TGF-β1, wherein the LAP region of latent TGF-β1 is not linked to a LTBPs, forming a small latent complex (SLC). In certain embodiments, the SLC exists in a soluble form. In some embodiments, the anti-latent TGF-β1 antibody binds to latent TGF-β1 (cell surface latent TGF-β1, LLC, or SLC) with a dissociation constant (KD) of 10 -8 nM or less, 10 -9 nM or less, or 10 -10 nM or less.

[0276] In one aspect, the 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, which is a component of latent TGF-β1. Activation of latent TGF-β1 can be detected, for example, by measuring mature TGF-β1 and / or measuring mature TGF-β1 activity using various techniques known in the art or described herein. In some embodiments, the 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), plasma kallikrein (PLK), matrix metalloproteinase (MMP) 2, and MMP9. In some embodiments, the anti-latent TGF-β1 antibody inhibits 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, which results in the release of mature TGF-β1. In some embodiments, the cleavage site for PLN and / or PLK is within a fragment consisting of amino acids 56 to 59 of the LAP polypeptide.

[0277] In preferred embodiments, the protease is selected from the group consisting of:

[0278] (i) plasmin (PLN), e.g., Sigma-Aldrich Catalog # 527624 (https: / / www.merckmillipore.com / JP / ja / product / Plasmin-EACA-and-Lysine-Free-Human-Plasma, EMD_BIO-527624);

[0279] (ii) plasma kallikrein (PLK), e.g., Enzyme Research Laboratories Catalog # HPKa1303 (https: / / enzymeresearch.com / product / human-kallikrein / );

[0280] (iii) matrix metalloproteinase 2 (MMP2), e.g., R&D Systems Catalog # 902-MP (https: / / www.rndsystems.com / products / recombinant-human-mmp-2-protein-cf_902-mp#product-datasheets); and

[0281] (iv) Matrix metalloproteinase 9 (MMP9), e.g., R&D Systems Catalog #909-MM (https: / / www.rndsystems.com / products / recombinant-mouse-mmp-9-protein-cf_909-mm).

[0282] In some embodiments, the anti-LTGF-βl antibody inhibits protease-mediated activation of LTGF-βl without inhibiting integrin-mediated activation of LTGF-βl or partially inhibits integrin-mediated activation of LTGF-βl. In some embodiments, the anti-LTGF-βl antibody inhibits protease-mediated activation of LTGF-βl but has less effect on inhibiting integrin-mediated activation of LTGF-βl.

[0283] In one aspect, the anti-LTGF-βl antibody inhibits protease-mediated release of mature TGF-βl from LTGF-βl without inhibiting or partially inhibiting protease-mediated cleavage of the LAP portion of LTGF-βl. In some embodiments, the anti-LTGF-βl antibody inhibits protease-mediated release of mature TGF-βl from LTGF-βl but has less effect on inhibiting protease-mediated cleavage of the LAP portion of LTGF-βl. In some embodiments, the anti-LTGF-βl antibody inhibits protease-mediated release of mature TGF-βl from LTGF-βl and allows protease cleavage of the LAP region while the anti-LTGF-βl antibody is bound to the LAP region of LTGF-βl. In some embodiments, the anti-LTGF-βl antibody does not block protease access to LTGF-βl, particularly to the cleavage site of PLN and / or PLK. In other embodiments, the anti-LTGF-βl antibody does not bind to the protease cleavage site of the LAP portion of LTGF-βl, particularly the cleavage site of PLN and / or PLK.

[0284] In some embodiments, an anti-latency TGF-β1 antibody that inhibits the release of mature TGF-β1 from latent TGF-β1 mediated by a protease 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-latency TGF-β1 antibody (1-i) inhibits the release of mature TGF-β1 mediated by MMP2 and / or MMP9 by inhibiting cleavage of the LAP portion of latent TGF-β1 by MMP2 and / or MMP9, and (1-ii) inhibits the release of mature TGF-β1 mediated by PLN and / or PLK without inhibiting cleavage of the LAP portion of latent TGF-β1 by PLN and / or PLK. Alternatively, an anti-latency TGF-β1 antibody (2-i) inhibits the release of mature TGF-β1 mediated by PLN and / or PLK by inhibiting cleavage of the LAP portion of latent TGF-β1 by PLN and / or PLK, and (2-ii) inhibits the release of mature TGF-β1 mediated by MMP2 and / or MMP9 without inhibiting cleavage of the LAP portion of latent TGF-β1 by MMP2 and / or MMP9. Alternatively, an anti-latency TGF-β1 antibody (3-i) inhibits the release of mature TGF-β1 mediated by PLN and / or PLK without inhibiting cleavage of the LAP portion of latent TGF-β1 by PLN and / or PLK, and (3-ii) inhibits the release of mature TGF-β1 mediated by MMP2 and / or MMP9 without inhibiting cleavage of the LAP portion of latent TGF-β1 by MMP2 and / or MMP9.

[0285] In some embodiments, an antibody that "inhibits activation of latent TGF-β1" includes an antibody that causes a decrease in TGF-β1 activation of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more. In other embodiments, an antibody that "inhibits the release of mature TGF-β1 from latent TGF-β1 mediated by a protease" includes an antibody that causes a decrease in the release of mature TGF-β1 from latent TGF-β1 mediated by a protease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more. In further embodiments, an antibody that inhibits the release of mature TGF-β1 from latent TGF-β1 mediated by a protease "without inhibiting cleavage of the LAP region of latent TGF-β1 mediated by a protease" includes an antibody that causes a decrease in cleavage of the LAP region of latent TGF-β1 mediated by a protease 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, 5% or less.

[0286] In some embodiments, the anti-LAP 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. When an anti-LAP TGF-β1 antibody "stabilizes" the structure of the LAP region, as used herein, the LAP region bound by the anti-LAP TGF-β1 antibody is held in some structure from which mature TGF-β1 cannot be released. In further embodiments, the latent TGF-β1 stabilized by the anti-LAP TGF-β1 antibody can be activated by integrins, preferably integrin αVβ8 and / or integrin αVβ6. In certain embodiments, the LAP region stabilized by the anti-LAP TGF-β1 antibody has been cleaved by a protease or has not been cleaved by a protease. In some embodiments, the anti-LAP TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1 and allows protease cleavage of the LAP region while the anti-LAP TGF-β1 antibody is bound to the LAP region of latent TGF-β1. In some embodiments, the anti-LAP 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 site of PLN and / or PLK. In other embodiments, the anti-LAP 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 site of MMP2 and / or MMP9.

[0287] In one aspect, the anti-LAP TGF-β1 antibody does not bind to mature TGF-β1. In some embodiments, the anti-LAP TGF-β1 antibody binds to latent TGF-β1 with a higher binding activity than to mature TGF-β1. In certain embodiments, the antibodies of the present application bind to latent TGF-β1 with 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-fold or more higher binding activity than to mature TGF-β1.

[0288] In one aspect, the anti-latent TGF-β1 antibody does not inhibit or does not significantly inhibit integrin-mediated activation of TGF-β1, i.e., integrin-mediated release of mature TGF-β1 from latent TGF-β1. In one aspect, the anti-latent TGF-β1 antibody has a minor effect on inhibiting integrin-mediated activation of TGF-β1, 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, an antibody that "does not inhibit or does not significantly inhibit integrin-mediated activation of TGF-β1" or "has a minor effect on inhibiting integrin-mediated activation of TGF-β1" includes an antibody that causes a decrease 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, 5% or less in integrin-mediated activation of TGF-β1, i.e., integrin-mediated release of mature TGF-β1 from latent TGF-β1.

[0289] In one aspect, anti-latent TGF-β1 antibodies elicit reduced or fewer toxicities and / or adverse effects associated with anti-TGF-β antagonists. In some embodiments, anti-latent TGF-β1 antibodies, such as those described herein, have superior safety- efficacy profiles compared to agents that elicit activity against mature TGF-β1 or agents that elicit activity against latent TGF-β1 but inhibit both protease- and integrin-mediated activation of latent TGF-β1. In some embodiments, anti-latent TGF-β1 antibodies of the present disclosure have reduced cardiotoxicity while having better or comparable efficacy than anti-mature TGF-β1 antibodies. Without being bound by any theory, anti-latent TGF-β1 antibodies of the present disclosure do not inhibit or do not significantly inhibit integrin-mediated TGF-β1 activation, or have less impact on inhibiting integrin-mediated TGF-β1 activation, and thus have reduced or fewer toxicities and / or adverse effects that arise from (i) integrin-mediated TGF-β1 activation or (ii) inhibition of TGF-β1 signaling at sites where TGF-β1 is activated by integrins. Thus, anti-latent TGF-β1 antibodies of the present disclosure can be administered to a subject in need thereof at a therapeutically effective dose without eliciting adverse effects, especially cardiotoxicity. Accordingly, such methods will broaden the dose range where both efficacy and safety / tolerability can be achieved in patients. Thus, the present invention provides methods of 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 or has less impact on inhibiting integrin-mediated TGF-β1 activation. The present invention encompasses use of an anti-latent TGF-β1 antibody for reducing toxicities and / or adverse effects associated with TGF-β1 inhibition in a subject. In some embodiments, toxicities and / or adverse effects can include cardiovascular toxicity, gastrointestinal toxicity, immunotoxicity, bone / cartilage toxicity, reproductive toxicity, and renal toxicity. In some embodiments, cardiovascular toxicity includes, but is not limited to, heart valve lesions, e.g., hemorrhage, inflammation, degeneration and proliferation of valve interstitial cells. In some embodiments, toxicities and / or adverse effects can include hemorrhage. In some embodiments, toxicities and / or adverse effects can include skin lesions or tumors. In some embodiments, toxicities and / or adverse effects can include tumor progression.

[0290] In some embodiments, anti-latent TGF-β1 antibodies of the present invention:

[0291] bind to latent TGF-β1;

[0292] bind to latent TGF-β1 to form an SLC;

[0293] binds to latent TGF-β1 to form an LLC;

[0294] binds to latent TGF-β1 in complex with GARP or LRRC33;

[0295] binds to cell surface latent TGF-β1;

[0296] binds to the LAP region of latent TGF-β1;

[0297] binds to LAP;

[0298] binds to latent TGF-β1 with a dissociation constant (KD) of 10 -8 nM or less, 10 -9 nM or less, or 10 -10 nM or less;

[0299] inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1;

[0300] does not inhibit protease-mediated cleavage of the LAP region of latent TGF-β1;

[0301] partially inhibits integrin-mediated activation of TGF-β1;

[0302] has little effect on inhibiting integrin-mediated activation of TGF-β1;

[0303] does not inhibit or does not significantly inhibit integrin-mediated release of mature TGF-β1 from latent TGF-β1; and / or

[0304] elicits reduced or fewer toxicities and / or adverse effects associated with anti-TGF-β1 antagonists, e.g., anti-mature TGF-β antibodies.

[0305] In further embodiments, the anti-latent TGF-β1 antibodies of the application are:

[0306] monoclonal antibodies;

[0307] human, humanized, or chimeric antibodies; and / or

[0308] IgG antibodies.

[0309] In some embodiments, the anti-latent TGF-β1 antibodies are monoclonal antibodies. In some embodiments, the anti-latent TGF-β1 antibodies are human antibodies. In some embodiments, the anti-latent TGF-β1 antibodies are humanized antibodies. In some embodiments, the anti-latent TGF-β1 antibodies are chimeric antibodies. In some embodiments, the anti-latent TGF-β1 antibodies are IgG antibodies.

[0310] In one aspect, the present application provides an anti-latent TGF-β1 antibody comprising:

[0311] (a) HVR-H1 comprising (or consisting of) an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, and 29;

[0312] (b) HVR-H2 comprising (or consisting of) an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 to 32 and 33;

[0313] (c) HVR-H3 comprising (or consisting of) an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 39 and 40;

[0314] (d) HVR-L1 comprising (or consisting of) an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 50 and 51;

[0315] (e) HVR-L2 comprising (or consisting of) an amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 53, and 54; and

[0316] (f) HVR-L3 comprising (or consisting of) an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 to 58 and 59,

[0317] wherein the antibody does not comprise HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 comprising SEQ ID NOs: 27, 30, 34, 48, 52, and 55, respectively.

[0318] In one aspect, the present application provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five, or six HVRs selected from the group consisting of:

[0319] (a) HVR-H1 comprising (or consisting of) an amino acid sequence of SEQ ID NO: 29;

[0320] (b) HVR-H2 comprising (or consisting of) an amino acid sequence of SEQ ID NO: 30;

[0321] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 36;

[0322] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 49;

[0323] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52; and

[0324] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0325] In one aspect, the present application provides an anti-Latent TGF-beta 1 antibody comprising at least one, two, three, four, five, or six HVRs selected from:

[0326] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 28;

[0327] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 32;

[0328] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37;

[0329] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 50;

[0330] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52; and

[0331] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0332] In one aspect, the present application provides an anti-Latent TGF-beta 1 antibody comprising at least one, two, three, four, five, or six HVRs selected from:

[0333] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27;

[0334] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30;

[0335] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 39;

[0336] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 48;

[0337] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 53; and

[0338] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0339] In one aspect, the present application provides an anti-Latent TGF-beta 1 antibody comprising at least one, two, three, four, five, or six HVRs selected from:

[0340] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27;

[0341] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 33;

[0342] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 40;

[0343] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 51;

[0344] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52; and

[0345] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0346] In one aspect, the present application provides an anti-Latent TGF-beta 1 antibody comprising:

[0347] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 29;

[0348] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30;

[0349] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 36;

[0350] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 49;

[0351] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52; and

[0352] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0353] In one aspect, the present application provides an anti-Latent TGF-beta 1 antibody comprising:

[0354] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 28;

[0355] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 32;

[0356] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37;

[0357] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 50;

[0358] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52; and

[0359] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0360] In one aspect, the present application provides an anti-Latent TGF-beta 1 antibody comprising:

[0361] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27;

[0362] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30;

[0363] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 39;

[0364] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 48;

[0365] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 53; and

[0366] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0367] In one aspect, the present application provides an anti-latent TGF-βl antibody comprising:

[0368] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27;

[0369] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 33;

[0370] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 40;

[0371] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 51;

[0372] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52; and

[0373] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0374] In one aspect, the present application provides an anti-latent TGF-βl antibody comprising HVR-H1, HVR-H2, and HVR-H3 of the VH sequence set forth in SEQ ID NO: 1 to 10 and 11, and HVR-L1, HVR-L2, and HVR-L3 of the VL sequence set forth in SEQ ID NO: 12 to 20 and 21 (or consisting thereof), wherein the HVRs are defined by: (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b), and / or (c), wherein the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 do not comprise SEQ ID NO: 27, 30, 34, 48, 52, and 55, respectively.

[0375] In one aspect, the application provides an anti-latent TGF-β1 antibody comprising (or consisting of) HVR-H1, HVR-H2, and HVR-H3 of a VH sequence set forth in SEQ ID NO: 4, and HVR-L1, HVR-L2, and HVR-L3 of a VL sequence set forth in SEQ ID NO: 16, wherein the HVRs are defined by: (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b), and / or (c).

[0376] In one aspect, the application provides an anti-latent TGF-β1 antibody comprising (or consisting of) HVR-H1, HVR-H2, and HVR-H3 of a VH sequence set forth in SEQ ID NO: 9, and HVR-L1, HVR-L2, and HVR-L3 of a VL sequence set forth in SEQ ID NO: 21, wherein the HVRs are defined by: (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b), and / or (c).

[0377] In one aspect, the application provides an anti-latent TGF-β1 antibody comprising (or consisting of) HVR-H1, HVR-H2, and HVR-H3 of a VH sequence set forth in SEQ ID NO: 11, and HVR-L1, HVR-L2, and HVR-L3 of a 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).

[0378] In one aspect, the application provides an anti-latent TGF-β1 antibody comprising (or consisting of) HVR-H1, HVR-H2, and HVR-H3 of a VH sequence set forth in SEQ ID NO: 10, and HVR-L1, HVR-L2, and HVR-L3 of a VL sequence set forth in SEQ ID NO: 20, wherein the HVRs are defined by: (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b), and / or (c).

