Cross-species anti-latent tgf-beta1 antibodies and methods of use

Abstract

The object of the present invention is to provide a cross-species anti-latent TGF-[beta] 1 antibody that inhibits protease-mediated activation of latent TGF-[beta] 1 and does not inhibit integrin-mediated activation of latent TGF-[beta] 1. In order to obtain the anti-latent TGF-[beta] 1 antibody of the present invention, an anti-latent TGF-[beta] 1 antibody that inhibits protease-mediated activation of latent TGF-[beta] 1 without inhibiting integrin-mediated activation of latent TGF-[beta] 1 is screened and then humanized, and further optimized. The invention also provides combination therapies comprising an anti-latent TGF-beta 1 antibody and one or more immune checkpoint inhibitors, preferably a PD-1 axis binding antagonist.

Description

[0001] This application is a divisional application of a patent application with an application date of August 28, 2020, Chinese patent application number 202080061243.X, and invention name “Cross-species anti-latent TGF-β1 antibodies and methods of use”. Technical Field

[0002] The present invention relates to anti-latent TGF-β1 antibodies and methods of using the same. Background Art

[0003] Transforming growth factor-β (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 a conserved structure and have pleiotropic functions in vitro and in vivo (NPL 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, despite being normally 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 regulating cell growth, migration, or phenotype.

[0004] 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 the type 2 TGF-β receptor (TGFR2) as homodimers (their active form); TGFR2 then recruits and activates the type 1 TGF-β receptor (TGFR1) to activate receptor signaling (NPL 5). However, the expression levels of the three isoforms vary from tissue to tissue (NPL 6), and their functions differ, as elucidated by the phenotypes of knockout mice (NPL 7-11).

[0005] Like other members of the TGF-β superfamily, TGF-β is synthesized as a precursor protein that forms a homodimer that interacts with its latency-associated peptide (LAP) and latent TGF-β binding protein (LTBP) to form a larger complex called the large latent complex (LLC). The TGF-β gene encodes a preproprotein sequence consisting of a signal peptide, a propeptide ending in a proprotein convertase (PPC) cleavage site, and the mature TGF-β sequence. Furin protease hydrolyzes the PPC cleavage site to produce individual TGF-β homodimers and propeptide-derived homodimers. These two homodimers remain non-covalently associated and are secreted. This latent complex keeps TGF-β in an inactive form that is unable to bind to its receptor (NPL 12, 13). The TGF-β activation process involves the release of LLC from the ECM, followed by further proteolysis of LAP to release active TGF-β to its receptor (NPL 3). Latent TGF-β is cleaved by multiple proteases, including plasmin (PLN), plasma kallikrein (PLK), matrix metalloproteinase (MMP) 2 and MMP9 (NPL 14), and 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 pro-MMPs. Pro-MMP2 is activated by a complex of membrane type 1 MMP (MT1-MMP / MMP14) and tissue inhibitor of metalloproteinase 2 (TIMP-2). Pro-MMP9 is activated by an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin generates active MMP-3 from its zymogen. Active MMP-3 cleaves the propeptide from the 92-kDa pro-MMP-9 to produce an 82-kDa enzyme with enzymatic activity. The cleavage site of the MMP has not been specifically determined; however, MMP3 has been reported to specifically cleave between 79 Ala and 80 Leu of latent TGF-β, thereby activating TGF-β (WO2005 / 023870). Alternatively, upon mechanical extension, integrins can activate TGF-β by binding to the RGD motif present in LAP to induce the release of mature TGF-β from its latent complex (NPL 16, 17).

[0006] Upon activation, dimeric TGF-β ligands bind to the extracellular domains of type I and type II receptors and induce close proximity, placing the intracellular serine / threonine kinase domains of the receptors in a conformation that promotes phosphorylation and subsequent activation of type I receptors. This activation of type I receptors results in signaling that propagates through at least two seemingly independent pathways: the SMAD-dependent classical pathway and the SMAD-independent or non-classical pathway. In the SMAD-dependent pathway, activation of TGFR1 (also known as ALK5) results in phosphorylation of SMAD proteins. SMAD2 and SMAD3 are substrates of TGFR1. Following phosphorylation by the receptors, SMADs translocate to the nucleus along with the common mediator SMAD4, where they interact with other transcription factors to regulate transcriptional responses (NPL18). In the nonclassical pathway, the activated TGF-β receptor complex transmits signals through other factors, such as tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4), TRAF6, TGF-β-activated kinase 1 (TAK1, also known as MAP3K7), p38 mitogen-activated protein kinase (p38 MAPK), RHO, phosphoinositide 3-kinase (PI3K), AKT (also known as protein kinase B), extracellular signal-regulated kinase (ERK), JUN N-terminal kinase (JNK), or nuclear factor-κB (NF-κB). Therefore, the cellular response to TGF-β signaling results from a dynamic combination of canonical and nonclassical signaling cascades.

[0007] Fibrosis, or the accumulation of ECM molecules that make up scar tissue, is a common feature of chronic tissue injury. Pulmonary fibrosis, renal fibrosis, and liver cirrhosis are the more common fibrotic diseases, which collectively represent a large unmet clinical need. TGF-β strongly promotes the production of extracellular matrix by mesenchymal cells while inhibiting epithelial cell growth, which contributes to the pathogenesis of sclerotic diseases. Overexpression of the active form of TGF-β1 in the liver of transgenic mice is 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 inhibits the overproduction of proteases in the lungs and prevents the destruction of lung tissue that leads to emphysema. Furthermore, TGF-β1-deficient mice display prenatal lethality (approximately 50% at 10.5 days post coitum) or their pups die shortly after birth, with extensive inflammatory lesions in many organs, including the lung (vasculitis, perivascular cuffing, and interstitial pneumonia) and heart (endocarditis and myocarditis), suggesting that TGF-β1 plays a crucial role in maintaining immune homeostasis (NPL 7).

[0008] Results of studies using TGF-β neutralizing antibodies and animal models suggest that cirrhotic diseases can be prevented or cured by inhibiting the action of TGF-β. Since TGF-β is produced as a precursor protein, there are several reported methods to prevent activation of the latent form. Another method to prevent activation of the latent form is to use inhibitors or antibodies that bind to latent TGF-β to block cleavage by proteases such as PLK and PLN. Several antibodies using this method of inhibiting TGF-β activation have been reported as preventing or treating liver fibrosis / cirrhosis (PTL 1). In addition, there have been documents mentioning anti-LAP antibodies for the treatment of cancer (PTL 2) and TGFβ1-binding immunoglobulins for the treatment of TGFβ1-related diseases (PTL 3).