[0379] 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 HVRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0380] In another aspect, an anti-latent TGF-β1 antibody comprises a heavy chain variable domain (VH) sequence 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: 4, 9, 11, or 10. In certain embodiments, an anti-latent TGF-β1 antibody comprises a heavy chain variable domain (VH) sequence 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: 2 to 10 or 11. 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 the anti-latent TGF-β1 antibody comprising that sequence retains the ability to bind to latent TGF-β1. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 4, 9, 11, or 10. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 2 to 10 or 11. 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 the VH sequence in SEQ ID NO: 4, 9, 11, or 10, including post-translational modifications of that sequence. Optionally, the anti-latent TGF-β1 antibody comprises the VH sequence in SEQ ID NO: 2 to 10 or 11, including post-translational modifications of that sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, 28, or 27; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, 32, or 33; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 36, 37, 39, or 40. In particular embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, 28, or 27; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30 to 32 or 33; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34 to 39 or 40.In certain embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, 28, or 27; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30 to 32 or 33; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34 to 39 or 40. Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0381] In another aspect, an anti-LTGF-βl antibody is provided, wherein the antibody comprises a heavy chain variable domain (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: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, an anti-LTGF-βl antibody is provided, wherein the antibody comprises a heavy chain variable domain (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: 1-10. 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 the anti-LTGF-βl antibody comprising that sequence retains the ability to bind to LTGF-βl. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 1-10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-LTGF-βl antibody comprises the VH sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including post-translational modifications of that sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, 12, 13, or 14; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, or 22. In particular embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, 12, 13, or 14; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, or 22. In particular embodiments, the VH comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, 12, 13, or 14; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, or 22.Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0382] In another aspect, an anti-LTGF-βl antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NOs: 1-11 and 12-21, respectively, including post-translational modifications of those sequences, wherein the antibody does not comprise the VH and VL of SEQ ID NOs: 1 and 12, respectively. In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NOs: 2-11 and 13-21, respectively. Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation. In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 16, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0383] In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 21, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0384] In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 17, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0385] In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 20, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0386] In some embodiments, the antibody comprises any of (a) to (h), including post-translational modifications of those sequences:

[0387] (a) a heavy chain sequence of SEQ ID NO: 82 and a light chain sequence of SEQ ID NO: 83;

[0388] (b) a heavy chain sequence of SEQ ID NO: 84 and a light chain sequence of SEQ ID NO: 85;

[0389] (c) a heavy chain sequence of SEQ ID NO: 86 and a light chain sequence of SEQ ID NO: 87;

[0390] (d) a heavy chain sequence of SEQ ID NO: 88 and a light chain sequence of SEQ ID NO: 89;

[0391] (e) a heavy chain sequence of SEQ ID NO: 90 and a light chain sequence of SEQ ID NO: 83;

[0392] (f) a heavy chain sequence of SEQ ID NO: 91 and a light chain sequence of SEQ ID NO: 85;

[0393] (g) a heavy chain sequence of SEQ ID NO: 92 and a light chain sequence of SEQ ID NO: 87; or

[0394] (h) a heavy chain sequence of SEQ ID NO: 93 and a light chain sequence of SEQ ID NO: 89.

[0395] Post-translational modifications include, but are not limited to, modification of a glutamine or glutamic acid in the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0396] In some embodiments, the antibodies of the present application comprise a heavy chain sequence and a light chain sequence disclosed herein.

[0397] The heavy chain sequence of each antibody consists of (i.e., is a combination of) the sequence (SEQ ID NO) of "VH" (N-terminal side) and the sequence (SEQ ID NO) of "CH" (C-terminal side) shown in Tables 1 and 3.

[0398] That is, the sequence (SEQ ID NO) of the heavy chain sequence can be deduced by joining the sequence (SEQ ID NO) of "VH" and the sequence (SEQ ID NO) of "CH".

[0399] The light chain sequence of each antibody consists of (i.e., is a combination of) the sequence (SEQ ID NO) of "VL" (N-terminal side) and the sequence (SEQ ID NO) of "CL" (C-terminal side) shown in Tables 1 and 3.

[0400] That is, the sequence of the light chain sequence (SEQ ID NO) can be deduced by concatenating the sequence of "VL" (SEQ ID NO) and the sequence of "CL" (SEQ ID NO).

[0401] Examples of such combinations are shown in Table 1.

[0402] [Table 1]

[0403]

[0404] In further aspects, the present application provides an antibody that binds to the same epitope as an anti-LTGF-βl antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as:

[0405] (1) an anti-LTGF-βl antibody comprising:

[0406] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 29,

[0407] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30,

[0408] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 36,

[0409] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 49,

[0410] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0411] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56;

[0412] (2) an anti-LTGF-βl antibody comprising:

[0413] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 28,

[0414] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 32,

[0415] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37,

[0416] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 50,

[0417] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0418] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56;

[0419] (3) an anti-latent TGF-β1 antibody comprising:

[0420] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27,

[0421] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30,

[0422] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 39,

[0423] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 48,

[0424] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 53, and

[0425] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56; or

[0426] (4) an anti-latent TGF-β1 antibody comprising:

[0427] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27,

[0428] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 33,

[0429] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 40,

[0430] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 51,

[0431] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0432] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0433] In certain embodiments, the present application provides an antibody that binds to the same epitope as an antibody comprising any of (a) to (h) below:

[0434] (a) the heavy chain sequence of SEQ ID NO: 82 and the light chain sequence of SEQ ID NO: 83;

[0435] (b) the heavy chain sequence of SEQ ID NO: 84 and the light chain sequence of SEQ ID NO: 85;

[0436] (c) the heavy chain sequence of SEQ ID NO: 86 and the light chain sequence of SEQ ID NO: 87;

[0437] (d) the heavy chain sequence of SEQ ID NO: 88 and the light chain sequence of SEQ ID NO: 89;

[0438] (e) the heavy chain sequence of SEQ ID NO: 90 and the light chain sequence of SEQ ID NO: 83;

[0439] (f) the heavy chain sequence of SEQ ID NO: 91 and the light chain sequence of SEQ ID NO: 85;

[0440] (g) the heavy chain sequence of SEQ ID NO: 92 and the light chain sequence of SEQ ID NO: 87; or

[0441] (h) the heavy chain sequence of SEQ ID NO: 93 and the light chain sequence of SEQ ID NO: 89.

[0442] In further aspects, the present application provides an antibody that binds to latent TGF-β1 of human, monkey, mouse, and / or rat. In certain embodiments, the present application provides an antibody that binds to latent TGF-β1 of human, monkey, and mouse. In certain embodiments, the present application provides an antibody that binds to latent TGF-β1 of human, monkey, and mouse forming SLC. In certain embodiments, the present application provides an antibody that binds to latent TGF-β1 of human, monkey, and mouse forming LLC. In certain embodiments, the present application provides an antibody that binds to latent TGF-β1 of human, monkey, and mouse forming LLC. In certain embodiments, the present application provides an antibody that binds to latent TGF-β1 of human, monkey, and mouse in complex with GARP or LRRC33. In certain embodiments, the present application provides an antibody that binds to cell surface latent TGF-β1 of human, monkey, and mouse.

[0443] In further aspects, the present application provides an antibody that binds to the same epitope as any one of the anti-LTGF-βl antibodies provided herein. The epitope can be present on human, monkey, mouse, and / or rat TGF-βl. For example, in certain embodiments, the present application provides an antibody that binds to the same epitope as a reference antibody, wherein the reference antibody is:

[0444] (1) an anti-LTGF-βl antibody comprising:

[0445] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 29,

[0446] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30,

[0447] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 36,

[0448] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 49,

[0449] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0450] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56;

[0451] (2) an anti-LTGF-βl antibody comprising:

[0452] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 28,

[0453] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 32,

[0454] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37,

[0455] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 50,

[0456] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0457] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56;

[0458] (3) an anti-latent TGF-βl antibody comprising:

[0459] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27,

[0460] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30,

[0461] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 39,

[0462] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 48,

[0463] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 53, and

[0464] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56; or

[0465] (4) an anti-latent TGF-βl antibody comprising:

[0466] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27,

[0467] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 33,

[0468] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 40,

[0469] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 51,

[0470] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0471] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0472] In certain embodiments, the present application provides an antibody that binds to the same epitope as a reference antibody, wherein the reference antibody comprises any one of (a) to (h):

[0473] (a) a heavy chain sequence of SEQ ID NO: 82 and a light chain sequence of SEQ ID NO: 83;

[0474] (b) a heavy chain sequence of SEQ ID NO: 84 and a light chain sequence of SEQ ID NO: 85;

[0475] (c) a heavy chain sequence of SEQ ID NO: 86 and a light chain sequence of SEQ ID NO: 87;

[0476] (d) a heavy chain sequence of SEQ ID NO: 88 and a light chain sequence of SEQ ID NO: 89;

[0477] (e) a heavy chain sequence of SEQ ID NO: 90 and a light chain sequence of SEQ ID NO: 83;

[0478] (f) a heavy chain sequence of SEQ ID NO: 91 and a light chain sequence of SEQ ID NO: 85;

[0479] (g) a heavy chain sequence of SEQ ID NO: 92 and a light chain sequence of SEQ ID NO: 87; or

[0480] (h) a heavy chain sequence of SEQ ID NO: 93 and a light chain sequence of SEQ ID NO: 89.

[0481] In further aspects, the application 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, there are provided antibodies that compete with the following for binding to human, monkey, mouse, and / or rat TGF-β1:

[0482] (1) an anti-latent TGF-β1 antibody comprising:

[0483] (a) an HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 29,

[0484] (b) an HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30,

[0485] (c) an HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 36,

[0486] (d) an HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 49,

[0487] (e) an HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0488] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56;

[0489] (2) an anti-latent TGF-βl antibody comprising:

[0490] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 28,

[0491] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 32,

[0492] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37,

[0493] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 50,

[0494] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0495] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56;

[0496] (3) an anti-latent TGF-βl antibody comprising:

[0497] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27,

[0498] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 30,

[0499] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 39,

[0500] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 48,

[0501] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 53, and

[0502] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56; or

[0503] (4) an anti-latent TGF-βl antibody comprising:

[0504] (a) HVR-H1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 27,

[0505] (b) HVR-H2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 33,

[0506] (c) HVR-H3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 40,

[0507] (d) HVR-L1 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 51,

[0508] (e) HVR-L2 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 52, and

[0509] (f) HVR-L3 comprising (or consisting of) the amino acid sequence of SEQ ID NO: 56.

[0510] In certain embodiments, the present application provides an antibody that competes for binding to human, monkey, mouse and / or rat TGF-β1 with an antibody comprising any of (a) to (h):

[0511] (a) a heavy chain sequence of SEQ ID NO: 82 and a light chain sequence of SEQ ID NO: 83;

[0512] (b) a heavy chain sequence of SEQ ID NO: 84 and a light chain sequence of SEQ ID NO: 85;

[0513] (c) a heavy chain sequence of SEQ ID NO: 86 and a light chain sequence of SEQ ID NO: 87;

[0514] (d) a heavy chain sequence of SEQ ID NO: 88 and a light chain sequence of SEQ ID NO: 89;

[0515] (e) a heavy chain sequence of SEQ ID NO: 90 and a light chain sequence of SEQ ID NO: 83;

[0516] (f) a heavy chain sequence of SEQ ID NO: 91 and a light chain sequence of SEQ ID NO: 85;

[0517] (g) a heavy chain sequence of SEQ ID NO: 92 and a light chain sequence of SEQ ID NO: 87; or

[0518] (h) a heavy chain sequence of SEQ ID NO: 93 and a light chain sequence of SEQ ID NO: 89.

[0519] In a further aspect of the application, 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 according to any of the above embodiments is a chimeric antibody. In one embodiment, the anti-latent TGF-β1 antibody according to any of the above embodiments is a humanized antibody. In one embodiment, the anti-latent TGF-β1 antibody according to any of the above embodiments is a human antibody. In some embodiments, the anti-latent TGF-β1 antibody according to any of the above embodiments is a chimeric monoclonal antibody. In some embodiments, the anti-latent TGF-β1 antibody according to any of the above embodiments is a humanized monoclonal antibody. In some embodiments, the anti-latent TGF-β1 antibody according to any of the above embodiments is a human monoclonal antibody. In another embodiment, the antibody is a full-length antibody, e.g., a complete IgGl, IgG2, IgG3, or IgG4 antibody or a complete antibody of other class or isotype as defined herein. In another embodiment, the antibody is, e.g., an IgGl, IgG2, IgG3, or IgG4 antibody or an antibody of other class or isotype as defined herein.

[0520] In one embodiment, the antibody fragment of the anti-latent TGF-β1 antibody is, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In one embodiment, the antibody fragment of the anti-latent TGF-β1 antibody includes any antigen binding molecule comprising a variable heavy chain and / or a variable light chain structure of an immunoglobulin.

[0521] In a further aspect, the anti-latent TGF-β1 antibody according to any of the above embodiments can incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0522] 1. Antibody Binding Activity

[0523] In certain embodiments, the antibodies provided herein have a dissociation constant (KD) of 1 micromolar or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M) to an epitope of latent TGF-β1.

[0524] In one embodiment, the binding activity of the antibody is measured by radiolabeled antigen binding assay (RIA) and expressed as KD. In one embodiment, the RIA is performed using the Fab form of the target antibody and its antigen. For example, the solution binding activity of the Fab to the antigen is determined by titrating the antibody with a minimum concentration of ( 125 The Fab binding activity was measured by equilibrating the Fab with an antigen labeled with Fab and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 micrograms / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 degrees Celsius). 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., consistent with 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; however, incubation can be continued for longer periods (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20™; Packard) is added and the plate is counted for tens of minutes in a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that give less than or equal to 20% maximal binding are selected for use in competitive binding assays.

[0525] In one embodiment, for measuring the binding activity of an antibody, a ligand capture method is used, for example using BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare, Uppsala, Sweden), which relies on a surface plasmon resonance analysis method as a measurement principle. BIACORE (registered trademark) control software is used for device operation. In one embodiment, according to the manufacturer's instructions, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used to immobilize a molecule for capturing a ligand, for example, an anti-tag antibody, an anti-IgG antibody, protein A, etc., on a sensor chip coated with carboxymethyl dextran (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with a 10 mM sodium acetate solution at an appropriate pH, and injected at an appropriate flow rate and for an appropriate injection time. A buffer containing 0.05% polysorbate 20 (also known as Tween (registered trademark)-20) is used as a measurement buffer, and the binding activity measurement is measured at a flow rate of 10-30 microliters / minute, and at a measurement temperature of preferably 25°C or 37°C. For measurements using an antibody captured by a ligand capture molecule as a ligand, the antibody is injected to capture a target amount of the antibody, and then a serial dilution of an antigen and / or Fc receptor (analyte) prepared using the measurement buffer is injected. For measurements using an antigen and / or Fc receptor captured by a ligand capture molecule as a ligand, the antigen and / or Fc receptor is injected to capture a target amount thereof, and then a serial dilution of an antibody (analyte) prepared using the measurement buffer is injected.

[0526] In one embodiment, the measurement results are analyzed using BIACORE (registered trademark) evaluation software. Kinetic parameter calculations are performed by simultaneously fitting the sensorgrams of association and dissociation using a 1:1 binding model, and the association rate (kon or ka), dissociation rate (koff or kd), and equilibrium dissociation constant (KD) can be calculated. For cases of weak binding activity, in particular for cases where dissociation is fast and kinetic parameters are difficult to calculate, a steady-state model can be used to calculate the equilibrium dissociation constant (KD). As an additional parameter on binding activity, the "amount of analyte bound per amount of ligand" can be calculated by dividing the amount of binding (resonance units: RU) of a specific concentration of analyte by the amount of ligand captured.

[0527] 2. Antibody Fragments

[0528] In certain embodiments, antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below. For a review of certain 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. For discussion of Fab fragments and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0529] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al. Nat. Med. 9: 129-134 (2003); and Hollinger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). See also Hudson et al. Nat. Med. 9: 129-134 (2003) for a description of triabodies and tetrabodies.

[0530] Single-domain antibodies are antibody fragments comprising 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, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

[0531] Antibody fragments can be made by various 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.

[0532] The present application also relates to antigen-binding molecules that bind to TGF-β1, including but not limited to, for example, minibodies (low-molecular-weight antibodies) and scaffold proteins. Any scaffold protein is acceptable in the present application as long as it is a peptide that has a stable three-dimensional structure and is capable of binding to at least one antigen. Such peptides include, for example, fragments of antibody variable regions, fibronectin, Protein A domains, LDL receptor A domains, lipocalins, and other molecules described by 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). When reference is made to such antibodies, for example, in the context of the present specification, "anti-latent TGF-β1 antibody" should be replaced with "anti-latent TGF-β1 antigen-binding molecule."

[0533] 3. Chimeric and Humanized Antibodies

[0534] In certain embodiments, an 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 mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Antigen-binding fragments of chimeric antibodies can also be provided.

[0535] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and binding capacity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody, while FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise 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 are derived), for example, to restore or improve antibody specificity or binding capacity.

[0536] Humanized antibodies and methods of making them are reviewed, for example, in the following references: Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and further described, for example, in the following references: 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 "surface reshaping"); 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 the "guided selection" approach to FR shuffling).