[0009] Reference List

[0010] Patent Literature

[0011] [PTL 1] WO 2011102483

[0012] [PTL 2] WO 2016115345

[0013] [PTL 3] WO 2017156500

[0014] Non-patent literature

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[0024] [NPL 10] Sanford, L. P. et al. Development 124, 2659-2670 (1997).

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[0026] [NPL 12] Dubois, C. M.et al. J. Biol. Chem. 270, 10618-10624 (1995)

[0027] [NPL 13] Nunes, I. et al. J. Am. Optom. Assoc. 69, 643-648 (1998)

[0028] [NPL 14] Annes, J. et al. J. Cell Sci. 116, 217-224 (2003).

[0029] [NPL 15] Schultz-Cherry, S. et al. J. Biol. Chem. 269, 26775-26782(1994).

[0030] [NPL 16] Munger, J. S. et al. Cell 96, 319-328 (1999).

[0031] [NPL 17] Shi, M. et al. Nature 474, 343-349 (2011).

[0032] [NPL 18] Shi, Y. & Massague, et al. Cell 113, 685-700 (2003).

[0033] [NPL 19] Sanderson, N. et al. Proc. Natl Acad. Sci.USA 92, 2572-2576(1995). Summary of the invention

[0034] Technical issues

[0035] The purpose of the present invention is to provide cross-species, humanized and optimized anti-latent TGF-β1 antibodies that inhibit protease-mediated activation of latent TGF-β1 without inhibiting integrin-mediated activation of latent TGF-β1. The present invention also provides a combination therapy comprising an anti-latent TGF-β1 antibody and one or more immune checkpoint inhibitors.

[0036] Solutions to technical problems

[0037] The present inventors conducted intensive research under the above circumstances and thus constructed a cross-species, humanized and optimized anti-latent TGF-β1 antibody that inhibits protease-mediated TGF-β1 activation without inhibiting integrin-mediated latent TGF-β1 activation. In addition, when administered in combination with one or more immune checkpoint inhibitors, the anti-latent TGF-β1 antibody exhibits an anti-tumor effect.

[0038] The present invention provides:

[0039] A1. An anti-latent TGF-β1 antibody comprising:

[0040] (a) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively;

[0041] (b) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively;

[0042] (c) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33 and 34, respectively; or

[0043] (d) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively.

[0044] A2. The anti-latent TGF-β1 antibody of A1, further comprising:

[0045] (a) HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 23, 24 and 25, respectively;

[0046] (b) HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 31, respectively;

[0047] (c) HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 36 and 37, respectively; and

[0048] (d) HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively.

[0049] A3. An anti-latent TGF-β1 antibody comprising:

[0050] (a) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 23, 24 and 25, respectively;

[0051] (b) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 31, respectively;

[0052] (c) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33 and 34, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 36 and 37, respectively; or

[0053] (d) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively.

[0054] A4. The anti-latent TGF-β1 antibody of any one of A1 to A3, comprising:

[0055] (a) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13, or (iii) a VH sequence as in (i) and a VL sequence as in (ii);

[0056] (b) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15, or (iii) a VH sequence as in (i) and a VL sequence as in (ii);

[0057] (c) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, or (iii) a VH sequence as in (i) and a VL sequence as in (ii); or

[0058] (d) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 18, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19, or (iii) a VH sequence as in (i) and a VL sequence as in (ii).

[0059] A5. The anti-latent TGF-β1 antibody of A4 comprises the VH sequence of SEQ ID NO: 12, 14, 16 or 18.

[0060] A6. The anti-latent TGF-β1 antibody of A4 or A5 comprises the VL sequence of SEQ ID NO: 13, 15, 17 or 19.

[0061] A7. The anti-latent TGF-β1 antibody of any one of A4 to A6, comprising:

[0062] (a) the VH sequence of SEQ ID NO: 12 and the VL sequence of SEQ ID NO: 13;

[0063] (b) the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 15;

[0064] (c) the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 17; or

[0065] (d) The VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 19.

[0066] A8. An anti-latent TGF-β1 antibody comprising:

[0067] (a) the VH sequence of SEQ ID NO: 12 and the VL sequence of SEQ ID NO: 13;

[0068] (b) the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 15;

[0069] (c) the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 17; or

[0070] (d) The VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 19.

[0071] A9. The anti-latent TGF-β1 antibody of any one of A1 to A8, which is a human antibody, a humanized antibody or a chimeric antibody.

[0072] A10. The anti-latent TGF-β1 antibody according to any one of A1 to A9, which is a full-length IgG antibody, preferably a full-length IgG1 antibody.

[0073] A11. The anti-latent TGF-β1 antibody of any one of A1 to A9, which is a bispecific antibody.

[0074] A12. 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 having reduced effector function compared to a wild-type IgG1 Fc region.

[0075] A13. The anti-latent TGF-β1 antibody of A12, wherein the modified IgG1 Fc region comprises an amino acid substitution at position EU235 and / or EU236 according to the EU index.

[0076] A14. The anti-latent TGF-β1 antibody of A12 or A13, wherein the modified IgG1 Fc region comprises amino acid substitutions of L235R and G236R according to the EU index.

[0077] A15. The anti-latent TGF-β1 antibody of any one of A12 to A14, wherein the modified IgG1 Fc region further has enhanced FcRn binding activity compared to the wild-type IgG1 Fc region.

[0078] A16. The anti-latent TGF-β1 antibody of A15, wherein the modified IgG1 Fc region comprises one or more amino acid substitutions at a position selected from the group consisting of EU428, EU434, EU438 and EU440 according to the EU index.

[0079] A17. The anti-latent TGF-β1 antibody of any one of A15 or A16, wherein the modified IgG1 Fc region comprises amino acid substitutions of M428L, N434A, Q438R and S440E.

[0080] A18. 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 and G236R.

[0081] A19. 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.

[0082] A20. The anti-latent TGF-β1 antibody of any one of A1 to A11, which is an antibody fragment.

[0083] A21. An anti-latent TGF-β1 antibody comprising:

[0084] (a) the full-length heavy chain sequence of SEQ ID NO: 47 and the full-length light chain sequence of SEQ ID NO: 60;

[0085] (b) the full-length heavy chain sequence of SEQ ID NO: 48 and the full-length light chain sequence of SEQ ID NO: 61;

[0086] (c) the full-length heavy chain sequence of SEQ ID NO: 49 and the full-length light chain sequence of SEQ ID NO: 62;

[0087] (d) the full-length heavy chain sequence of SEQ ID NO: 50 and the full-length light chain sequence of SEQ ID NO: 63;

[0088] (e) the full-length heavy chain sequence of SEQ ID NO: 51 and the full-length light chain sequence of SEQ ID NO: 64;

[0089] (f) the full-length heavy chain sequence of SEQ ID NO: 52 and the full-length light chain sequence of SEQ ID NO: 65;

[0090] (g) the full-length heavy chain sequence of SEQ ID NO: 53 and the full-length light chain sequence of SEQ ID NO: 66; or

[0091] (h) the full-length heavy chain sequence of SEQ ID NO: 54 and the full-length light chain sequence of SEQ ID NO: 67.

[0092] A22. The anti-latent TGF-β1 antibody of any one of A1 to A21, wherein the latent TGFβ-1 is human latent TGFβ-1, mouse latent TGFβ-1 or cynomolgus monkey latent TGFβ-1

[0093] A23. The anti-latent TGF-β1 antibody of any one of A1 to A22, wherein the anti-latent TGF-β1 antibody binds to human latent TGF-β1, mouse latent TGF-β1, and cynomolgus monkey latent TGF-β1.

[0094] A24. The anti-latent TGF-β1 antibody of any one of A1 to A23, wherein the anti-latent TGF-β1 antibody binds to the latency associated peptide (LAP) region of latent TGF-β1.

[0095] A25. An immunoconjugate comprising the anti-latent TGFβ-1 antibody of any one of A1 to A24 and a cytotoxic agent.

[0096] A26. An isolated nucleic acid encoding the anti-latent TGFβ-1 antibody of any one of A1 to A24.

[0097] A27. A vector comprising the nucleic acid of A26.

[0098] A28. A host cell comprising the nucleic acid of A26 or the vector of A27.

[0099] A29. A method for producing an anti-latent TGFβ-1 antibody, comprising culturing the host cell of A28 to produce the antibody.

[0100] A30. The method of A29 further comprises recovering the antibody from the host cell.

[0101] B1. An immunoconjugate of the anti-latent TGFβ-1 antibody of any one of A1 to A24 or A25 for use as a medicament.

[0102] B2. An immunoconjugate of the anti-latent TGFβ-1 antibody of any one of A1 to A24 or A25 for use in treating fibrosis or cancer.

[0103] B3. Use of an anti-latent TGFβ-1 antibody of any one of A1 to A24 or an immunoconjugate of A25 in the manufacture of a medicament for treating fibrosis or cancer.

[0104] B4. An anti-latent TGFβ-1 antibody of any one of A1 to A24 or an immunoconjugate of A25, used in combination with an additional therapeutic agent, preferably an immune checkpoint inhibitor, for the treatment of cancer.

[0105] B5. The anti-latent TGFβ-1 antibody or immunoconjugate of B4, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0106] B6. The anti-latent TGFβ-1 antibody or immunoconjugate of B4, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0107] B7. The anti-latent TGFβ-1 antibody or immunoconjugate of any one of B4 to B6, wherein the immune checkpoint inhibitor is administered simultaneously with the anti-latent TGFβ-1 antibody or immunoconjugate.

[0108] B8. The anti-latent TGFβ-1 antibody or immunoconjugate of any one of B4 to B6, wherein the immune checkpoint inhibitor is administered before or after administration of the anti-latent TGFβ-1 antibody or immunoconjugate.

[0109] C1. A pharmaceutical preparation comprising the anti-latent TGFβ-1 antibody of any one of A1 to A24 or the immunoconjugate of A25, and a pharmaceutically acceptable carrier.

[0110] C2. The pharmaceutical preparation of C1, further comprising an additional therapeutic agent, preferably an immune checkpoint inhibitor.

[0111] C3. The pharmaceutical preparation of C2, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0112] C4. The pharmaceutical preparation of C2, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0113] C5. The pharmaceutical preparation of any one of C1 to C4, for use in treating fibrosis or cancer.

[0114] C6. The pharmaceutical formulation of any one of C1 to C4, used in combination with another therapeutic agent, preferably an immune checkpoint inhibitor, for treating cancer.

[0115] C7. The pharmaceutical preparation of C6, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD1 antibody or an anti-PD-L1 antibody.

[0116] C8. The pharmaceutical preparation of C6, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0117] C9. The pharmaceutical preparation of any one of C6 to C8, wherein the immune checkpoint inhibitor is administered simultaneously with the pharmaceutical preparation.

[0118] C10. The pharmaceutical preparation of any one of C6 to C8, wherein the immune checkpoint inhibitor is administered before or after administration of the pharmaceutical preparation.

[0119] D1. A pharmaceutical preparation comprising an immune checkpoint inhibitor and a pharmaceutically acceptable carrier, used in combination with the anti-latent TGFβ-1 antibody of any one of claims A1 to A24 or the immunoconjugate of claim A25 for treating cancer.

[0120] D2. The pharmaceutical preparation of D1, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD1 antibody or an anti-PD-L1 antibody.

[0121] D3. The pharmaceutical preparation of D1, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0122] D4. The pharmaceutical preparation of any one of D1 to D3, wherein the anti-latent TGFβ-1 antibody or immunoconjugate is administered simultaneously with the pharmaceutical preparation.

[0123] D5. The pharmaceutical preparation of any one of D1 to D3, wherein the anti-latent TGFβ-1 antibody or immunoconjugate is administered before or after administration of the pharmaceutical preparation.

[0124] E1. A method of treating an individual having fibrosis or cancer, comprising administering to the individual an effective amount of an anti-latent TGFβ-1 antibody of any one of A1 to A24 or an immunoconjugate of A25.

[0125] E2. The method of E1, further comprising administering to the individual an additional therapeutic agent, preferably an immune checkpoint inhibitor.