[0537] Human framework regions that can be used include, but are not limited to, framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., 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, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0538] 4. Human Antibodies

[0539] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0540] Human antibodies can be made by administering the antigen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with a human variable region in response to antigenic challenge. Such animals are typically encompassed by the term "nude mice" as used herein. Such animals have generally been modified to contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions. TM

[0541] ​Human antibodies can also be made by using various techniques such as those involving the hybridoma method. Human myeloma and mouse-human heteromyeloma cells lines have been described for the production of human monoclonal antibodies. (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 produced by 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, e.g., in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4): 265-268 (2006) (describing 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).

[0542] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0543] 5. Library-derived Antibodies

[0544] Antibodies of the application can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. For a review of such methods, see, e.g., Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further described in, e.g., 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).

[0545] In certain phage display methods, all or a portion of the VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined at random in a phage library, which can then be screened for antigen-binding phage as described in Winter et al. Ann. Rev. Immunol., 12: 433-455 (1994). Phage will typically display antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-binding activity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive libraries can be cloned (e.g., from a human) to provide a single source of antibodies to a wide range of non-self and self antigens without any immunization as described in Griffiths et al. EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made by cloning unrearranged V-gene segments from stem cells; and using PCR primers with random sequence to encode the highly variable CDR3 regions and to complete the rearrangement in vitro as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, e.g., U.S. Patent No. 5,750,373, and U.S. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0546] Antibodies or antibody fragments isolated from human antibody libraries herein are considered to be human antibodies or human antibody fragments.

[0547] 6. Multispecific Antibodies

[0548] In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for 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 can bind to two different epitopes of TGF-β1. Bispecific antibodies can also be used to target cytotoxic agents to cells which express TGF-β1. Bispecific antibodies can be prepared as full length antibodies. Antibody fragments of a bispecific antibody can also be provided.

[0549] Techniques for making multispecific antibodies include, but are not limited to, recombinant co- expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see, e.g., Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009 / 089004 Al); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using “diabody” technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

[0550] Also included among the antibodies herein are engineered antibodies having three or more functional antigen binding sites, including “octopus antibodies” (see, e.g., US 2006 / 0025576 Al).

[0551] Also included among the antibody fragments herein are “Dual Acting Fabs” or “DAFs,” which comprise an antigen binding site that binds to TGF-β1 as well as another, different antigen (see, e.g., US 2008 / 0069820).

[0552] 7. Antibody Variants

[0553] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can 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 sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0554] a) Substitution, Insertion, and Deletion Variants

[0555] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are provided in Table 2 under the heading of "exemplary substitutions," and further described below in reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for a desired activity, such as retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0556] [Table 2]

[0557] Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu (L) norleucine; lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Trp; Leu; Val; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala; norleucine Leu

[0558] Amino acids can be grouped according to common side-chain properties:

[0559] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

[0560] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0561] (3) acidic: Asp, Glu;

[0562] (4) basic: His, Lys, Arg;

[0563] (5) residues that influence chain orientation: Gly, Pro;

[0564] (6) aromatic: Trp, Tyr, Phe.

[0565] Non-conservative substitutions will require exchanging a member of one of these classes for a member from another.

[0566] One type of substitutional variant involves substituting one or more amino acids in a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant antibody will have at least about 80% sequence identity, but can have more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, with the parent antibody. Usually, the selected substitution(s) will be in HVRs. The resulting variant antibody will have at least one, but usually more than one, of the following modifications (e.g., improvements) relative to the parent antibody: increased binding affinity for the antigen, increased biological activity (e.g., increased cytotoxicity), decreased immunogenicity, and / or improved production yield.

[0567] Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody binding activity. Such changes can be made in HVR "hotspots," i.e., residues encoded by codons that mutate at a relatively high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, with the resulting variant VHand / or VLbeing tested for binding activity. Binding activity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of binding activity 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 created. The library is then screened to identify any antibody variants with the desired binding activity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomly grouped. HVR residues involved in antigen binding can be specifically identified, e.g., using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0568] In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding activity can be made in HVRs. Such alterations can be outside of antigen contacting residues of HVRs. In certain embodiments of the variant VHand VLsequences provided above, each HVR remains unchanged, or contains no more than one, two, or three amino acid substitutions.

[0569] A useful method for identification of residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as 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 arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions can be introduced at the amino acid positions demonstrating functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened for appropriate binding affinity.

[0570] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include a fusion with a polypeptide at the N- or C-terminus of the antibody.

[0571] b) Glycosylation Variants

[0572] In certain embodiments, an antibody provided herein is altered 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, for example, changing the number of carboxylic acid residues in the

[0573] When the antibody comprises an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is typically linked to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications can be made to the oligosaccharide in an antibody of the application in order to create antibody variants that have certain improved properties.

[0574] In one embodiment, antibody variants are provided having carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibody can be from 1 % to 80%, from 1 % to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycosylation structures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 can be located about + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants can have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Relevant “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing afucosylated antibodies include protein fucosylation deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 Al, Presta, L; and WO 2004 / 056312 Al, Adams et al., inter alia in Example 11), and knockout cell lines such as CHO cells knocked out for the a-1,6-fucosyltransferase gene FUT8 (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0575] Further provided are antibody variants with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, e.g., WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants can have improved CDC function. Such antibody variants are described in, e.g., WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0576] c) Fc Region Variants

[0577] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. Fc region variants can comprise human Fc region sequences (e.g., a human IgGl, 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, a human Fc variant can comprise a chimeric human Fc region sequence (e.g., a human IgGl / 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.

[0578] In certain embodiments, the present application contemplates antibody variants that have some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo 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 conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody is deficient in FcyR binding (hence, likely deficient in ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression 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. Patent 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); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods can be used (see, for example, ACT1 TMNon-radioactive cytotoxicity assays (Cell Technology, Inc. Mountain View, CA); and CytoTox 96 (registered trademark) 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 the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (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)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0579] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine ( U.S. Patent No. 7,332,581 ).

[0580] "Reduced effector function" refers to a reduction in the effector function of the Fc region of an antibody, which varies with the antibody isotype. Examples of reduced effector function include: reduced C1q binding; reduced complement-dependent cytotoxicity (CDC); reduced Fc receptor binding (e.g., reduced Fcγ receptor binding); reduced antibody-dependent cell-mediated cytotoxicity (ADCC); reduced phagocytosis; reduced downregulation of cell surface receptors (e.g., B cell receptors); and reduced B cell activation.

[0581] A "reduction" in effector function / activity means that the effector function / activity is 95% or less, preferably 90% or less, 85% or less, 80% or less, 75% or less, more preferably 70% or less, 65% or less, 60% or less, 55% or less, 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, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the function / activity of an intact IgG (or an antibody comprising an intact Fc region). For example, the function / activity (e.g., Fcy receptor binding activity) of an IgG is preferably reduced by a factor of at least about 10-fold or more, about 50-fold or more, about 100-fold or more, compared to the function / activity (e.g., Fcy receptor binding activity) of an intact IgG (or an antibody comprising an intact Fc region).

[0582] Certain antibody variants with increased or decreased binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0583] In certain embodiments, antibody variants comprise an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0584] In some embodiments, changes are made in the Fc region which result in altered (i.e., either increased or decreased) C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0585] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US 2005 / 0014934 A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following 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., substitutions at Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0586] 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 for other examples of Fc region variants.

[0587] d) Cysteine-engineered Antibody Variants

[0588] In certain embodiments, it may be desirable to produce antibodies engineered with cysteine, for example, "thioMAbs," wherein one or more residues of an antibody are replaced with cysteine ​​residues. In a particular embodiment, the substituted residues are present at accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are thereby located at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to produce immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine-engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0589] e) Antibody Derivatives

[0590] In certain embodiments, the antibodies provided herein can be further modified to comprise additional non-proteinaceous moieties known in the art and readily available to the skilled worker. Moieties suitable for derivatization of the antibody 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), co-polymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic acid copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. The polymers can have any molecular weight and can be branched or linear. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on such considerations as the particular properties or functions that the antibody is intended to improve, whether the antibody derivative will be used for therapy under defined conditions, etc.

[0591] In another embodiment, conjugates of the antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells, but heat the non-proteinaceous moiety to a temperature that kills cells in the vicinity of the antibody-non-proteinaceous moiety conjugate.

[0592] B. Recombinant Methods and Compositions

[0593] Recombinant methods and compositions can be used to produce antibodies, e.g., 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 can encode an amino acid sequence comprising a VL of the antibody and / or an amino acid sequence comprising a VH of the antibody (e.g., a light chain and / or a heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antibody and an amino acid sequence comprising a VH of the antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making an anti-latent TGF-β1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above in conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0594] For recombinant production of an anti-latent TGF-β1 antibody, a nucleic acid encoding the antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific to genes encoding the heavy and light chains of the antibody).

[0595] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not needed. 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 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) Antibodies can be isolated from bacterial cell paste in a soluble fraction following expression, and can be further purified.

[0596] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0597] Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0598] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0599] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (such as 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, such as those described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -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 certain mammalian host cell lines suitable for production of antibodies, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0600] C. Assays

[0601] The anti-latent TGF-β1 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0602] 1. Binding Assays and Other Assays

[0603] In one aspect, the antibodies of the application are tested for antigen binding affinity / activity, e.g., by known methods such as ELISA, Western blot, surface plasmon resonance (e.g., BIACORE (registered trademark)), or similar techniques (e.g., KinExa or OCTET (registered trademark)), etc.

[0604] In another aspect, a competition assay can be used to identify antibodies that compete for binding to latent TGF-β1 with any of the anti-latent TGF-β1 antibodies described herein (preferably hT0947AE20-SG181, hT0947AE27-SG181, hT0947AE34-SG181, hT0947AE37-SG181, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, or hT0947AE37-SG191). In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as any of the anti-latent TGF-β1 antibodies described herein (preferably hT0947AE20-SG181, hT0947AE27-SG181, hT0947AE34-SG181, hT0947AE37-SG181, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, or hT0947AE37-SG191). Detailed exemplary methods for mapping the epitopes to which antibodies bind 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 methods.

[0605] In certain embodiments, such competing antibodies block (e.g., reduce) binding of a reference antibody to latent TGF-β1 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more when such competing antibodies are present in excess. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as the anti-latent TGF-β1 antibodies described herein. In further aspects, the reference antibody is hT0947AE20-SG181, hT0947AE27-SG181, hT0947AE34-SG181, hT0947AE37-SG181, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, or hT0947AE37-SG191.

[0606] In an exemplary competition assay, immobilized latent TGF-β1 is incubated in a solution comprising a first labeled antibody (reference antibody) that binds to latent TGF-β1 (e.g., hT0947AE20-SG181, hT0947AE27-SG181, hT0947AE34-SG181, hT0947AE37-SG181, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, or hT0947AE37-SG191) and a second unlabeled antibody whose ability to compete with the first antibody for binding to latent TGF-β1 is being tested. This second antibody can be present in a hybridoma supernatant. As a control, immobilized latent TGF-β1 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to latent TGF-β1, excess unbound antibody is removed, and the amount of label associated with the immobilized latent TGF-β1 is measured. If the amount of label associated with the immobilized latent TGF-β1 in the test sample is significantly reduced relative to the control sample, then the second antibody is competing 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).

[0607] 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, and the like. For example, cells expressing latent TGF-β1 can be contacted with an anti-latent TGF-β1 antibody directly conjugated with PE or APC, or with an unconjugated anti-latent TGF-β1 antibody, followed by a second antibody conjugated with PE or APC, and staining of cell surface latent TGF-β1 can be detected. See, e.g., Oida et al., PLoS One. 2010 Nov 24; 5(11):e15523; Su et al., Hum Mol Genet. 2015 Jul 15; 24(14):4024-36.

[0608] 2. Activity Assays

[0609] In one aspect, assays are provided for identifying anti-latent TGF-β1 antibodies having biological activity. Biological activity can include, for example, inhibition of activation of latent TGF-β1, inhibition of release of mature TGF-β1 from latent TGF-β1, inhibition of protease-mediated release of mature TGF-β1 from latent TGF-β1, inhibition of protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibition of protease-mediated cleavage of the LAP region of latent TGF-β1, inhibition of protease-mediated release of mature TGF-β1 from latent TGF-β1 without blocking protease access to latent TGF-β1, inhibition of protease-mediated release of mature TGF-β1 from latent TGF-β1 while allowing protease to cleave the LAP region of latent TGF-β1, inhibition of protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibition or partial inhibition of integrin-mediated TGF-β1 activation, and the like. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0610] In certain embodiments, such biological activity of antibodies of the application are tested.

[0611] In some embodiments, after contacting a latent TGF-β1 activator (e.g., a protease, integrin, other non-protease activators, etc.) with latent TGF-β1 in the presence or absence of the test antibody, the test antibody is determined to determine whether it inhibits activation of latent TGF-β1, i.e., inhibits the release of mature TGF-β1 from latent TGF-β1, by detecting mature TGF-β1 using methods 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 integrin). It is also known that activation of latent TGF-β1, i.e., release of mature TGF-β1 from latent TGF-β1, also occurs in the absence of an activator (spontaneous activation of latent TGF-β1). In some embodiments, whether the test antibody inhibits the spontaneous activation of latent TGF-β1 is determined by detecting mature TGF-β1 using the above method after incubation of latent TGF-β1 with or without the test antibody. In some embodiments, when the amount of mature TGF-β1 detected in the presence of the test antibody (or after contact with the test antibody) is reduced compared to the amount detected in the absence of the test antibody, the test antibody is identified as an antibody that can inhibit the activation of latent TGF-β1. In one example, the amount of mature TGF-β1, whether it is reduced or increased, can be measured based on the concentration of mature TGF-β1 (e.g., g / ml, mg / ml, micrograms / ml, ng / ml or pg / ml, etc.). In another example, the amount of mature TGF-β, whether it is reduced or increased, can be measured based on the optical density (OD) (e.g., wavelength in mm or nm, etc.) of a marker directly or indirectly associated with mature TGF-β.

[0612] In certain embodiments, inhibition of TGF-β1 activation comprises a decrease in the amount of mature TGF-β1 in an assay by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more compared to a negative control under similar conditions. In some embodiments, it refers to inhibition of TGF-β1 activation, i.e., inhibition of the release of mature TGF-β1 by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more.

[0613] In some embodiments, testing whether an 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 the activity of mature TGF-β1, e.g., the activity of binding to TGF-β1 receptors, or the activity of mediating signal transduction in cells expressing TGF-β1 receptors, etc. In some embodiments, the binding of mature TGF-β1 to TGF-β1 receptors can be detected using a receptor binding assay. In some embodiments, the activity of mediating TGF-β1 signal transduction can be determined by detecting the activation of the TGF-β1 / Smad pathway. Cells that can be used for such assays can be cells expressing endogenous TGF-β1 receptors or cells generated by transfecting cells with a TGF-β1 receptor gene. For example, HEK-Blue TGF-β cells, or those genetically modified, transiently or stably, to express a transgene encoding a TGF-β1 receptor, can be used. The activity of mediating TGF-β1 signal transduction can be detected at any level in the signal transduction pathway, e.g., by examining the phosphorylation of Smad polypeptides, examining the expression of TGF-β1 regulated genes, including reporter genes, or measuring the proliferation of TGF-β1 dependent cells. TM TGF-β1 receptors, or those genetically modified, transiently or stably, to express a transgene encoding a TGF-β1 receptor, can be used. The activity of mediating TGF-β1 signal transduction can be detected at any level in the signal transduction pathway, e.g., by examining the phosphorylation of Smad polypeptides, examining the expression of TGF-β1 regulated genes, including reporter genes, or measuring the proliferation of TGF-β1 dependent cells.

[0614] In some embodiments, the activity of mediating TGF-β1 signal transduction can also be determined by detecting the activation of the TGF-β1 / Smad pathway, by examining the 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, the activity of mediating TGF-β1 signal transduction can be determined by examining the ability of TGF-β to inhibit cell migration in a “wounded” monolayer culture of BAE cells, by examining the ability of TGF-β to inhibit cell growth, by examining the ability of TGF-β to inhibit plasminogen activator (PA) activity, by examining 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)

[0615] Inhibition of TGF-β1 activation can also be detected and / or measured using the methods described and exemplified in the working examples. Using these or other suitable types of assays, test antibodies that are capable of inhibiting the activation of TGF-β1 can be screened. In certain embodiments, inhibition of TGF-β1 activation comprises at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or greater reduction in TGF-β1 activation in the assay as compared to a negative control under similar conditions. In some embodiments, it refers to inhibition of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater 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 greater reduction in the amount of mature TGF-β1 detected in the assay as compared to a negative control under similar conditions. In some embodiments, it refers to a reduction in the amount of mature TGF-β1 of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater.