[0126] E3. The method of E1 or E2, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0127] E4. The method of E3, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0128] E5. The method of any one of E1 to E4, wherein the immune checkpoint inhibitor is administered simultaneously with the anti-latent TGFβ-1 antibody or immunoconjugate.

[0129] E6. The method of any one of E1 to E4, wherein the immune checkpoint inhibitor is administered before or after administration of the anti-latent TGFβ-1 antibody or immunoconjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] [ Figure 1A ] Figure 1A Results of antibody binding to cell surface latent TGF-β1 on BaF3 cells are shown. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191 and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0131] [ Figure 1B ] Figure 1B Shows the antibody and FreeStyle TM Results of cell surface latent TGF-β1 binding on 293-F cells. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191 and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0132] [ Figure 2A ] Figure 2A Results showing antibody binding to mouse mature TGF-β1. GC1008-F1332m represents an anti-mature TGF-β antibody as a positive control. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0133] [ Figure 2B ] Figure 2B Results showing antibody binding to human mature TGF-β1. GC1008-F1332m represents an anti-mature TGF-β antibody as a positive control. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0134] [ Figure 2C ] Figure 2C Results of antibody binding to mouse LAP are shown. GC1008-F1332m represents an anti-mature TGF-β antibody. IC17-hIgG1 represents an anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0135] [ Figure 3A ] Figure 3A Results of antibody activity against spontaneous mouse latent TGF-β1 activation are shown. mSLC represents recombinant mouse latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0136] [ Figure 3B ] Figure 3B Results of antibody activity against spontaneous human latent TGF-β1 activation are shown. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0137] [ Figure 4A ] Figure 4AResults of antibody activity against plasmin (PLN)-mediated activation of mouse latent TGF-β1 are shown. mSLC represents recombinant mouse latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0138] [ Figure 4B ] Figure 4B Results of antibody activity against plasmin (PLN)-mediated activation of latent human TGF-β1 are shown. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0139] [ Figure 5A ] Figure 5A Results for antibody activity against kallikrein (PLK)-mediated activation of latent mouse TGF-β1 are shown. mSLC represents recombinant mouse latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0140] [ Figure 5B ] Figure 5B Results for antibody activity against kallikrein (PLK)-mediated activation of latent human TGF-β1 are shown. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0141] [ Fig. 6A ] Fig. 6AResults are shown for antibody activity against matrix metalloproteinase (MMP) 2-mediated activation of human latent TGF-β1. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0142] [ Figure 6B ] Figure 6B Results of antibody activity against matrix metalloproteinase (MMP) 9-mediated activation of latent human TGF-β1 are shown. hSLC represents recombinant human latent TGF-β1. IC17-hIgG1 represents anti-KLH antibody as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0143] [ Fig. 7A ] Fig. 7A Results of antibody activity against plasmin (PLN)-mediated cleavage of latent mouse TGF-β1 are shown. Cam stands for camostat, a protease inhibitor used as a control. AE04 (hT0947AE04-SG191), AE07 (hT0947AE07-SG191), AE08 (hT0947AE08-SG191), and AE09 (hT0947AE09-SG191) represent anti-latent TGF-β1 antibodies.

[0144] [ Figure 7B ] Figure 7B Results are shown for antibody activity against plasmin (PLN)-mediated cleavage of latent human TGF-β1. Cam stands for camostat, a protease inhibitor used as a control. AE04 (hT0947AE04-SG191), AE07 (hT0947AE07-SG191), AE08 (hT0947AE08-SG191), and AE09 (hT0947AE09-SG191) represent anti-latent TGF-β1 antibodies.

[0145] [ Figure 8 ] Figure 8Results are shown for antibody activity against integrin-mediated mouse TGF-β1 activation in mouse PBMCs. IC17-hIgG1 represents anti-KLH antibody as a negative control. GC1008-F1332m represents anti-mature TGF-β antibody as a positive control. RGE represents RGE peptide. RGD represents RGD peptide as a positive control. hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191 represent anti-latent TGF-β1 antibodies.

[0146] [ Fig. 9 ] Fig. 9 Tumor growth curves of the isotype control treatment group (solid circles), anti-PD-L1 treatment group (solid squares), hT0947AE04-mF18 treatment group (solid triangles), and hT0947AE04-mF18 combined with anti-PD-L1 treatment group (crosses) are shown. Each point represents the mean tumor volume of each group. (N=10)

[0147] [ Fig.10 ] Fig.10 Tumor growth curves (crosses) are shown for the isotype control treatment group (solid circles), anti-PD-L1 treatment group (solid squares), hT0947AE04-mF18 treatment group (solid triangles), and hT0947AE04-mF18 combined with anti-PD-L1 treatment group. Each point represents the mean tumor volume of each group. (N=10)

[0148] [ Fig.11 ] Fig.11 Tumor growth curves of the vehicle-treated group (solid circles), anti-PD-L1-treated group (solid squares), hT0947AE04-mF18 combined with anti-PD-L1-treated group (X), hT0947AE07-SG181 combined with anti-PD-L1-treated group (solid triangles), and hT0947AE08-SG181 combined with anti-PD-L1-treated group (open circles) are shown. Each point represents the mean tumor volume of each group. (N=10)

[0149] [ Fig.12 ] Fig.12 Results for hydroxyproline content in kidney are shown. The monoclonal antibodies were evaluated in a mouse model of renal fibrosis induced by unilateral ureteral obstruction (UUO). The sham group represents a non-disease-induced control group. IC17dk-SG181 is an anti-KLH antibody used as a negative control. hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191 represent anti-latent TGF-β1 antibodies.