[0616] In some embodiments, after the protease is contacted with latent TGF-β1 in the presence or absence of a test antibody, it is determined whether the test antibody inhibits cleavage of the LAP portion of latent TGF-β1 by detecting cleavage products 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, in the case where a protein tag (e.g., a FLAG tag, etc.) is added to the N-terminus of the LAP region of latent TGF-β1, when protease-mediated cleavage occurs, the portion with the protein tag is cleaved off. Thus, cleavage products of latent TGF-β1 can be detected by detecting latent TGF-β1 (or the LAP region of latent TGF-β1) that does not contain the protein tag, and / or uncleaved latent TGF-β1 can be detected by detecting latent TGF-β1 with the protein tag.

[0617] For another example, in the case where a protein tag (e.g., a FLAG tag, etc.) is added to the N-terminus of the LAP region of latent TGF-β1, and in the case where the location of the cleavage site of the protease is not close to the N-terminus of the LAP region of latent TGF-β1, when protease-mediated cleavage occurs, the LAP region with the protein tag is shortened. Thus, cleavage products of latent TGF-β1 can be detected by detecting latent TGF-β1 (or the shortened LAP region of latent TGF-β1) with the protein tag that has a shortened LAP region.

[0618] In some embodiments, a test antibody is identified as an antibody that can inhibit cleavage of latent TGF-β1 when the amount of cleavage product of latent TGF-β1 detected in the presence of the test antibody (or after contact with the test antibody) is reduced compared to the amount detected in the absence of the test antibody. Conversely, a test antibody is identified as an antibody that does not inhibit cleavage of latent TGF-β1 when the amount of cleavage product of latent TGF-β1 in the presence of the test antibody (or after contact with the test antibody) is not significantly reduced compared to the amount detected in the absence of the test antibody. In some embodiments, a test antibody is identified as an antibody that can inhibit cleavage of latent TGF-β1 when an increased 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. Conversely, a test antibody is identified as an antibody that does not inhibit cleavage of latent TGF-β1 when the amount of uncleaved latent TGF-β1 in the presence of the test antibody (or after contact with the test antibody) is not significantly increased compared to the amount detected in the absence of the test antibody. In certain embodiments, whether a test antibody blocks protease access to latent TGF-β1 is determined by a method that detects a protein interaction between the protease and latent TGF-β1, such as an ELISA or surface plasmon resonance (e.g., BIACORE (registered trademark)) or similar technology (e.g., KinExa or OCTET (registered trademark)). A test antibody is identified as an antibody that can block protease access to latent TGF-β1 when the interaction between the protease and latent TGF-β1 is reduced 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.

[0619] In certain embodiments, non-inhibition of cleavage of latent TGF-β1 comprises an increase in the amount of cleavage product of latent TGF-β1 in the assay of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more compared to a negative control under similar conditions. In some embodiments, non-inhibition of cleavage of latent TGF-β1 comprises an increase in the amount of uncleaved latent TGF-β1 in the assay of at least 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, 5% or less compared to a negative control under similar conditions.

[0620] In some embodiments, anti-latent TGF-β1 antibodies can be subjected to other biological activity assays, e.g., to assess their effectiveness as therapeutic agents. Such assays are known in the art and depend on the target antigen and the intended use of the antibody. For example, the biological effects of TGF-β1 blockade by an anti-latent TGF-β1 antibody can be assessed in a mouse model of renal fibrosis induced by unilateral ureteral obstruction (UUO) (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.) and / or syngeneic tumor models (e.g., as described in Mariathasan S, et al. TGF-beta attenuates tumour 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 can be subjected to the biological activity assays described herein.

[0621] 3. Screening Methods

[0622] In one aspect, a method for screening antibodies of the application includes various assays described herein and known in the art. For example, a method for screening an anti-latent TGF-β1 antibody includes:

[0623] (a) contacting a biological sample comprising latent TGF-β1 and a protease with a test antibody;

[0624] (b) detecting (i) whether the test antibody inhibits cleavage of the LAP region of latent TGF-β1 and (ii) whether the test antibody inhibits activation of latent TGF-β1; and

[0625] (c) selecting a test antibody that inhibits activation of latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP portion of latent TGF-β1.

[0626] Alternatively, instead of steps (b) and (c) above, a method for screening an anti-latent TGF-β1 antibody includes, e.g., the following steps (b) and (c):

[0627] (b) measuring (i) the amount of uncleaved latent TGF-β1 and (ii) the amount of mature TGF-β1; and

[0628] (c) if the amount of uncleaved latent TGF-β1 is not significantly increased and the amount of mature TGF-β1 is decreased as compared to in the absence of the test antibody, then the test antibody is selected that inhibits the release of mature TGF-β1 from latent TGF-β1 mediated by a protease without inhibiting the cleavage of the LAP region of latent TGF-β1 mediated by a protease.

[0629] Alternatively, instead of the above steps (b) and (c), the method for screening an anti-latent TGF-β1 antibody comprises, for example, the following steps (b) and (c):

[0630] (b) measuring (i) the amount of cleavage product of latent TGF-β1 and (ii) the level of activity of mature TGF-β1; and

[0631] (c) if the amount of cleavage product is not significantly decreased and the level of activity of mature TGF-β1 is decreased as compared to in the absence of the test antibody, then the test antibody is selected that inhibits the activation of latent TGF-β1 mediated by a protease without inhibiting the cleavage of the LAP region of latent TGF-β1 mediated by a protease.

[0632] Further, the present application provides a method for producing an anti-latent TGF-β1 antibody, which comprises, for example, the following steps (d) and (e) in addition to the above steps (a) to (c):

[0633] (d) obtaining amino acid sequence information of the anti-latent TGF-β1 antibody selected in step (c); and

[0634] (e) introducing a gene encoding the anti-latent TGF-β1 antibody into a host cell.

[0635] In one embodiment, the method for producing an anti-latent TGF-β1 antibody further comprises, after the above (e), (f) culturing the host cell, thereby producing the anti-latent TGF-β1 antibody.

[0636] In this context, the term "not significantly increased / decreased", e.g., in the phrases "the amount of uncleaved latent TGF-β1 is not significantly increased" and "the amount of (cleaved products of latent TGF-β1) is not significantly decreased" means that the level / extent of increase / decrease can be zero, or can not be zero but close to zero, or can be very low enough to be technically negligible or practically / essentially considered as zero by one of skill in the art. For example, in immunoblot analysis, when a researcher is unable to detect or observe any significant signal / band (or a relatively high or strong signal) of uncleaved latent TGF-β1, it is considered that the amount of uncleaved latent TGF-β1 is "not significantly increased", or the amount of (cleaved products of latent TGF-β1) is "not significantly decreased". In addition, the term "not significantly increased / decreased" is used interchangeably with the term "not substantially increased / decreased".

[0637] In some embodiments, testing whether an antibody inhibits cleavage of the LAP region of latent TGF-β1, and testing whether an antibody inhibits activation of latent TGF-β1 can be determined by various assays described herein and known in the art.

[0638] D. Immunoconjugates

[0639] The present application also provides immunoconjugates comprising an anti-latent TGF-β1 antibody herein conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0640] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent EP 0 425 235 Bl); an auristatin, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or a derivative thereof (see U.S. Patent 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 Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see 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; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

[0641] 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 fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, phytolaca A chain, a- sarcin, gelonin, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saporin, goseplin, mitogellin, retstrictin, phenomycin, enomycin, and the trichothecals.

[0642] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes 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 Pb and Lu. When the radioconjugate is used for detection, it can comprise a radioactive atom for scintigraphic studies, for example, Tc-99m or I-123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine- 123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. 123 I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0643] A variety of bifunctional protein coupling agents can be used to couple the antibody to the cytotoxic agent, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), 4-(N- maleidomethyl) cyclohexane-l-carboxylate succinimidyl ester (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), azetidines (such as bis-(p-azidophenylthioethyl) ether, bi-(4-azidosalycilamide), diisocyanates (such as toluene 2,6-diisocyanate), and bis-diazo compounds (such as bis-(p-diazoazobenzoyl)-ethylenediamine). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for coupling radionucleidides to the antibodies. See WO94 / 11026. The linker can be a "cleavable linker" facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

[0644] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linker agents, including but not limited to, commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A) 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).

[0645] E. Methods and Compositions for Diagnosis and Detection

[0646] In certain embodiments, any of the anti-latent TGF-β1 antibodies provided herein can be used to detect the presence of TGF-β1, e.g., the presence of latent TGF-β1 in a biological sample. The term "detecting" 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, ascitic fluid, breast milk, colostrum, breast secretions, lymph, urine, sweat, tears, gastric fluid, synovial fluid, peritoneal fluid, ocular lens fluid, and mucus.

[0647] In one embodiment, an anti-latent TGF-β1 antibody for use in a diagnostic or detection method is provided. In a further aspect, a method of detecting the presence of TGF-β1, e.g., latent TGF-β1, in a biological sample is provided. For example, a method of detecting the presence of latent TGF-β1 comprises:

[0648] (a) contacting a biological sample with an anti-latent TGF-β1 antibody of the application described herein under conditions that allow the anti-latent TGF-β1 antibody to bind to latent TGF-β1; and

[0649] (b) detecting whether a complex is formed between the anti-latent TGF-β1 antibody and latent TGF-β1.

[0650] Such methods can be in vitro or in vivo methods. In one embodiment, the anti-latent TGF-β1 antibody is used to select a subject suitable for treatment with the anti-latent TGF-β1 antibody, e.g., in the case of TGF-β1, e.g., latent TGF-β1 is a biomarker for selecting a patient. That is, the anti-latent TGF-β1 antibody can be used as a diagnostic agent targeting TGF-β1.

[0651] More specifically, the anti-latent TGF-β1 antibody can be used for the diagnosis of fibrosis, preferably myocardial fibrosis, kidney fibrosis, eye fibrosis, bone marrow fibrosis, liver fibrosis, and lung / pulmonary fibrosis. The anti-latent TGF-β1 antibody of the application can also be used for the diagnosis of cancer.

[0652] In some embodiments, the present application provides a method of 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, comprising contacting a biological sample containing latent TGF-β1 with an anti-latent TGF-β1 antibody of the application under conditions that allow the antibody to bind to latent TGF-β1.

[0653] In certain embodiments, labeled anti-latent TGF-β1 antibodies are provided, e.g., for detection / diagnostic purposes. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are detected indirectly (such as enzymes or ligands, e.g., by enzymatic reaction or molecular interaction). Exemplary labels include, but are not limited to, radioactive isotopes 32 P、 14 C、 125 I、 3 H and 131 I; fluorophores such as rare earth chelates or fluoresceins and their derivatives, rhodamines and their derivatives, dansyl, umbelliferone; luciferases, e.g., firefly luciferase and bacterial luciferase (US 4,737,456); luciferin; 2,3-dihydrophthalazinediones; horseradish peroxidase (HRP); alkaline phosphatase; beta-galactosidase; glucoamylase; lysozyme; saccharide oxidizing enzymes, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidizing enzymes such as uricase and xanthine oxidase; coupling to an enzyme that employs hydrogen peroxide to oxidize a dye precursor, such as HRP, lactoperoxidase, or microperoxidase; biotin / avidin; a spin label; a phage label; stable free radicals; and the like.

[0654] F. Pharmaceutical Formulations

[0655] Pharmaceutical formulations of anti-latent TGF-β1 antibodies as described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers are nontoxic to recipients at the dosages and concentrations employed; and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polyols. Peptides;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 dextrins;Chelating agents such as EDTA;Sugars such as sucrose, mannitol, trehalose or sorbitol;Salt-forming counterions such as sodium;Metal complexes (e.g., zinc-protein complexes);And / or nonionic surfactants such as polyethylene glycol (PEG).Exemplary pharmaceutical carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0656] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0657] In one aspect, the present invention provides a pharmaceutical formulation comprising an anti-latent TGF-β1 antibody for use in treating fibrosis, preferably myocardial fibrosis, renal fibrosis, ocular fibrosis, myelofibrosis, liver fibrosis, and lung / pulmonary fibrosis. The present invention also provides a pharmaceutical formulation comprising an anti-latent TGF-β1 antibody for use in treating cancer.

[0658] The formulations herein can also contain more than one active ingredient necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it can be desirable to further provide an immune checkpoint inhibitor.

[0659] The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively; in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules); or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0660] Sustained-release preparations can 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, for example, films or microcapsules.

[0661] The formulations to be used for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through a sterile filtration membrane.

[0662] G. Therapeutic Methods and Compositions

[0663] Any of the anti-latent TGF-β1 antibodies provided herein can be used in a method of treatment. In one aspect, an anti-latent TGF-β1 antibody for use as a medicament is provided. In a further aspect, an anti-latent TGF-β1 antibody for use in the treatment of cancer or fibrosis, such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis, among others, is provided. In a further aspect, an anti-latent TGF-β1 antibody for use in the treatment of fibrosis is provided. In a further aspect, an anti-latent TGF-β1 antibody for use in the treatment of kidney fibrosis is provided. In a further aspect, an anti-latent TGF-β1 antibody for use in the treatment of liver fibrosis is provided. In a further aspect, an anti-latent TGF-β1 antibody for use in the treatment of lung / pulmonary fibrosis is provided. In certain embodiments, an anti-latent TGF-β1 antibody for use in a method of treatment is provided. In certain embodiments, the present application provides an anti-latent TGF-β1 antibody for use in a method of treating an individual having cancer or fibrosis, such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis, among others, the method comprising administering to the individual an effective amount of the anti-latent TGF-β1 antibody. In certain embodiments, the present application provides an anti-latent TGF-β1 antibody for use in a method of treating an individual having fibrosis, the method comprising administering to the individual an effective amount of the anti-latent TGF-β1 antibody. In certain embodiments, the present application provides an anti-latent TGF-β1 antibody for use in a method of treating an individual having kidney fibrosis, the method comprising administering to the individual an effective amount of the anti-latent TGF-β1 antibody. In certain embodiments, the present application provides an anti-latent TGF-β1 antibody for use in a method of treating an individual having liver fibrosis, the method comprising administering to the individual an effective amount of the anti-latent TGF-β1 antibody. In certain embodiments, the present application provides an anti-latent TGF-β1 antibody for use in a method of treating an individual having lung / pulmonary fibrosis, the method comprising administering to the individual an effective amount of the anti-latent 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., a therapeutic agent as described below. In further embodiments, the present application provides an anti-latent TGF-β1 antibody for use in inhibiting activation of latent TGF-β1 by proteases. In certain embodiments, the present application provides an anti-latent TGF-β1 antibody for use in a method of inhibiting activation of latent TGF-β1 by proteases in an individual, the method comprising administering to the individual an effective amount of the anti-latent TGF-β1 antibody to inhibit activation of latent TGF-β1 by proteases. The "individual" according to any of the above embodiments is preferably a human.

[0664] In further aspects, the present application provides use of an anti-latent TGF-β1 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer or fibrosis (such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis), etc. In one embodiment, the medicament is for the treatment of fibrosis. In one embodiment, the medicament is for the treatment of kidney fibrosis. In one embodiment, the medicament is for the treatment of liver fibrosis. In one embodiment, the medicament is for the treatment of lung / pulmonary fibrosis. In further embodiments, the medicament is for use in a method of treating cancer or fibrosis (such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis), etc., the method comprising administering to an individual having cancer or fibrosis (such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis), etc., an effective amount of the medicament. In further embodiments, the medicament is for use in a method of treating fibrosis, the method comprising administering to an individual having fibrosis an effective amount of the medicament. In further embodiments, the medicament is for use in a method of treating kidney fibrosis, the method comprising administering to an individual having kidney fibrosis an effective amount of the medicament. In further embodiments, the medicament is for use in a method of treating liver fibrosis, the method comprising administering to an individual having liver fibrosis an effective amount of the medicament. In further embodiments, the medicament is for use in a method of treating lung / pulmonary fibrosis, the method comprising administering to an individual having lung / pulmonary fibrosis an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., a therapeutic agent as described below. In further embodiments, the medicament is for inhibiting protease-mediated activation of latent TGF-β1. In further embodiments, the medicament is for use in a method of inhibiting protease-mediated activation of latent TGF-β1 in an individual, the method comprising administering to the individual an effective amount of the medicament to inhibit protease-mediated activation of latent TGF-β1. The "individual" according to any of the above embodiments can be a human.

[0665] In a further aspect, the present application provides a method for treating cancer or fibrosis, such as renal fibrosis, liver fibrosis, and lung / pulmonary fibrosis, etc. In a further aspect, the present application provides a method for treating fibrosis. In a further aspect, the present application provides a method for treating renal fibrosis. In a further aspect, the present application provides a method for treating liver fibrosis. In a further aspect, the present application provides a method for treating lung / pulmonary fibrosis. In one embodiment, the method comprises administering to an individual having such cancer or fibrosis, such as renal fibrosis, liver fibrosis, and lung / pulmonary fibrosis, etc., an effective amount of an anti-latent TGF-β1 antibody. In one embodiment, the method comprises administering to an individual having such fibrosis an effective amount of an anti-latent TGF-β1 antibody. In one embodiment, the method comprises administering to an individual having such renal fibrosis an effective amount of an anti-latent TGF-β1 antibody. In one embodiment, the method comprises administering to an individual having such liver fibrosis an effective amount of an anti-latent TGF-β1 antibody. In one embodiment, the method comprises administering to an individual having such lung / pulmonary fibrosis an effective amount of an anti-latent 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 as described below. In some embodiments, the administration of the antibody and the agent is concomitant. The "individual" according to any of the above embodiments can be a human.

[0666] In a further aspect, the present application 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.

[0667] In a further aspect, the present application provides a pharmaceutical formulation comprising any of the anti-latent TGF-β1 antibodies provided herein, e.g., for use in any of the above treatment methods. 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, a 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.

[0668] The antibodies of the present application (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and if desired for local treatment, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be performed by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short term or long term. Various administration schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0669] The antibodies of the present application will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context 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 delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibodies need not, but may, be presented separately in their pure form, but preferably, are presented as a pharmaceutical formulation.

[0670] For preventing or treating disease, the appropriate dosage of an antibody of the present application (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the molecule is for preventive or therapeutic use, the patient's health and previous medical history, and the judgment of the prescribing physician. The antibody is suitably administered to the patient once or in a series of treatments as a series of doses.

[0671] Formulations (e.g., pharmaceutical formulations) containing an antibody of the present disclosure can be administered by parenteral routes of administration, e.g., injection (such as subcutaneous injection, intravenous injection, intramuscular injection, etc.), transdermal, transmucosal, nasal, and pulmonary routes. In some embodiments, the formulation is administered by subcutaneous injection. In some embodiments, the formulation is for subcutaneous administration, e.g., subcutaneous injection.

[0672] In some embodiments, the formulation of the present disclosure has a viscosity of less than 70 mPa-s (millipascal-seconds), less than 60 mPa-s, less than 50 mPa-s, less than 40 mPa-s, less than 30 mPa-s, less than 20 mPa-s, less than 15 mPa-s. In some embodiments, the viscosity is 3 mPa-s to 8 mPa-s. In some embodiments, the viscosity is 3 mPa-s to 7 mPa-s. In some embodiments, the viscosity is 3 mPa-s to 6 mPa-s. In some embodiments, the viscosity is 3 mPa-s to 5 mPa-s. In some embodiments, the viscosity is 3 mPa-s to 4 mPa-s. In some embodiments, the viscosity is 3 mPa-s to 3.5 mPa-s. The viscosity of the formulation can be measured by a rotational viscometer method using a cone-plate type viscometer, for example, according to Japanese Pharmacopoeia, General Tests, 2.53 Viscosity Determination.

[0673] In some embodiments, the concentration of the antibody in the formulation is 50 to 350 mg / ml. In some embodiments, the concentration of the antibody in the formulation is 100 to 300 mg / ml. In some embodiments, the concentration of the antibody in the formulation is 100 to 250 mg / ml. In some embodiments, the concentration of the antibody in the formulation is 100 to 200 mg / ml.

[0674] It should be understood that any of the articles described herein can include an immunoconjugate of the present application in place of or in addition to an anti-latent TGF-β1 antibody.

[0675] IV. Articles of Manufacture, Kits

[0676] A. Articles of Manufacture

[0677] In another aspect of the application, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of any of the conditions described above (e.g., fibrosis, such as renal fibrosis, liver fibrosis, and lung / pulmonary fibrosis, or cancer) is provided. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition, which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition (e.g., fibrosis, such as renal fibrosis, liver fibrosis, and lung / pulmonary fibrosis, or cancer) and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody or immunoconjugate of the application. The label or package insert indicates that the composition is used for treating the condition of choice (e.g., fibrosis, such as renal fibrosis, liver fibrosis, and lung / pulmonary fibrosis, or cancer). Moreover, the article of manufacture can comprise (a) a first container wherein there is a composition contained within said first container, wherein the composition comprises an antibody / immunoconjugate of the application; and (b) a second container wherein there is a composition contained within said second container, wherein the composition comprises an additional therapeutic agent or other treatment agent. The article of manufacture in this embodiment of the application can further comprise a package insert indicating that the compositions can be used to treat the particular condition (e.g., fibrosis, such as renal fibrosis, liver fibrosis, and lung / pulmonary fibrosis, or cancer). Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0678] It should be understood that any of the above articles of manufacture can include an immunoconjugate of the application instead of or in addition to an anti-latent TGF-β1 antibody.

[0679] B. Kits

[0680] The present disclosure provides kits for use in methods of treating, preventing, and / or diagnosing a disorder described herein, particularly treating an individual having fibrosis (such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis) or cancer, containing an anti-latent TGF-β1 antibody, an immunoconjugate comprising an anti-latent TGF-β1 antibody, an isolated nucleic acid encoding an anti-latent TGF-β1 antibody, or a vector comprising a nucleic acid of the disclosure, or produced by a method of the disclosure. The kit can additionally contain any therapeutic agent, such as an immune checkpoint inhibitor, including an anti-PD-L1 antibody. The kit can be packaged with additional pharmaceutical carriers or diluents disclosed herein, or instructions describing how to use the kit, etc. As with the articles described herein, the kit can contain: a material for the treatment of fibrosis (such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis) or cancer; a container and a label on the container or a package insert associated with the container; a composition, by itself or in combination with another composition, effective for the treatment of fibrosis (such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis) or cancer; a sterile access port, and the like. The kit can further include a label or package insert stating that the composition can be used for the treatment of fibrosis (such as kidney fibrosis, liver fibrosis, and lung / pulmonary fibrosis) or cancer. Alternatively or additionally, the kit can further include a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0681] <Anti-latent TGF-β1 antibody pH-dependent binding>

[0682] In the present context, "acidic pH" means a pH which can be selected from, for example, pH 4.0 to pH 6.5. In one embodiment, acidic pH means, but is not limited to, pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, or pH 6.5. In a particular embodiment, the term acidic pH means pH 5.8.

[0683] In the present context, "neutral pH" refers to a pH selected from, for example, 6.7 to pH 10.0. In one embodiment, neutral pH refers to, but is not limited to, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.5, or pH 10.0. In a specific embodiment, the term neutral pH refers to pH 7.4.

[0684] In an alternative aspect, the anti-LTBP3 antibody of the present disclosure is an anti-LTBP3 antibody that binds to latent TGF-β1 in a pH-dependent manner. In the context of the present disclosure, an anti-LTBP3 antibody that binds to latent TGF-β1 in a pH-dependent manner refers to an antibody that has a reduced binding affinity for latent TGF-β1 at an acidic pH compared to its binding affinity for latent TGF-β1 at a neutral pH. For example, a pH-dependent anti-LTBP3 antibody includes an antibody that has a higher affinity for latent TGF-β1 at a neutral pH than at an acidic pH. In one embodiment, the anti-LTBP3 antibody of the present disclosure has an affinity for latent TGF-β1 at a neutral pH that is at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10,000-fold or more than the affinity at an acidic pH.

[0685] In one embodiment, an anti-latency TGF-β1 antibody that binds to latent TGF-β1 in a pH-dependent manner refers to an antibody whose binding affinity to latent TGF-β1 is reduced at pH 5.8 compared to the binding affinity to latent TGF-β1 at pH 7.4. For example, a pH-dependent anti-latency TGF-β1 antibody includes an antibody that has a higher affinity to latent TGF-β1 at pH 7.4 than at pH 5.8. In one embodiment, the anti-latency TGF-β1 antibody of the present disclosure has an affinity to latent TGF-β1 at pH 7.4 that is at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10,000-fold or more than the affinity at pH 5.8.

[0686] The binding affinity can be measured using a surface plasmon resonance method such as BIACORE (registered trademark), but is not particularly limited. When measured using BIACORE (registered trademark), in some embodiments, the following buffer and temperature conditions are preferably used: 20 mM ACES, 150 mM NaCl, 0.05% Tween-20, and 0.005% NaN3. The association rate constant (kon) and the dissociation rate constant (koff) can be calculated by simultaneously fitting the association and dissociation sensorgrams based on a simple one-to-one Langmuir binding model using BIACORE (registered trademark) T200 evaluation software (GE Healthcare). The equilibrium dissociation constant (KD) is calculated as the ratio of koff / kon. In order to screen antibodies whose binding affinity varies depending on pH, there is no particular limitation, and ELISA, KinExA TM ) and the like, as well as surface plasmon resonance methods such as BIACORE (registered trademark) can be used. The pH-dependent latent TGF-β1 binding ability refers to the property of binding to latent TGF-β1 in a pH-dependent manner. At the same time, whether an antibody can bind to latent TGF-β1 multiple times can be assessed by, for example, the method described in WO2009 / 125825.

[0687] In one embodiment, it is preferable that the anti-latent TGF-β1 antibody of the present disclosure has a small dissociation constant (KD) for latent TGF-β1 at neutral pH. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at neutral pH is, for example, 0.3 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at neutral pH is, for example, 0.1 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at neutral pH is, for example, 0.03 nM or less, but is not limited thereto.

[0688] In one embodiment, it is preferable that the anti-latent TGF-β1 antibody of the present disclosure has a small dissociation constant (KD) for latent TGF-β1 at neutral pH. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at neutral pH is, for example, 0.3 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at neutral pH is, for example, 0.1 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at neutral pH is, for example, 0.03 nM or less, but is not limited thereto.

[0689] In one embodiment, it is preferable that the anti-latent TGF-β1 antibody of the present disclosure has a large dissociation constant (KD) for latent TGF-β1 at acidic pH. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at acidic pH is, for example, 3 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at acidic pH is, for example, 10 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at acidic pH is, for example, 30 nM or more, but is not limited thereto.

[0690] In one embodiment, it is preferable that the anti-latent TGF-β1 antibody of the present disclosure has a large dissociation constant (KD) for latent TGF-β1 at pH 5.8. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at pH 5.8 is, for example, 3 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at pH 5.8 is, for example, 10 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of the present disclosure for latent TGF-β1 at pH 5.8 is, for example, 30 nM or more, but is not limited thereto.

[0691] In some embodiments, the dissociation constant (KD) of human latent TGF-β1 at pH 5.8 is 2.0 x 10 -7 M or more, 3.0 x 10 -7 M or more, 4.0 x 10 -7 M or more, 5.0 x 10 -7 M or more.

[0692] In some embodiments, the dissociation constant (KD) of human latent TGF-β1 at pH 5.8 is 2.0 x 10 -7 M to 3.0 x 10 -7 M, 2.0 x 10 -7 M to 4.0 x 10 -7 M or 2.0 x 10 -7 M to 5.0 x 10 -7 M.

[0693] In one embodiment, it is preferable that the anti-latent TGF-β1 antibody of the present disclosure has a greater binding affinity to latent TGF-β1 at neutral pH than at acidic pH.

[0694] In one embodiment, the dissociation constant ratio between acidic pH and neutral pH of the anti-latent TGF-β1 antibody of the present disclosure [KD(acidic pH) / KD(neutral pH)] is, for example, 30 or more, but is not limited thereto. In one embodiment, the dissociation constant ratio between acidic pH and neutral pH of the anti-latent TGF-β1 antibody of the present disclosure [KD(acidic pH) / KD(neutral pH)] is, for example, 3 or more, 5 or more, 10 or more, 50 or more, 90 or more, 100 or more, 150 or more, 200 or more, for example, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, but is not limited thereto.

[0695] In one embodiment, the anti-latent TGF-β1 antibody of the present disclosure has a dissociation constant ratio between pH 5.8 and pH 7.4 [KD(pH 5.8) / KD(pH 7.4)] of 30 or more, but not limited thereto. In one embodiment, the anti-body of the present disclosure has a dissociation constant ratio between pH 5.8 and pH 7.4 [KD(pH 5.8) / KD(pH 7.4)] of, for example, 3 or more, 5 or more, 10 or more, 50 or more, 90 or more, 100 or more, 150 or more, 200 or more, for example, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, but not limited thereto.

[0696] In some embodiments, the antibody of the present disclosure has a dissociation constant ratio between pH 5.8 and pH 7.4 [KD(pH 5.8) / KD(pH 7.4)] for human latent TGF-β-1 of, for example, 100 or more, 120 or more, 130 or more, or 160 or more.

[0697] In some embodiments, the antibody of the present disclosure has a dissociation constant ratio between pH 5.8 and pH 7.4 [KD(pH 5.8) / KD(pH 7.4)] for human latent TGF-β-1 of, for example, 100 to 170, 120 to 170, 130 to 170, or 160 to 170.

[0698] In one embodiment, it is preferable that the anti-latent TGF-β1 antibody of the present disclosure has a large dissociation rate constant (koff) at an acidic pH. In one embodiment, the antibody of the present disclosure has a dissociation rate constant at an acidic pH of, for example, 0.003 (1 / s) or more, but not limited thereto. In one embodiment, the antibody of the present disclosure has a dissociation rate constant at an acidic pH of, for example, 0.005 (1 / s) or more, but not limited thereto. In one embodiment, the antibody of the present disclosure has a dissociation rate constant at an acidic pH of, for example, 0.01 (1 / s) or more, but not limited thereto.

[0699] In one embodiment, it is preferable that the anti-latent TGF-β1 antibody of the present disclosure has a large dissociation rate constant (koff) at pH 5.8. In one embodiment, the dissociation rate constant of the antibody of the present disclosure at pH 5.8 is, for example, 0.003 (1 / s) or more, but is not limited thereto. In one embodiment, the dissociation rate constant of the antibody of the present disclosure at pH 5.8 is, for example, 0.005 (1 / s) or more, but is not limited thereto. In one embodiment, the dissociation rate constant of the antibody of the present disclosure at pH 5.8 is, for example, 0.01 (1 / s) or more, but is not limited thereto.

[0700] Examples

[0701] The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments can be practiced in light of the general description provided above.

[0702] Example 1: Expression and Purification of Latent TGF-β1

[0703] The sequences used for expression and purification were: FLAG®-tagged human latent TGF-β1 (SEQ ID NO: 64) and FLAG®-tagged mouse latent TGF-β1 (SEQ ID NO: 65), and FLAG®-tagged cynomolgus monkey latent TGF-β1 (SEQ ID NO: 66). Each of these FLAG®-tagged latent TGF-β1s has, from its N-terminus to C-terminus, a signal sequence derived from rat serum albumin (SEQ ID NO: 67), a FLAG®-tag, and the sequence of latent TGF-β1. The Cys residue at the thirtieth (30) position in each of these FLAG®-tagged latent TGF-β1s is substituted with Ser, which corresponds to the “C33S mutation” (see, e.g., Yoshinaga K, et al. Perturbation of transforming growth factor (TGF)-beta 1 association with latent TGF-beta binding protein yields inflammation and tumors. Proc Natl Acad Sci USA. 2008; 105(48): 18758-18763).

[0704] FLAG (registered trademark)-tagged human latent TGF-β1 (hereinafter referred to as "human latent TGF-β1 (SLC)" or "human latent TGF-β1"), FLAG (registered trademark)-tagged mouse latent TGF-β1 (hereinafter referred to as "mouse latent TGF-β1 (SLC)" or "mouse latent TGF-β1"), or FLAG (registered trademark)-tagged cynomolgus monkey latent TGF-β1 (hereinafter referred to as "monkey latent TGF-β1 (SLC)" or "monkey latent TGF-β1") were transiently co-expressed with a plasmid expressing human furin using FreeStyle 293 (trademark)-F or Expi293F (trademark) cell lines (Thermo Fisher Scientific). The conditioned medium expressing human, mouse, or monkey latent TGF-β1 (SLC) was applied to a column packed with anti-FLAG (registered trademark) M2 affinity resin (Sigma), and the latent TGF-β1 (SLC) was eluted with FLAG (registered trademark) peptide (Sigma). The fraction containing human, mouse, or monkey latent TGF-β1 (SLC) was collected and then subjected to a SUPERDEX (registered trademark) 200 gel filtration column (GE healthcare) equilibrated with 1x PBS. The fraction containing human, mouse, or monkey latent TGF-β1 (SLC) was then combined and stored at -80°C.

[0705] Example 2. Humanization and Optimization of Anti-TGF-β1 Antibodies

[0706] (2-1) Humanization

[0707] The parental anti-TGF-β antibody TBA0947 (chimeric antibody) was humanized as follows. First, the variable regions of the heavy and light chains of the humanized antibody were designed using the variable regions of TBA0947 and human germline frameworks. Then, the polynucleotides of each of the designed heavy chain variable region and light chain variable region were cloned into expression vectors containing the heavy chain constant region SG181 sequence (SEQ ID NO: 22) and the light chain constant region SK1 sequence (SEQ ID NO: 25), respectively. The humanized antibodies were transiently expressed in FreeStyle 293-F cells (Thermo Fisher Scientific), and BIACORE (registered trademark) analysis was performed. The humanized antibodies that showed at least similar BIACORE (registered trademark) binding activity to the parental antibody were selected.