[0150] [ Fig.13 ] Fig.13 Tumor growth curves of the vehicle-treated group (solid circle), anti-mouse PD-L1 antibody-treated group (solid square), hT0947AE07-SG191 (10 mg / kg) combined with anti-mouse PD-L1 antibody-treated group (solid triangle), and hT0947AE07-SG191 (30 mg / kg) combined with anti-mouse PD-L1 antibody-treated group (open circle) are shown. Each point represents the average tumor volume of the corresponding group. (N=10 per group) DETAILED DESCRIPTION

[0151] I. Definitions

[0152] "Acceptor human framework" for the purposes of this article 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. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework has identity in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0153] The term "binding activity" refers to the strength of the sum of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In this article, "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 the members of the binding pair reflect a monovalent 1:1 interaction, the binding activity is particularly referred to as intrinsic binding affinity. When the members of the binding pair are capable of monovalent binding and multivalent binding, the binding activity is the sum of each binding strength. The binding activity of a molecule X and its partner Y can generally be represented by a dissociation constant (KD) or "analyte binding amount per unit amount of ligand" (hereinafter referred to as "binding amount"). It will be understood by those skilled in the art that, in general, the lower the value of the dissociation constant (KD), the higher the binding activity, and the higher the value of "analyte binding amount per unit amount of ligand" or "binding amount", the higher the binding activity. The binding activity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

[0154] "Binding activity matured", "affinity matured" antigen-binding molecule or antibody, "binding activity increased (enhanced)", or "affinity increased (enhanced)" antigen-binding molecule or antibody refers to an antibody with one or more changes (e.g., substitutions) in one or more hypervariable regions (HVRs), which result in an increase in the antigen-binding activity of the antigen-binding molecule or antibody to the antigen, compared to a parent antigen-binding molecule or parent antibody that does not carry such changes.

[0155] The terms "anti-latent TGF-β1 antibody" and "antibody that binds to latent TGF-β1" refer to an antibody that is capable of binding to latent TGF-β1 with sufficient binding activity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting latent TGF-β1. In one embodiment, an "antibody that binds to latent TGF-β1" is an antibody that specifically binds to latent TGF-β1. In one embodiment, the extent of binding activity of the anti-latent TGF-β1 antibody to unrelated, non-latent TGF-β1 proteins is less than about 10% of the binding activity of the antibody to latent TGF-β1, for example, as measured by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (KD) of the antibody that binds to TGF-β1 is 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 smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M). In certain embodiments, the anti-latent TGF-β1 antibody binds to a latent TGF-β1 epitope that is conserved among latent TGF-β1 from different species.

[0156] The term "antibody" is used in the broadest sense herein and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen binding activity. The term "antibody" also includes any antigen binding molecule comprising an immunoglobulin variable heavy chain and / or variable light chain structure.

[0157] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen that 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.

[0158] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0159] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is generally characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative diseases, and various types of head and neck cancer. In one example, the cancer is resistant to immune checkpoint inhibitors and / or has limited response to immune checkpoint inhibitors.

[0160] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is from a different source or species.

[0161] The "class" of an antibody refers to the type of constant domain or constant region possessed by the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0162] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 212radioisotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0163] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0164] An "effective amount" of an agent (eg, a pharmaceutical formulation) is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0165] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Immunological Target Protein Sequences, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0166] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, in VH (or VL), HVR and FR sequences usually appear in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0167] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0168] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the originally transformed cell are included herein.

[0169] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0170] "People's consensus framework" is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, human immunoglobulin VL or VH sequences are from a subgroup of variable domain sequences. Typically, the sequence subgroup is a subgroup such as Kabat et al., Immunology Target Protein Sequences, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI such as Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III such as Kabat et al. (supra).

[0171] 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, wherein 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 may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

[0172] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain whose sequence has high variability ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Typically, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0173] (a) Hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0174] (b) CDRs occur at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Protein Sequences of Immunological Targets, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0175] (c) Antigenic contacts occur at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

[0176] (d) A combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0177] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0178] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0179] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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.

[0180] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0181] 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 that is different from its natural chromosomal location.

[0182] "Isolated nucleic acid encoding an anti-latent TGF-β1 antibody" or "nucleic acid encoding an anti-latent TGF-β1 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), such nucleic acid molecules are included in a single vector or in separate vectors, and such nucleic acid molecules are present at one or more locations in a host cell.

[0183] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody group, that is, the individual antibodies constituting the group are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or mutations generated during the production process of monoclonal antibody preparations, such variants are usually present in small amounts. In contrast to polyclonal antibody preparations (which typically include different antibodies for different determinants (epitopes)), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody group, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies described herein.

[0184] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0185] "Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From N-terminus 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-terminus 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 chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be designated as one of two types, called κ (kappa) and λ (lambda).

[0186] 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.

[0187] "Percentage (%) of amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the skill of the art (e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.)). One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0188] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington, D.C. 20559, and it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. in South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. In the case of using ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity % of a given amino acid sequence A to, with or for a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a specific amino acid sequence identity % to, with or for a given amino acid sequence B) is calculated as follows:

[0189] 100 times the fraction X / Y

[0190] 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 when 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 be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.

[0191] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and which contains no additional components which would be unacceptably toxic to a subject to which the preparation would be administered.

[0192] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0193] Unless otherwise indicated, the term "TGF-β1" as used herein 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 produced by processing in cells. The term also encompasses naturally occurring TGF-β1 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human TGF-β1 preproprotein is shown in SEQ ID NO: 68 (NCBI RefSeq: NP_000651.3), and the nucleic acid sequence encoding an exemplary human TGF-β1 is shown in SEQ ID NO: 69 (NCBI RefSeq: NM_000660.6). The amino acid sequence of an exemplary mouse TGF-β1 preproprotein is shown in SEQ ID NO: 70 (NCBI RefSeq: NP_035707.1), and the nucleic acid sequence encoding an exemplary mouse TGF-β1 is shown in SEQ ID NO: 71 (NCBI Reference Sequence: NM_011577.2). The amino acid sequence of an exemplary cynomolgus monkey TGF-β1 preproprotein is shown in SEQ ID NO: 72 (NCBI RefSeq: XP_005589396.1), and the nucleic acid sequence encoding an exemplary cynomolgus monkey TGF-β1 is shown in SEQ ID NO: 73 (NCBI RefSeq: XM_005589339.2). The term "TGF-β1" includes latent TGF-β1 and mature TGF-β1.

[0194] As used herein, the term "latent TGF-β1" refers to any TGF-β1 that forms a latent TGF-β1 complex ("latent cell surface TGF-β1", LLC or SLC (see below)) and / or is unable to bind to its receptor. Transforming growth factor-β1 (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 that interacts with its latency-associated peptide (LAP) and latent TGF-β binding protein (LTBP) to form a larger complex, called the large latent complex (LLC). The amino acid sequence of an exemplary latent human TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30-390 of SEQ ID NO: 68. An exemplary amino acid sequence of mouse latent TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30-390 of SEQ ID NO: 70. An exemplary amino acid sequence of cynomolgus monkey latent TGF-β1 (TGF-β homodimer and its LAP) is amino acids 30-390 of SEQ ID NO: 72.