[0708] (2-2) Optimization

[0709] An anti-human latent TGF-β1 antibody hT0947AE07-SG191 was developed from TBA0947 by improving the antigen binding affinity / activity. Although hT0947AE07-SG191 has preferable antigen binding affinity / activity, it showed rapid clearance in cynomolgus monkey PK study (Fig. 14-1 to 14-6). For the PK study in cynomolgus monkeys, a human IgG1-based heavy chain constant region designated as SG191 (SEQ ID NO 23) was used because it showed enhanced human FcRn binding to improve the PK profile.

[0710] To further improve its pharmacokinetic profile, a pH-dependent antibody T0947H0053H0156-SG181 / T0947L001L0049-SK1 was developed from hT0947AE07-SG181 by subjecting its heavy chain CDRs and light chain CDRs to histidine scanning. The pH-dependent binding was evaluated by BIACORE (registered trademark) analysis. In the BIACORE (registered trademark) analysis, a human IgG1-based heavy chain constant region designated as SG181 (SEQ ID NO 22) was used for stable capture onto a BIACORE (registered trademark) sensor chip. Although the pH-dependent antibody showed dissociation of human latent TGF-β1 at pH 5.8 in the BIACORE (registered trademark) analysis, the binding affinity to human latent TGF-β1 was not strong enough at pH 7.4 with a KD of 1.34E-08 M (Fig. 2). Therefore, antibody variants were designed to improve the affinity to human TGF-β1 while maintaining the pH-dependent profile.

[0711] First, T0947H0053H0156-SG181 / T0947L001L0049-SK1 was subjected to comprehensive mutagenesis of all residues in its heavy chain CDRs. Each amino acid was substituted with 18 other naturally occurring amino acids, excluding the original amino acid and cysteine. A total of 648 antibody variants were designed by comprehensive mutagenesis. The designed antibody variants were transiently expressed in Expi293 (trademark) cells (Thermo Fisher Scientific) and purified from the culture supernatant. All the antibodies produced were evaluated in BIACORE (registered trademark) analysis against human and mouse latent TGF-β1, in vitro neutralization assays against plasmin (PLN) human and mouse latent TGF-β1, and in antibody expression titers.

[0712] Successive rounds of design of combinations of mutations in CDRs were performed to identify preferred amino acid sequences in CDRs by utilizing data for each variant evaluated by BIACORE (registered trademark) in vitro experiments and in the neutralization assay of latent TGF-β1 as described above. All designed antibodies were produced by transient expression in Expi293 cells and evaluated in BIACORE (registered trademark) analysis against human and mouse latent TGF-β1 and in neutralization assays against PLN of human and mouse latent TGF-β1 and antibody expression titer.

[0713] In the first round, 40 antibody variants were designed and produced. For each produced antibody, pH-dependent binding to human and mouse latent TGF-β1 was assessed by BIACORE (registered trademark). Based on BIACORE (registered trademark) results and neutralization activity, antibodies with combined mutations were designed for the second round.

[0714] From the 2nd to the 6th round, 80 antibody variants were designed and tested per round. Although a total of 480 antibody variants were assessed, the KD against human latent TGF-β1 at pH 7.4 could not be sufficiently improved while maintaining the pH-dependent binding characteristics. Therefore, to explore more antibody mutations, T0947H0053H1184-SG181 / T0947L001L0083-SK1 were selected from all antibody variants evaluated by the 1st to 6th round of antibody design and another comprehensive mutagenesis was performed on the heavy chain CDRs thereof. Then, 450 antibody variants with single mutations from T0947H0053H1184-SG181 / T0947L001L0083-SK1 were evaluated by BIACORE (registered trademark) and in vitro TGF-β1 neutralization assays. Potential antibody mutations were used for combination in further rounds of antibody design.

[0715] In the 7th and 8th rounds, 80 antibodies were designed and tested for BIACORE (registered trademark) and neutralization activity per round. In the 9th and 10th rounds, 120 antibodies were designed and tested for BIACORE (registered trademark) and neutralization activity per round. In the 11th to 13th rounds, 80 antibodies were designed and tested for BIACORE and neutralization activity per round.

[0716] After the 13th round, based on BIACORE (registered trademark) binding properties and neutralizing activity, hT0947AE20-SG181, hT0947AE22-SG181, hT0947AE23-SG181, hT0947AE24-SG181, hT0947AE25-SG181, and hT0947AE26-SG181 were selected from all antibody variants designed and tested through the 1st to 13th rounds and the comprehensive mutagenesis. Through the antibody optimization process, the KD of these antibodies was significantly improved relative to the parent pH-dependent anti-human latent TGF-β1 antibody T0947H0053H0156-SG181 / T0947L001L0049-SK1, and these antibodies showed strong neutralizing activity against human latent TGF-β1 to PLN (Fig. 1). These results indicated that antibodies with potential in vivo efficacy could be generated through antibody optimization. However, the KD of these antibodies to human latent TGF-β1 at pH 7.4 was 2.5 to 5 times that of the non-pH-dependent anti-human latent TGF-β1 antibody hT0947AE07-SG181 (Figs. 2 and 3), indicating that the affinity of these antibodies could still be improved. Therefore, several more rounds of antibody engineering were applied.

[0717] To design antibodies with better antigen binding affinity / activity to human latent TGF-β1, the method for evaluating BIACORE (registered trademark) results used in the next round of antibody design was modified. For the antibodies designed in the 1st to 13th rounds, BIACORE (registered trademark) results were evaluated based on KD at neutral pH and pH dependence to latent human TGF-β1. However, for the antibodies designed in the 14th to 17th rounds, BIACORE (registered trademark) results were evaluated based on KD to human latent TGF-β1 at neutral pH.

[0718] After the 13th round, hT0947AE22-SG181, which showed rapid dissociation from human latent TGF-β1 at pH 5.8, was selected for another round of comprehensive mutagenesis of its heavy and light chain CDRs. 1134 variants were generated for in vitro characterization. Through the use of the in vitro characterization results of antibodies generated by comprehensive mutagenesis, additional rounds of antibody design and characterization were performed until the 17th round. In the 14th to 17th rounds, 150, 96, 160, and 180 antibody variants were designed and generated, respectively. In these 14th to 17th rounds, antibody optimization was performed to improve the affinity of the KD values evaluated in BIACORE (registered trademark) rather than the pH-dependent binding characteristics. In addition, AC-SINS and PEG-SPR were performed on all antibody variants designed in the 14th to 17th rounds to find antibody variants with preferred physicochemical properties, as the antibodies of the previous rounds had increased hydrophobicity. Antibodies were designed to maintain AC-SINS and PEG-SPR data at a level similar to that of clinical antibodies that have been approved by the FDA. As shown in FIG. 3, from the 14th to 17th rounds, the KD for human latent TGF-β1 can be improved while maintaining the pH-dependent binding characteristics. From all the antibodies generated through the successive rounds of these antibody design and in vitro characterization processes, the pH-dependent anti-latent TGF-β1 antibodies hT0947AE20-SG181, hT0947AE22-SG181, hT0947AE23-SG181, hT0947AE24-SG181, hT0947AE25-SG181, hT0947AE26-SG181, hT0947AE27-SG181, hT0947AE34-SG181, and hT0947AE37-SG181 were selected from all the antibody variants evaluated through all the rounds of antibody design.

[0719] (2-3) Optimization of the amino acid sequence of the antibody

[0720] The amino acid sequences of the anti-latent TGF-β1 antibodies are listed in Table 3.

[0721] [Table 3]

[0722]

[0723]

[0724] The heavy chain HVR (CDR) sequences and framework (FR) sequences of each antibody are shown in Table 4-1.

[0725] [Table 4-1]

[0726]

[0727] The light chain HVR (CDR) sequences and framework (FR) sequences for each antibody are shown in Table 4-2.

[0728] [Table 4-2]

[0729]

[0730] In vitro characterization of antibody screening during antibody optimization

[0731] BIACORE (Registered Trademark) screening assay for binding affinity and pH-dependent interaction of anti-latent TGF-β1 antibodies to recombinant human latent TGF-β1

[0732] Binding affinity of anti-latent TGF-β1 antibodies to recombinant human latent TGF-β1 was measured using a BIACORE (Registered Trademark) T200 instrument (GE Healthcare). For BIACORE (Registered Trademark) screening, antibodies with human IgG1 constant regions (SG181, SEQ ID NO: 22 and SK1, SEQ ID NO: 25) were prepared. Mouse anti-human Fc (GE Healthcare) was immobilized onto all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies were captured onto the anti-human Fc sensor surface to a capture level of approximately 20 RU, and then recombinant human latent TGF-β1 was injected over the flow cells. All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. The sensor surface was regenerated with 3M MgCl2 for each cycle. Binding affinity was determined by processing the data and fitting it to a 1:1 binding model using BIACORE (Registered Trademark) T200 Evaluation Software version 3.0 (GE Healthcare).

[0733] Screening of anti-latent TGF-β1 antibodies for pH-dependent interaction to recombinant human latent TGF-β1 was determined by the modified BIACORE (Registered Trademark) assay described above. Briefly, an additional dissociation phase at pH 5.8 was integrated into the BIACORE (Registered Trademark) assay immediately after the dissociation phase at pH 7.4. Dissociation rates at pH 5.8 buffer were determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0734] Affinity capture self-interaction nanoparticle spectroscopy (AC-SINS)

[0735] For the AC-SINS assay, antibodies were prepared with human IgGl constant region (SG181, SEQ ID NO: 22 and SK1, SEQ ID NO: 25). In the AC-SINS assay, 0.025 mg / mL concentration of anti-latent TGF-β1 antibody was added to pre-coated gold nanoparticles (Ted Pella Inc. #15705) with 80% polyclonal goat anti-human IgG Fc (Jackson ImmunoResearch #109-005-098) as capture and 20% goat non-specific antibody (Jackson ImmunoResearch #005-000-003). The antibody was incubated with the pre-coated gold nanoparticles for 2 hours at room temperature and wavelength shift was measured using a SpectraMax (registered trademark) M2 (Molecular Devices).

[0736] Polyethylene glycol (PEG) induced antibody precipitation assay

[0737] For the PEG SPR assay, antibodies were prepared with human IgGl constant region (SG181, SEQ ID NO 22: and SK1, SEQ ID NO: 25). In the PEG-SPR assay, an equal volume of 0.05 mg / mL concentration of anti-latent TGF-β1 antibody was added to a 25% PEG solution. The mixture was mixed on a shaker at 700 rpm for 1 minute and then incubated at room temperature for 2 hours. The samples were transferred to a MultiScreen HTS GV filter plate and centrifuged at 2500 rpm for 5 minutes. The flow-through containing the soluble sample was analyzed with a BIACORE (registered trademark) 8K+ instrument (GE Healthcare). Protein A / G was immobilized onto all flow cells of a CM5 sensor chip using an amine coupling kit (GE Healthcare). The assay temperature was set to 25 °C and the running buffer consisted of ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. The samples were injected at 10 uL / min onto the protein A / G sensor surface for 30 seconds. The sensor surface was regenerated with 10 mM glycine-HCl pH 1.5 for each cycle. The binding response of each sample was analyzed using BIACORE (registered trademark) 8K Evaluation Software 1.1.1.7442 (GE Healthcare).

[0738] Solubility of each sample was analyzed by normalizing the binding response in PEG to PBS.

[0739] Latent TGF-β1 neutralization assay against plasmin (PLN)

[0740] For latent TGF-β1 neutralization assay, antibodies with human IgGl constant region (SG181, SEQ ID NO: 22 and SK1, SEQ ID NO: 25) were prepared. Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example 1 were each incubated with human plasmin (Calbiochem) in the presence or absence of anti-latent TGF-β1 antibody at 37°C for 1 hour. The antibody was pre-incubated with mouse or human latent TGF-β1 (SLC) at room temperature for 30 minutes, and then incubated with plasmin. Anti-KLH antibody IC17-hIgGl (heavy chain SEQ ID NO: 68, light chain SEQ ID NO: 69) 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 (registered trademark) ELISA kit, R&D systems) according to the manufacturer's procedure.

[0741] Example 3. Immunogenicity Assessment of Anti-Latent TGF-β1 Antibodies

[0742] CD4 T cells that secrete IL-2 before showing active proliferation, as described in WO2018 / 124005 (Kubo C. et al.), were used to evaluate the immunogenic potential of the antibodies. Specifically, CD8 T cells were prepared from human peripheral blood mononuclear cells (PBMC) and cultured with the antibodies for 67 hours. + The proportion of T cells was evaluated as an index of the immunogenic potential of the antibodies. Specifically, CD8 T cells were prepared from human peripheral blood mononuclear cells (PBMC) and cultured with the antibodies for 67 hours. - CD25low PBMC, and the cells were cultured in the presence of the antibodies for 67 hours.

[0743] Figure 4 shows the results of determining the proportion of IL-2-secreting cells in the cultured cell population. The frequency of positive donors of hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, and hT0947AE37-SG191 was similar or slightly higher than that of the negative control. On the other hand, the frequency of positive donors of hT0947AE22-SG191, hT0947AE23-SG191, hT0947AE24-SG191, hT0947AE25-SG191, and hT0947AE26-SG191 was higher than that of the negative control. These results indicate that hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, and hT0947AE37-SG191 are considered to have low immunogenic potential.

[0744] Example 4. BIACORE® Binding Affinity Assessment of Anti-Latent TGF-β1 Antibodies to Recombinant Human, Cynomolgus Monkey, and Mouse Latent TGF-β1 Example 5. Characterization of Anti-Latent TGF-β1 Antibodies

[0745] For BIACORE (registered trademark) affinity evaluation, antibodies with human IgGl constant region (SG191, SEQ ID NO: 23 and SK1, SEQ ID NO: 25) were prepared. The binding affinity of anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0497AE20-SG191, hT0497AE27-SG191, hT0497AE34-SG191 and hT0497AE37-SG191) to human, cynomolgus monkey and mouse latent TGF-β1 was measured using a BIACORE (registered trademark) 8K instrument (GE Healthcare). Mouse anti-human Fc (GE Healthcare) was immobilized onto all flow cells of a CM5 sensor chip using an amine coupling kit (GE Healthcare). Antibodies were captured onto the anti-human Fc sensor surface to a capture level of about 20 RU, and then recombinant latent TGF-β1 of human, cynomolgus monkey and mouse was injected over the flow cells. All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3. The sensor surface was regenerated with 3M MgCl2 for each cycle. The binding affinity was determined by processing the data and fitting them to a 1:1 binding model using BIACORE (registered trademark) 8K evaluation software version 1.1.1.7442 (GE Healthcare).

[0746] The binding affinity of anti-latent TGF-β1 antibodies to human, cynomolgus monkey and mouse latent TGF-β1 at pH 7.4 and pH 5.8 is shown in Figures 5 and 6. The pH-dependent anti-latent TGF-β1 antibodies showed dissociation at acidic pH 5.8 with kd of 3.93E-02 to 6.54E-02 as shown in Figure 6, while antibody hT0947AE07-SG191 showed slower dissociation at pH 5.8 with kd of 1.49E-03.

[0747] Figure 7 shows the ratio of koff and KD of hT0947AE07-SG191, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191 and hT0947AE37-SG191 (pH 5.8 / pH 7.4) (calculated from the results of Figures 5 and 6).

[0748] (5-3) Anti-Latent TGF-β1 Antibodies Inhibit Plasmin (PLN)-Mediated Activation of Latent TGF-β1

[0749] (5-1) Anti-latent TGF-β1 antibodies binding to cell surface latent TGF-β1

[0750] For FACS analysis, antibodies with human IgG1 constant regions (SG191, SEQ ID NO: 23 and SK1, SEQ ID NO: 25) were prepared. Ba / F3 cells expressing mouse latent TGF-β1 or FreeStyle cells expressing human latent TGF-β1 were used. TM 293-F cells (ThermoFisher) were assayed for binding activity of anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0497AE20-SG191, hT0497AE27-SG191, hT0497AE34-SG191, and hT0497AE37-SG191) to cell surface latent TGF-β1 by FACS. Anti-latent TGF-β1 antibodies (10 μg / mL each) were incubated with each cell line at 4°C for 30 minutes and washed with FACS buffer (2% PBS, 2 mM EDTA in PBS). The anti-KLH antibody IC17dK-SG181 (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71) with a human IgG1 Fc region (which does not bind to mouse latent TGF-β1 nor to human latent TGF-β1) was used as a negative control antibody. Goat F(ab')2 anti-human IgG, mouse ads-PE (Southern Biotech, catalog number 2043-09) were then added and incubated at 4°C for 30 minutes and washed with FACS buffer. Data were acquired on a FACS Verse (Becton Dickinson), and then analyzed and calculated using FlowJo (trademark) software (TreeStar) and GraphPad Prism software (GraphPad). As shown in Figure 8, all anti-latent TGF-β1 antibodies bound to mouse cell surface latent TGF-β1 and FreeStyle IgG expressed on Ba / F3 cells. TM Binds to latent TGF-β1 expressed on the surface of human cells in 293-F cells.