[0195] The complex formed by the TGF-β homodimer and its LAP is called the small latent complex (SLC). This latent complex keeps TGF-β in an inactive form and cannot bind to its receptor. SLC can be covalently linked to another protein, the latent TGF-β binding protein (LTBP), to form a large latent complex (LLC). Four different LTBP isoforms are known, LTBP-1, LTBP-2, LTBP-3 and LTBP-4. LTBP-1, LTBP-3 and LTBP-4 have been reported to bind to SLC (see, e.g., Rifkin et al., J Biol Chem. 2005 Mar 4; 280(9):7409-12). SLC can also be covalently linked to other additional proteins, such as glycoprotein A repeat dominant protein (GARP) or leucine-rich repeat 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). For LLC, LLC has been reported to covalently associate with the extracellular matrix (ECM) through the N-terminus of LTBP (see, e.g., Saharinen et al., Cytokine Growth Factor Rev. 1999 Jun;10(2):99-117.). In some embodiments, latent TGF-β1 associated with the ECM on the cell surface is referred to as "cell surface latent TGF-β1".

[0196] As used herein, the term "active TGF-β1", "mature TGF-β1" or "active mature TGF-β1" 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 the release of LLC from the ECM, followed by further proteolysis of LAP to release active TGF-β to its receptor. A wide range of proteases are known, including plasmin (PLN), prekallikrein (PLK), matrix metalloproteinase (MMP) 2, MMP9, MMP13, MMP14, thrombin, tryptase and calpain, which can 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 floor-spondin (F-spondin) can also activate latent TGF-β. Alternatively, upon mechanical extension, integrins (preferably integrin αVβ8 and / or integrin αVβ6) can activate TGF-β by binding to the RGD motif present in LAP and inducing the release of mature TGF-β from its latent complex form.

[0197] As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be used for prevention or performed during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease development or slow disease progression.

[0198] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies 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, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be separated using VH or VL domains from antibodies that bind to the antigen to screen libraries 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).

[0199] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0200] II. Compositions and Methods

[0201] In one aspect, the present invention is based in part on anti-latent TGF-β1 antibodies and their uses. In certain embodiments, antibodies that bind to TGF-β1 are provided. The antibodies of the present invention can be used, for example, to diagnose or treat fibrosis, preferably myocardial fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, skin fibrosis, ocular fibrosis and bone marrow fibrosis. The antibodies of the present invention can also be used, for example, for the diagnosis or treatment of cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, lymphoproliferative diseases such as leukemia, and various types of head and neck cancer.

[0202] A. Exemplary Anti-Latent TGF-β1 Antibodies

[0203] In one aspect, the invention provides an isolated antibody that binds to latent TGF-β1. In a further embodiment, the anti-latent TGF-β1 antibody binds to the latency associated protein (LAP) region of latent TGF-β1. An example of a LAP region comprises amino acids 30-278 of human TGF-β1 preproprotein (SEQ ID NO: 1). As described above, LAP is a component of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody is present at 10 -8 nM or less, 10 -9 nM or less, or 10 -10 Binds to latent TGF-β1 with a dissociation constant (KD) of nM or less.

[0204] In one aspect, the anti-latent TGF-β1 antibody binds to a complex formed with latent TGF-β1, LLC, and / or latent TGF-β1 and GARP or LRRC33. In certain embodiments, the anti-latent TGF-β1 antibody binds to latent TGF-β1 on the cell surface, which is latent TGF-β1 associated with the extracellular matrix (ECM) on the cell surface. On the other hand, the anti-latent TGF-β1 antibody binds to latent TGF-β1, wherein the LAP region of latent TGF-β1 is not connected to LTBP to form a small latent complex (SLC). In certain embodiments, the SLC exists in a soluble form. In some embodiments, the anti-latent TGF-β1 antibody is present in a soluble form at 10 -8 nM or less, 10 -9 nM or less, or 10 -10 Binds to latent TGF-β1 (latent TGF-β1, LLC or SLC on the cell surface) with a dissociation constant (KD) of nM or less.

[0205] On the one hand, anti-latent TGF-β1 antibodies inhibit latent TGF-β1 activation. 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. Latent TGF-β1 activation 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, anti-latent TGF-β1 antibodies inhibit the release of mature TGF-β1 from latent TGF-β1. As described above, mature TGF-β1 has been reported to be released from latent TGF-β1 by activators (e.g., proteases, integrins, and other non-protease activators). Non-limiting examples of proteases that activate latent TGF-β1 include plasmin (PLN), prekallikrein (PLK), matrix metalloproteinase (MMP) 2, and MMP9. In some embodiments, 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, resulting in the release of mature TGF-β1. In some embodiments, the cleavage site of PLN and / or PLK is located within a fragment consisting of amino acids 56-59 of the LAP polypeptide.

[0206] On the one hand, the anti-latent TGF-β1 antibody inhibits the protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibiting the protease-mediated cleavage of the LAP portion of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody inhibits the protease-mediated release of mature TGF-β1 from latent TGF-β1 and allows the protease to cleave the LAP region, while the anti-latent TGF-β1 antibody binds to the LAP region of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody does not block the protease from approaching the latent TGF-β1, especially the cleavage site of PLN and / or PLK. In other embodiments, the anti-latent TGF-β1 antibody does not bind to the protease cleavage site of the LAP portion of latent TGF-β1, especially the cleavage site of PLN and / or PLK.

[0207] In some embodiments, the anti-latent TGF-β1 antibody that inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1 is an antibody that (i) inhibits cleavage of the LAP region mediated by one or more proteases, but (ii) does not inhibit cleavage of the LAP region mediated by other proteases. For example, the anti-latent TGF-β1 antibody (1-i) inhibits MMP2- and / or MMP9-mediated release of mature TGF-β1 by inhibiting MMP2- and / or MMP9-mediated cleavage of the LAP portion of latent TGF-β1, and (1-ii) inhibits PLN- and / or PLK-mediated release of mature TGF-β1 without inhibiting PLN- and / or PLK-mediated cleavage of the LAP portion of latent TGF-β1. Alternatively, the anti-latent TGF-β1 antibody (2-i) inhibits PLN- and / or PLK-mediated release of mature TGF-β1 by inhibiting PLN- and / or PLK-mediated cleavage of the LAP portion of latent TGF-β1, and (2-ii) inhibits MMP2- and / or MMP9-mediated release of mature TGF-β1 without inhibiting MMP2- and / or MMP9-mediated cleavage of the LAP portion of latent TGF-β1. Alternatively, the anti-latent TGF-β1 antibody (3-i) inhibits PLN- and / or PLK-mediated release of mature TGF-β1 without inhibiting PLN- and / or PLK-mediated cleavage of the LAP portion of latent TGF-β1, and (3-ii) inhibits MMP2- and / or MMP9-mediated release of mature TGF-β1 without inhibiting MMP2- and / or MMP9-mediated cleavage of the LAP portion of latent TGF-β1.

[0208] In some embodiments, antibodies that "inhibit activation of latent TGF-β1" include antibodies that cause a reduction in TGF-β1 activation of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more. In other embodiments, antibodies that "inhibit protease-mediated release of mature TGF-β1 from latent TGF-β1" include antibodies that cause a reduction in protease-mediated release of mature TGF-β1 from latent TGF-β1 of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% or more. In a further embodiment, antibodies that inhibit protease-mediated release of mature TGF-β1 from latent TGF-β1 "without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1" include antibodies that cause a 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less reduction in protease-mediated cleavage of the LAP region of latent TGF-β1.

[0209] In some embodiments, the anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1. When the anti-latent TGF-β1 antibody "stabilizes" the structure of the LAP region, as used herein, the LAP region surrounded by the anti-latent TGF-β1 antibody remains in a specific structure, whereby mature TGF-β1 cannot be released. In a further embodiment, the latent TGF-β1 stabilized by the anti-latent TGF-β1 antibody can be activated by integrins (preferably integrin αVβ8 and / or integrin αVβ6). In certain embodiments, the LAP region stabilized by the anti-latent TGF-β1 antibody has been cleaved or not cleaved by a protease. In some embodiments, the anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of the latent TGF-β1 and allows the protease to cleave the LAP region while the anti-latent TGF-β1 antibody binds to the LAP region of the latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1 without blocking proteases from accessing latent TGF-β1, especially PLN and / or PLK cleavage sites. In other embodiments, the anti-latent TGF-β1 antibody stabilizes the structure of the LAP region of latent TGF-β1 without blocking proteases from accessing latent TGF-β1, especially MMP2 and / or MMP9 cleavage sites.

[0210] In one aspect, the anti-latent TGF-β1 antibody does not bind to mature TGF-β1. In some embodiments, the anti-latent TGF-β1 antibody binds to latent TGF-β1 with a higher binding activity than mature TGF-β1. In certain embodiments, the antibodies of the invention bind to latent TGF-β1 with a binding activity that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times higher than mature TGF-β1.

[0211] In one aspect, the anti-latent TGF-β1 antibody does not inhibit or does not significantly inhibit integrin-mediated TGF-β1 activation, i.e., integrin-mediated release of mature TGF-β1 from latent TGF-β1. Preferably, the integrin herein is integrin αVβ8 and / or integrin αVβ6. In some embodiments, antibodies that "do not inhibit or do not significantly inhibit integrin-mediated TGF-β1 activation" include antibodies that cause integrin-mediated TGF-β1 activation (i.e., integrin-mediated release of mature TGF-β1 from latent TGF-β1) to be reduced by 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less.

[0212] In one aspect, anti-latent TGF-β1 antibodies result in reduced or decreased toxicity and / or side effects associated with anti-TGF-β antagonists. In some embodiments, anti-latent TGF-β1 antibodies, such as those described herein, have superior safety-efficacy characteristics compared to agents that induce activity on mature TGF-β1 or agents that induce activity on latent TGF-β1 but inhibit protease- and integrin-mediated activation of latent TGF-β1. In some embodiments, the anti-latent TGF-β1 antibodies of the present disclosure have reduced cardiotoxicity while having better or comparable efficacy than anti-mature TGF-β1 antibodies. Without being bound by any theory, the anti-latent TGF-β1 antibodies of the present disclosure do not inhibit or significantly inhibit integrin-mediated TGF-β1 activation, and therefore have reduced or decreased toxicity and / or side effects caused by: (i) integrin-mediated TGF-β1 activation or (ii) inhibition of TGF-β1 signaling at sites where TGF-β1 is activated by integrins. Therefore, the anti-latent TGF-β1 antibodies of the present disclosure can be administered to subjects in need at therapeutically effective doses without causing side effects, especially cardiac toxicity. Therefore, this method will expand the dose range within which patients can obtain efficacy and safety / tolerance. Therefore, the present invention provides a method for treating a disease associated with TGF-β1 signaling by administering to a subject an effective amount of an anti-latent TGF-β1 antibody that does not inhibit or does not significantly inhibit integrin-mediated TGF-β1 activation. The present invention encompasses the use of anti-latent TGF-β1 antibodies for reducing toxicity and / or side effects associated with TGF-β1 inhibition in a subject. In some embodiments, toxicity and / or side effects may 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, for example, bleeding, inflammation, degeneration, and proliferation of valvular interstitial cells. In some embodiments, toxicity and / or side effects may include bleeding. In some embodiments, toxicity and / or side effects may include skin lesions or tumors. In some embodiments, toxicity and / or side effects may include tumor progression.

[0213] In some embodiments, the anti-latent TGF-β1 antibodies of the invention:

[0214] Binds to latent TGF-β1;

[0215] Forms SLC binding with latent TGF-β1;

[0216] Forms LLC binding with latent TGF-β1;

[0217] Binds to the complex formed by latent TGF-β1 and GARP or LRRC33;

[0218] Binds to latent TGF-β1 on the cell surface;

[0219] Binds to the LAP region of latent TGF-β1;

[0220] Combined with LAP;

[0221] Take 10 -8 nM or lower, 10 -9 nM or lower, or 10 -10 Binds to latent TGF-β1 with a dissociation constant (KD) of nM or lower;

[0222] Inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1;

[0223] It does not inhibit protease-mediated cleavage of the LAP region of latent TGF-β1;

[0224] does not inhibit or does not significantly inhibit integrin-mediated release of mature TGF-β1 from latent TGF-β1; and / or

[0225] Resulting in reduced or decreased toxicity and / or side effects associated with anti-TGF-β1 antagonists (eg, anti-mature TGF-β antibodies).