[0751] (5-2) Anti-latent TGF-β1 antibodies inhibit spontaneous latent TGF-β1 activation

[0752] For latent TGF-β1 neutralization assays, antibodies with human IgGl constant region (SG191, SEQ ID NO: 23 and SK1, SEQ ID NO: 25) were prepared. Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example 1 were each incubated in the presence or absence of one of the anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0497AE20-SG191, hT0497AE27-SG191, hT0497AE34-SG191 and hT0497AE37-SG191) at 37°C for 1 hour. Anti-KLH antibody IC17dk-SG181 (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71) was used as a negative control. Spontaneous latent TGF-β1 activation and inhibition of antibody-mediated spontaneous latent TGF-β1 activation were analyzed by mature TGF-β1 ELISA (Human TGF-β1 Quantikine (registered trademark) ELISA Kit, R&D systems) according to the manufacturer's procedure.

[0753] As shown in Figure 9, the anti-latent TGF-β1 antibodies inhibited spontaneous activation of latent TGF-β1.

[0754] (5-4) Anti-Latent TGF-β1 Antibodies Inhibit Plasma Kallikrein (PLK)-Mediated Activation of Latent TGF-β1

[0755] For latent TGF-β1 neutralization assay, antibodies with human IgGl constant region (SG191, SEQ ID NO: 23 and SK1, SEQ ID NO: 25) were prepared. Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example 1 were each incubated with human plasmin (Calbiochem) in the presence or absence of one of the anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0497AE20-SG191, hT0497AE27-SG191, hT0497AE34-SG191 and hT0497AE37-SG191) at 37°C for 1 hour. The antibodies were pre-incubated with mouse or human latent TGF-β1 (SLC) at room temperature for 30 minutes, and then incubated with plasmin. Anti-KLH antibody IC17dk-SG181 (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71) 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 (registered trademark) ELISA kit, R&D systems) according to the manufacturer's procedure. As shown in FIG. 10, the anti-latent TGF-β1 antibodies inhibited plasmin-mediated activation of latent TGF-β1.

[0756] (5-5) Anti-Latent TGF-β1 Antibodies Inhibit MMP2 and MMP9-Mediated Activation of Human Latent TGF-β1

[0757] For latent TGF-β1 neutralization assay, antibodies with human IgGl constant region (SG191, SEQ ID NO: 23 and SK1, SEQ ID NO: 25) were prepared. Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example 1 were each incubated with human kallikrein (Enzyme Research Laboratories) in the presence or absence of one of the anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0497AE20-SG191, hT0497AE27-SG191, hT0497AE34-SG191 and hT0497AE37-SG191) at 37°C for 2 hours. The antibodies were pre-incubated with mouse or human latent TGF-β1 (SLC) at room temperature for 30 minutes, and then incubated with kallikrein. Anti-KLH antibody IC17dk-SG181 (heavy chain SEQ ID NO 70, light chain SEQ ID NO 71) 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 (registered trademark) ELISA kit, R&D systems) according to the manufacturer's procedure. As shown in FIG. 11, the anti-latent TGF-β1 antibodies inhibited kallikrein-mediated activation of latent TGF-β1.

[0758] (5-6) Anti-Latent TGF-β1 Antibodies Inhibit Integrin-Mediated Activation of Latent TGF-β1 in Mouse PBMCs

[0759] For latent TGF-β1 neutralization assay, antibodies with human IgGl constant region (SG191, SEQ ID NO: 23 and SK1, SEQ ID NO: 25) were prepared. Human latent TGF-β1 (SLC) prepared in Example 1 was incubated with activated metalloproteinase 2 (MMP2) or MMP9 (R&D systems) in the presence or absence of one of the anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0497AE20-SG191, hT0497AE27-SG191, hT0497AE34-SG191 and hT0497AE37-SG191) for 2 hours at 37°C. The antibodies were pre-incubated with human latent TGF-β1 (SLC) for 30 minutes at room temperature, then incubated with MMP2 or MMP9. Anti-KLH antibody IC17dk-SG181 (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71) 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 (registered trademark) ELISA Kit, R&D systems) according to the manufacturer's procedure. As shown in Figure 11, the anti-latent TGF-β1 antibodies inhibited both MMP2-mediated human latent TGF-β1 activation and MMP9-mediated human latent TGF-β1 activation.

[0760] With Minor Modifications ​

[0761] Mouse PBMC and HEK-Blue TM TGF-β Cell Co-culture Assay to Detect Integrin-mediated Latent TGF-β1 Activation. Mouse PBMC were isolated from mouse blood by using Histopaque-1083 density gradient medium (Sigma-Aldrich). HEK-Blue TM TGF-β Cells (Invivogen) express a Smad3 / 4-binding element (SBE)-inducible SEAP reporter, allowing detection of biologically active TGF-β1 (both mouse and human TGF-β1) by monitoring activation of Smad3 / 4. Active TGF-β1 stimulates the production of SEAP and its secretion into the cell supernatant. The amount of secreted SEAP was assessed by using QUANTI-Blue TM reagent (Invivogen).

[0762] HEK-Blue TMTGF-β cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 50 U / mL streptomycin, 50 microgram / mL penicillin, 100 microgram / mL Normocin (Trade Mark), 30 microgram / mL blasticidin, 200 microgram / mL HygroGold (Trade Mark) and 100 microgram / mL Zeocin (Registered Trademark). During the functional assay, cell medium was changed to assay medium (RPMI1640 with 10% FBS) and seeded to 96-well plates. Then, one of the anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0947AE20-SG191, hT0947AE34-SG191 or hT0947AE37-SG191 ) and mouse PBMCs were applied to the wells and incubated with HEK-Blue TM TGF-β cells were incubated overnight. Then, cell supernatant was mixed with QUANTI-Blue TM and the optical density (OD) at 620 nm was measured in a colorimetric plate reader. The RGD peptide (GRRGDLATIH (SEQ ID NO: 101 ), GenScript) is known to bind to integrins and was used as a decoy integrin ligand to inhibit integrin-mediated TGF-β1 activation. Therefore, the RGD peptide was used as a positive control. In addition, the RGE control peptide (GRRGELATIH (SEQ ID NO: 102), GenScript) which is not a decoy integrin ligand was used as a negative control. The anti-KLH antibody IC17dk-SG181 (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71 ) was used as a negative control. The anti-mature TGF-β antibody GC1008-F1332m (heavy chain SEQ ID NO: 76, light chain SEQ ID NO: 77) was used as a positive control. F1332m is a human IgG1 heavy chain constant region which includes amino acid substitutions to reduce effector function.

[0763] As shown in Figure 12, the anti-latent TGF-β1 antibodies had less impact on inhibiting integrin-mediated TGF-β1 activation in mouse PBMCs compared to those of the positive control.

[0764] (5-7) Inhibition of integrin-mediated activation of latent TGF-β1 by anti-latent TGF-β1 antibodies in Detroit 562 Inhibition has a small effect

[0765] Detroit 562 and HEK-Blue TM TGF-β cell co-culture assays to detect integrin-mediated latent TGF-β1 activation. Detroit 562 were purchased from ATCC (Virginia, US). HEK-Blue TMTGF-β cells (Invivogen) express a Smad3 / 4-binding element (SBE)-inducible SEAP reporter, allowing the detection of bioactive TGF-β1 (both mouse and human TGF-β1) by monitoring the activation of Smad3 / 4. Active TGF-β1 stimulates the production of SEAP and its secretion into the cell supernatant. The amount of secreted SEAP was assessed by using QUANTI-Blue reagent (Invivogen) according to the manufacturer’s instructions. TM The amount of secreted SEAP was assessed by using QUANTI-Blue reagent (Invivogen) according to the manufacturer’s instructions.

[0766] HEK-Blue TGF-β cells were seeded in 96-well plates at a density of 50,000 cells / well in 100 μΐ of DMEM medium supplemented with 10% FBS, 50 U / mL streptomycin, 50 μg / mL penicillin, 100 μg / mL Normocin (trade mark), 30 μg / mL blasticidin, 200 μg / mL HygroGold (trade mark) and 100 μg / mL Zeocin (registered trade mark). The next day, 100 μΐ of assay medium (RPMI 1640 with 10% FBS) was added to each well and the cells were incubated for 2 hours at 37°C. Then, 100 μΐ of the anti-latent TGF-β1 antibody (hT0947AE07-SG191, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191 or hT0947AE37-SG191) was added to each well and incubated for 2 hours at 37°C. Finally, 100 μΐ of the HEK-Blue TGF-β cells was added to each well and incubated overnight at 37°C. TM 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 (trade mark), 30 μg / mL blasticidin, 200 μg / mL HygroGold (trade mark) and 100 μg / mL Zeocin (registered trade mark). During the functional assay, cell medium was changed to assay medium (RPMI 1640 with 10% FBS) and seeded to 96-well plates. Then, one of the anti-latent TGF-β1 antibodies (hT0947AE07-SG191, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191 or hT0947AE37-SG191) and Detroit 562 were applied to the wells and incubated with HEK-Blue TGF-β cells overnight. TM Cell supernatants were then mixed with QUANTI-Blue reagent (Invivogen) and incubated for 30 minutes at 37°C. The absorbance was measured at 650 nm using a microplate reader. TMMixed and the optical density (OD) at 620 nm was measured in a colorimetric plate reader. The RGD peptide (GRRGDLATIH (SEQ ID NO: 101), GenScript) is known to bind to integrin and was used as a decoy integrin ligand to inhibit integrin-mediated TGF-β1 activation. Therefore, the RGD peptide was used as a positive control. In addition, the RGE control peptide (GRRGELATIH (SEQ ID NO: 102), GenScript) which is not a decoy integrin ligand was used as a negative control. Anti-KLH antibody IC17-SG105 (heavy chain SEQ ID NO: 74, light chain SEQ ID NO: 75) was used as a negative control. Anti-mature TGF-β1 antibody 21D1-F1332m (heavy chain SEQ ID NO: 78, light chain SEQ ID NO: 79) and anti-integrin avb6 antibody 3G9-mF18 (heavy chain SEQ ID NO: 80, light chain SEQ ID NO: 81) were used as positive controls. F1332m is a human IgG1 heavy chain constant region which includes amino acid substitutions to reduce effector function.

[0767] As shown in Figure 13, the anti-latent TGF-β1 antibodies had less of an effect on inhibiting integrin-mediated TGF-β1 activation in Detroit 562 compared to those of the positive controls.

[0768] Example 6. Pharmacokinetics of anti-latent TGF-β1 antibodies

[0769] (6-1) PK testing of pH-dependent antibodies using cynomolgus monkeys

[0770] The pharmacokinetics of anti-human latent TGF-β1 antibodies (hT0947AE07-SG191, hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, and hT0947AE37-SG191) were evaluated in cynomolgus monkeys. HT0947AE07-SG191 was administered to cynomolgus monkeys at 0.4 mg / kg and 2 mg / kg by single-dose intravenous administration on day 0. The other antibodies were administered to cynomolgus monkeys at 0.8 mg / kg and 2 mg / kg or 4 mg / kg by single-dose intravenous administration on day 0. An anti-CD40 antibody was administered to cynomolgus monkeys at 5 mg / kg, 0.5 mg / kg, 0.5 mg / kg, 0.5 mg / kg, and 5 mg / kg by intravenous administration on days 0, 7, 14, 21, and 29, respectively. Blood samples were collected over time. The collected blood was immediately centrifuged at 1700 x g and 4°C for 5 minutes to obtain plasma. The isolated plasma was stored in a refrigerator set to -70°C or lower until measurement.

[0771] Measurement of concentration in cynomolgus monkey plasma was performed by ELISA.

[0772] As shown in FIGS. 14-1 to 14-6, the pH-dependent anti-human latent TGF-β1 antibodies hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, and hT0947AE37-SG191 showed slower clearance compared to the non-pH dependent hT0947AE07-SG191.

[0773] (6-2) PK test of pH-dependent antibodies using mice

[0774] The pharmacokinetics of anti-TGF-β1 antibodies (hT0947AE04-SG181 (heavy chain SEQ ID NO: 103, light chain SEQ ID NO: 104), hT0947AE07-SG191, hT0947AE20-SG181, hT0947AE27-SG181, hT0947AE34-SG181, and hT0947AE37-SG181) were evaluated in mice. Antibodies were administered to mice at 2 mg / kg and 10 mg / kg by single dose tail vein administration, and blood samples were collected over time. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set to -20°C or lower until measurement.

[0775] Measurement of concentration in mouse plasma was performed by ELISA.

[0776] As shown in FIGS. 14-7 to 14-10, the pH-dependent anti-human latent TGF-β1 antibodies hT0947AE20-SG181, hT0947AE27-SG181, hT0947AE34-SG181, and hT0947AE37-SG181 showed slower clearance compared to the non-pH dependent hT0947AE04-SG181 and hT0947AE07-SG191.

[0777] Example 7. In vivo efficacy of anti-latent TGF-β1 antibodies in a UUO-induced mouse model of kidney fibrosis

[0778] The in vivo efficacy of monoclonal antibodies hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, and hT0947AE37-SG191 was evaluated in a unilateral ureteral obstruction (UUO) mouse model known to induce progressive renal fibrosis.

[0779] (7-1) Establishment of a UUO-induced mouse model of kidney fibrosis

[0780] The in vivo efficacy of the monoclonal antibodies hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191 and hT0947AE37-SG191 was evaluated in a mouse model of unilateral ureteral obstruction (UUO) that induces progressive renal fibrosis.

[0781] C57BL / 6J mice 7-week-old male mice were purchased from Charles river (Kanagawa, Japan) and acclimated for 1 week before the start of the treatment. All mice were maintained under a regular light-dark cycle (12:12 light:dark), with free access to food (CE-2, CLEA Japan, Tokyo, Japan) and water.

[0782] UUO surgery was performed under isoflurane anesthesia. The left side of the abdomen was shaved and a vertical incision was made through the skin. A second incision was made through the peritoneum and the skin was also retracted to expose the kidney. Using forceps, the kidney was brought to the surface and the left ureter was ligated twice with surgical silk under the kidney. The ligated kidney was gently placed back in its correct anatomical position and the peritoneum and skin were sutured. An analgesic was added to reduce animal suffering. In the sham-operated group, only the peritoneum and skin were incised and sutured.

[0783] (7-2) Evaluation of in vivo efficacy

[0784] All monoclonal antibodies were administered by subcutaneous injection one day after the UUO surgery. The anti-KLH antibody IC17-mF18 (heavy chain SEQ ID NO: 72, light chain SEQ ID NO: 73) was used as a negative control in this study. The anti-KLH antibody (IC17-mF18) was administered to the sham-operated group. Animals were weighed 7 days after surgery and then sacrificed by exsanguination under isoflurane anesthesia. Blood samples were collected from the heart or posterior vena cava, transferred into tubes containing heparin and centrifuged (10,000 g, 10 min, 4°C) to prepare plasma samples. The kidneys were quickly removed. Part of the kidney tissue was quickly frozen in liquid nitrogen or dry ice for molecular analysis.

[0785] Hydroxyproline content in the kidney was measured to assess extramatrix deposition into the tissue, which is an amino acid contained in collagen. Wet kidney tissues were dried at 110°C overnight and weighed. Then, 6N HC1 (100 μΐ / 1 mg of dry tissue) was added to the dried tissues and boiled at 110°C overnight. The samples were cleaned by a filter, and 10 μΐ of each sample was inoculated into a 96-well plate. The plate with the samples was dried at 60°C, and hydroxyproline was measured using a hydroxyproline assay kit (BioVision) or mass spectrometry analysis. The results of this experiment are shown in FIG. 15. A significant increase in hydroxyproline content was observed in the disease-induced kidney, and all antibodies (hT0947AE20-SG191, hT0947AE27-SG191, hT0947AE34-SG191, and hT0947AE37-SG191) inhibited kidney fibrosis. Data are expressed as mean + / - standard error of the mean (SEM). Statistical analysis was performed using Student's t-test or Dunnett's multiple comparison test. Differences were considered statistically significant when the P value was < 0.05.

[0786] Example 8. In vivo efficacy of anti-latent TGF-β1 antibodies in aGBM model

[0787] In vivo efficacy of monoclonal antibodies hT0947AE07-mF18, hT0947AE20-mF18, hT0947AE34-mF18, and hT0947AE37-mF18 was evaluated in a model of glomerulonephritis induced by anti-GBM nephrotoxic serum, which is known to cause glomerulosclerosis (aGBM mouse model).

[0788] (8-1) Establishment of aGBM mouse model

[0789] Seven-week-old female C57BL / 6JJcl mice were presensitized by subcutaneous injection of sheep IgG. Five days after presensitization, nephrotoxic serum (NTS) containing anti-GBM antibody was intravenously injected for 3 days. All mice were maintained under a regular light-dark cycle (12:12 light:dark), with free access to food (CE-2, CLEA Japan, Tokyo, Japan) and water.

[0790] (8-2) Evaluation of in vivo efficacy

[0791] All monoclonal antibodies were administered by subcutaneous injection at 30 mg / kg, twice a week, from one day before NTS injection. Anti-KLH antibody IC17-mF18 (heavy chain SEQ ID NO: 72, light chain SEQ ID NO: 73) was used as a negative control in this study. Kidneys were collected 14 days after the first NTS injection. To analyze glomerulosclerosis, a glomerulus classification model was created by training the annotated sclerotic and non-sclerotic glomeruli in some mice using the classifier of Halo AI (DenseNetAI V2 (Plugin)), and then applied to all mice.

[0792] The results of this experiment are shown in Figure 16. A significant increase in glomerulosclerosis was observed in disease control mice, and all anti-latent TGF-β1 antibodies inhibited the number of sclerotic glomeruli. Data are expressed as mean + / - standard error of the mean (SEM). Statistical analysis was performed using Student’s t-test or Dunnett’s multiple comparison test. Differences were considered statistically significant when P value < 0.05.

[0793] Example 9. In vivo efficacy of anti-latent TGF-β1 antibodies in Alport mouse model

[0794] The in vivo efficacy of monoclonal antibodies hT0947AE20-mF18 and hT0947AE37-mF18 was evaluated in a Col4a3 knockout mouse model known to cause progressive renal dysfunction (Alport mouse model).

[0795] (9-1) Establishment of Col4a3 knockout mice

[0796] Col4a3 knockout mouse strains were established by zinc finger nuclease (ZFN)-mediated gene editing. A pair of ZFNs was designed to induce a mutation in exon 48, which encodes the first part of the NC1 domain, which is a functional domain of the normal collagen chain. All mice were maintained under a regular circadian light cycle (12:12 light:dark), with free access to food (CE-2, CLEA Japan, Tokyo, Japan) and water.

[0797] (9-2) Evaluation of in vivo efficacy

[0798] All monoclonal antibodies were administered twice a week by subcutaneous injection from 14 weeks of age to 20 weeks of age. Anti-KLH antibody IC17dk-mF18 (heavy chain SEQ ID NO: 72, light chain SEQ ID NO: 73) was used as a negative control in this study. To analyze the progression of kidney disease in Alport mice, blood was collected from the jugular vein under isoflurane anesthesia at 14, 16, 18, and 20 weeks of age and from the abdominal vena cava at 21 weeks of age, transferred to a tube containing heparin, and centrifuged (10,000g, 10 minutes, 4°C) to prepare plasma samples. Biochemical parameters in plasma were measured by using a TBA-120FR automatic analyzer (Canon Medical Systems Corporation, Tochigi, Japan).

[0799] The results of this experiment are shown in Figure 17. A significant increase in plasma creatinine was observed in the disease control mice, and both hT0947AE20-mF18 and hT0947AE37-mF18 showed improvement in kidney function. Data are expressed as mean + / - standard error of the mean (SEM). Statistical analysis was performed using the Student’s t-test or Dunnett’s multiple comparison test. Differences were considered statistically significant when the P value was <0.05.

[0800] Example 10. Viscosity of pH-dependent anti-latent TGF-β1 antibodies

[0801] For viscosity measurement, antibodies with human IgGl constant regions (SG191, SEQ ID NO: 23 and SK1, SEQ ID NO: 25) were prepared. Anti-latent TGF-β1 antibodies (hT0497AE07-SG191, hT0497AE20-SG191, hT0497AE27-SG191, hT0497AE34-SG191, and hT0497AE37-SG191) were prepared at 150 mg / mL in formulation buffer (20 mM His, 150 mM Arg, 162.1 mM Asp, 20 mM Met, pH 6.0). The viscosity of the mAb solution was measured at 23°C by an electromagnetic rotation (EMS) method using an EMS viscometer (Kyoto Electronics Manufacturing, Kyoto, Japan). An aluminum ball was placed in a glass bottle containing the sample, and the viscosity of each sample solution was obtained at a rotation speed of 1000 rpm. The viscosity of the liquid sample can be calculated from the rotation speed of the aluminum ball measured using a flash lamp and a CCD camera.

[0802] The viscosity of anti-latent TGF-β1 antibodies at an antibody concentration of 150 mg / mL is shown in Figure 18. The pH-dependent antibodies hT0947AE20-SG191, hT0947AE27-SG191 and hT0947AE37-SG191 show lower viscosity compared to the antibody hT0947AE07-SG191, which indicates that these pH-dependent antibodies are preferably developed for high-concentration antibody drug products for subcutaneous administration.

[0803] Example 11. Thermal acceleration stability study of pH-dependent anti-latent TGF-β1 antibodies

[0804] Anti-latent TGF-β1 antibodies (hT0497AE20-SG191, hT0497AE34-SG191 and hT0497AE37-SG191) were concentrated to 130 mg / mL in formulation buffer (20 mM His, 150 mM Arg, 162.1 mM Asp, 20 mM Met, 0.5 mg / mL PS80, pH 6.0) using Amicon (registered trademark) Ultra 15 mL 30K (Millipore # UFC903024). The samples were kept at 40°C and time points were taken at day 14 and day 28. The samples were then analyzed by size exclusion chromatography (SEC; Phenomenex # 00H-4788-K0) with a running buffer containing 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0.

[0805] The results of the SEC analysis are shown in Figure 19. hT0497AE34-SG191 and hT0497AE37-SG191 did not show a significant increase of high molecular weight species (HMW) even after 4 weeks of treatment at 40°C, whereas hT0497AE20-SG191 showed an increase of HMW after 2 and 4 weeks of treatment at 40°C.

[0806] Example 12. In vivo efficacy of anti-latent TGF-β1 antibodies in CDAHFD mouse model

[0807] The in vivo efficacy of the monoclonal antibody hT0947AE37-SG191 was evaluated in a CDAHFD (choline deficient, L-amino acid defined, high fat diet)-induced mouse NASH / liver fibrosis model.

[0808] (12-1) Establishment of a CDAHFD-induced mouse NASH / liver fibrosis model

[0809] C57BL / 6JmsSlc mice 5-week-old male mice were purchased from Japan SLC (Shizuoka, Japan) and acclimated for 1 week before the start of treatment. All mice were maintained on a regular light-dark cycle (12:12 light:dark), with free access to food and water. Test diets, choline-deficient, L-amino acid-defined high-fat diet (CDAHFD; #A06071302, Research Diets New Brunswick, NJ, USA) were fed to mice for 2 or 8 weeks. During the study, 3 groups were fed CDAHFD (n=10), and 1 group (n=5) was fed CRF-1 (Oriental Yeast, Tokyo, Japan) as a normal control group.

[0810] (12-2) Evaluation of Anti-fibrotic Effect

[0811] All monoclonal antibodies were administered by subcutaneous injection 2 days before sacrifice. Anti-KLH antibody IC17dk was used as a negative control in this study. Animals were weighed at 2 or 8 weeks after feeding CDAHFD, and then sacrificed by exsanguination under isoflurane anesthesia. Blood samples were collected from the heart or posterior vena cava, transferred to tubes containing EDTA-3K, and centrifuged (10,000g, 10 minutes, 4°C) to prepare plasma samples. The liver was quickly removed and weighed. Part of the liver tissue was quickly frozen in liquid nitrogen or dry ice for molecular analysis.

[0812] Total RNA was extracted from liver tissue using TRIzol (trademark) RNA isolation reagent (ThermoFisher) and cDNA was synthesized using Transcriptor Universal cDNA Master (Roche Diagnostics). Gene expression was measured using a 7900HT Fast Real-Time PCR System (ThermoFisher). Primers and Taq-Man probes for genes were purchased from Applied Biosystems. Mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference for each sample. Relative mRNA expression values were calculated using a double ΔCt analysis.

[0813] The results of this experiment are shown in Figure 20. A significant increase in collagen type 1 alpha 1 mRNA was observed in disease control mice, and hTO947AE37-SG191 (AE37) showed a decrease. Data are expressed as mean + / - standard error of the mean (SEM). Statistical analysis was performed using Student’s t-test or Dunnett’s multiple comparison test. Differences were considered statistically significant when P value < 0.05.

[0814] Example 13. In vivo efficacy of anti-latent TGF-β1 antibodies in BLM mouse model

[0815] The in vivo efficacy of the monoclonal antibody hT0947AE37-SG191 was evaluated in a bleomycin-induced pulmonary fibrosis model.

[0816] (13-1) Establishment of a bleomycin-induced pulmonary fibrosis model in mice

[0817] C57BL / 6J mice 8-week-old male mice were purchased from THE JACKSON LABORATORY JAPAN, INC (Kanagawa, Japan) and acclimated for 1 week before the start of the treatment. All mice were maintained under a regular light-dark cycle (12:12 light:dark) with free access to food CE-2 (CLEA Japan, Tokyo, Japan) and water. Bleomycin (Nihonkayaku, Tokyo, Japan) was administered subcutaneously a total of 10 times (5 consecutive days, 2 days off, and then 5 consecutive days).

[0818] (13-2) Evaluation of in vivo efficacy

[0819] All monoclonal antibodies were administered once a week by subcutaneous injection at day 14 and day 21 after disease induction. In this study, vehicle or anti-KLH antibody IC17dk was administered as a negative control. At day 28 after disease induction, animals were weighed and then sacrificed by exsanguination under isoflurane anesthesia. Blood samples were collected from the heart or posterior vena cava, transferred into tubes containing heparin, and centrifuged (10,000 g, 10 min, 4°C) to prepare plasma samples. The lungs were quickly removed and weighed. Part of the lung tissue was quickly frozen in liquid nitrogen or dry ice for molecular analysis.

[0820] The hydroxyproline content (an amino acid contained in collagen) in the lungs was measured to evaluate the extracellular matrix deposition of the tissue. Wet lung tissue was dried overnight by a freeze dryer and weighed. Then, 6N HC1 (100 μl / 1 mg of dry tissue) was added to the dried tissue and boiled at 110°C overnight. The sample was cleaned by a filter, and measured using a hydroxyproline assay kit (QuickZyme) or mass spectrometry analysis.

[0821] Total RNA was extracted from lung tissue using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized using Transcriptor Universal cDNA Master (Roche Diagnostics). Gene expression was measured using the 7900HT Fast Real-Time PCR System (ThermoFisher). Primers and Taq-Man probes for genes were purchased from Applied Biosystems. Mouse mitochondrial ribosomal protein L19 (MRPL19) was used as an endogenous reference for each sample. Relative mRNA expression values were calculated using the double ΔCt analysis.

[0822] The results of this experiment are shown in Figure 21. A significant increase in collagen type 1 alpha 1 mRNA, Serpinel mRNA, and hydroxyproline content was observed in disease-induced lungs, and hTO947 AE37-SG191 (AE37) showed a reduction. Data are expressed as mean + / - standard error of the mean (SEM). Statistical analysis was performed using Student’s t-test or Dunnett’s multiple comparison test. Differences were considered statistically significant when P value < 0.05.

[0823] The disclosures of International Publication Nos. WO 2019 / 163927 and WO 2021 / 039945 are incorporated by reference in their entireties.

[0824] The disclosure of Japanese Patent Application No. 2023-019304 is incorporated by reference in its entirety.

[0825] Although the present application is described in detail herein by way of illustration and example for purposes of clarity and understanding, such description is not intended to be limiting in any way. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

[0826] Industrial Applicability

[0827] The present disclosure provides anti-latent TGF-β1 antibodies that cross species, are humanized, and optimized, which inhibit protease-mediated activation of latent TGF-β1 without inhibiting integrin-mediated activation of latent TGF-β1 or partially inhibit integrin-mediated activation of latent TGF-β1, or which inhibit protease-mediated activation of latent TGF-β1 but have a lesser impact on inhibiting integrin-mediated activation of latent TGF-β1. In addition, the present disclosure provides anti-TGF-β1 antibodies that have pH-dependent binding affinity for latent TGF-β1. Anti-latent TGF-β1 antibodies are expected to be useful in treating TGF-β1 -related diseases, such as fibrosis and cancer. For example, anti-latent TGF-β1 antibodies can be used to treat kidney fibrosis, liver fibrosis, and lung fibrosis.

Claims

1. An anti-latent TGF-βl antibody comprising: (a) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 36, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 49, 52, and 56, respectively; (b) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 28, 32, and 37, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 52, and 56, respectively; (c) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 27, 30, and 39, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 48, 53, and 56, respectively; or (d) HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 27, 33, and 40, respectively; and HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 51, 52, and 56, respectively.

2. The anti-latent TGF-βl antibody of claim 1, comprising: (a) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, or (iii) a VH sequence as in (i) and a VL sequence as in (ii); (b) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 21, or (iii) a VH sequence as in (i) and a VL sequence as in (ii); (c) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, or (iii) a VH sequence as in (i) and a VL sequence as in (ii); or (d) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20, or (iii) a VH sequence as in (i) and a VL sequence as in (ii).

3. The anti-latent TGF-βl antibody of claim 1, comprising: (a) a VH sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 16; (b) a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 21; (c) a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 17; or (d) a VH sequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO:

20.

4. The anti-latent TGF-βl antibody of claim 1, which is a human, humanized, or chimeric antibody.

5. The anti-latent TGF-βl antibody of claim 1, which is an IgG antibody.

6. The anti-latent TGF-βl antibody of claim 1, wherein the anti-latent TGF-βl antibody comprises a modified IgGl Fc region having reduced effector function compared to a wild-type IgGl Fc region.

7. The anti-latent TGF-βl antibody of claim 1, wherein the anti-latent TGFβl antibody binds to latent TGF-βl in a pH-dependent manner.

8. An anti-latent TGF-βl antibody, comprising: (a) a heavy chain sequence of SEQ ID NO: 82 and a light chain sequence of SEQ ID NO: 83; (b) a heavy chain sequence of SEQ ID NO: 84 and a light chain sequence of SEQ ID NO: 85; (c) a heavy chain sequence of SEQ ID NO: 86 and a light chain sequence of SEQ ID NO: 87; (d) a heavy chain sequence of SEQ ID NO: 88 and a light chain sequence of SEQ ID NO: 89; (e) a heavy chain sequence of SEQ ID NO: 90 and a light chain sequence of SEQ ID NO: 83; (f) a heavy chain sequence of SEQ ID NO: 91 and a light chain sequence of SEQ ID NO: 85; (g) a heavy chain sequence of SEQ ID NO: 92 and a light chain sequence of SEQ ID NO: 87; or (h) a heavy chain sequence of SEQ ID NO: 93 and a light chain sequence of SEQ ID NO:

89.

9. An immunoconjugate, comprising: the anti-latent TGFβ-1 antibody of claim 1, and a cytotoxic agent.

10. An isolated nucleic acid encoding the anti-latent TGFβ-1 antibody of claim 1.

11. A vector comprising the nucleic acid of claim 10.

12. A host cell comprising the nucleic acid of claim 10.

13. A method of producing an anti-latent TGF-βl antibody, the method comprising culturing the host cell of claim 12, thereby producing the antibody.

14. An immunoconjugate, comprising: the anti-latent TGFβ-1 antibody of claim 8, and a cytotoxic agent.

15. An isolated nucleic acid encoding the anti-latent TGFβ-1 antibody of claim 8.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 15.

18. A method of producing an anti-latent TGF-βl antibody, the method comprising culturing the host cell of claim 17, thereby producing the antibody.

19. A pharmaceutical preparation comprising: the anti-latent TGF-βl antibody of claim 1, and a pharmaceutically acceptable carrier.

20. A pharmaceutical preparation comprising: the anti-latent TGF-βl antibody of claim 8, and a pharmaceutically acceptable carrier.

21. A pharmaceutical preparation comprising: the immunoconjugate of claim 9, and a pharmaceutically acceptable carrier.

22. A pharmaceutical preparation comprising: the immunoconjugate of claim 14, and a pharmaceutically acceptable carrier.

23. The pharmaceutical preparation of claim 19 for use in treating fibrosis or cancer.

24. The pharmaceutical preparation of claim 20 for use in treating fibrosis or cancer.

25. The pharmaceutical preparation of claim 21 for use in treating fibrosis or cancer.

26. The pharmaceutical preparation of claim 22 for use in treating fibrosis or cancer.

27. A method of treating fibrosis or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of claim 1.

28. A method of treating fibrosis or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of claim 8.

29. A method of treating fibrosis or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the immunoconjugate of claim 9.

30. A method of treating fibrosis or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the immunoconjugate of claim 14.

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