[0226] In a further embodiment, the anti-latent TGF-β1 antibody of the invention is:

[0227] Monoclonal antibodies;

[0228] Human, humanized or chimeric antibodies;

[0229] Full length IgG antibody; and / or

[0230] Antibody fragments.

[0231] In one aspect, the invention provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five or six HVRs selected from:

[0232] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20;

[0233] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21;

[0234] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22;

[0235] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23;

[0236] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and

[0237] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25.

[0238] In one aspect, the invention provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five or six HVRs selected from:

[0239] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26;

[0240] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27;

[0241] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28;

[0242] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29;

[0243] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and

[0244] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31.

[0245] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising at least one, two, three, four, five or six HVRs selected from the following:

[0246] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32;

[0247] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33;

[0248] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34;

[0249] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35;

[0250] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and

[0251] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37.

[0252] In one aspect, the present invention provides a latent TGF-β1 antibody comprising at least one, two, three, four, five or six HVRS selected from the following:

[0253] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38;

[0254] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39;

[0255] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40;

[0256] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41;

[0257] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and

[0258] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0259] In one aspect, the present invention provides a latent TGF-β1 antibody comprising:

[0260] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20;

[0261] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21;

[0262] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22;

[0263] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23;

[0264] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; and

[0265] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25.

[0266] In one aspect, the present invention provides a latent TGF-β1 antibody comprising:

[0267] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26;

[0268] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27;

[0269] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28;

[0270] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29;

[0271] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and

[0272] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31.

[0273] In one aspect, the present invention provides an anti-latent TGF-β1 antibody, comprising:

[0274] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32;

[0275] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33;

[0276] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34;

[0277] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35;

[0278] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and

[0279] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37.

[0280] In one aspect, the present invention provides an anti-latent TGF-β1 antibody, comprising:

[0281] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38;

[0282] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39;

[0283] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40;

[0284] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41;

[0285] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and

[0286] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0287] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2 and HVR-H3 of the VH sequence shown in SEQ ID NO: 12, and HVR-L1, HVR-L2 and HVR-L3 of the VL sequence shown in SEQ ID NO: 13, wherein the HVRs are defined by (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b) and / or (c).

[0288] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2 and HVR-H3 of the VH sequence shown in SEQ ID NO: 14, and HVR-L1, HVR-L2 and HVR-L3 of the VL sequence shown in SEQ ID NO: 15, wherein the HVRs are defined by (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b) and / or (c).

[0289] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2 and HVR-H3 of the VH sequence shown in SEQ ID NO: 16, and HVR-L1, HVR-L2 and HVR-L3 of the VL sequence shown 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).

[0290] In one aspect, the present invention provides an anti-latent TGF-β1 antibody comprising HVR-H1, HVR-H2 and HVR-H3 of the VH sequence shown in SEQ ID NO: 18, and HVR-L1, HVR-L2 and HVR-L3 of the VL sequence shown in SEQ ID NO: 19, wherein the HVRs are defined by (a) Chothia; (b) Kabat; (c) MacCallum; or (d) a combination of (a), (b) and / or (c).

[0291] 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 of any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0292] In another aspect, the 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: 12, 14, 16 or 18. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-latent TGF-β1 antibody comprising the 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: 12, 14, 16 or 18. In certain embodiments, the substitutions, insertions or deletions occur in regions outside of the HVR (i.e., in the FR). Optionally, the anti-latent TGF-β1 antibody comprises a VH sequence in SEQ ID NO: 12, 14, 16 or 18, including post-translational modifications of the sequence. In a specific embodiment, VH comprises one, two or three HVRs selected from the following: (a) HVR-H1 comprising an amino acid sequence of SEQ ID NO: 20, 26, 32 or 38, (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 21, 27, 33 or 39, and (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 22, 28, 34 or 40. Post-translational modifications include, but are not limited to, modifying the glutamine or glutamic acid at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0293] In another aspect, an anti-latent TGF-β1 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13, 15, 17 or 19. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to the reference sequence, but the anti-latent TGF-β1 antibody comprising the 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: 13, 15, 17 or 19. In certain embodiments, the substitution, insertion or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-latent TGF-β1 antibody comprises a VL sequence of SEQ ID NO: 13, 15, 17 or 19, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from the following: (a) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 23, 29, 35 or 41; (b) HVR-L2 comprising an amino acid sequence of SEQ ID NO: 24, 30, 36 or 42; and (c) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 25, 31, 37 or 43. Post-translational modifications include, but are not limited to, modifying the glutamine or glutamic acid at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0294] In another aspect, an anti-latent TGF-β1 antibody is provided, wherein the antibody comprises a VH as in any embodiment provided above and a VL as in any embodiment provided above. In one embodiment, the antibody comprises the VH and VL sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifying the glutamine or glutamic acid at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0295] In one embodiment, the antibody comprises the VH and VL sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifying the glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0296] In one embodiment, the antibody comprises the VH and VL sequences shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of the glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0297] In one embodiment, the antibody comprises the VH and VL sequences shown in SEQ ID NO: 18 and SEQ ID NO: 19, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of the glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0298] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-latent TGF-β1 antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as the antibodies shown below are provided:

[0299] (1) An anti-latent TGF-β1 antibody comprising:

[0300] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20,

[0301] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21,

[0302] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22,

[0303] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23,

[0304] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and

[0305] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25;

[0306] (2) An anti-latent TGF-β1 antibody comprising:

[0307] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26,

[0308] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27,

[0309] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28,

[0310] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29,

[0311] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30, and

[0312] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31;

[0313] (3) A latent TGF-β1 antibody comprising:

[0314] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32,

[0315] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33,

[0316] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34,

[0317] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35,

[0318] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, and

[0319] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37; or

[0320] (4) A latent TGF-β1 antibody comprising:

[0321] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38,

[0322] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39,

[0323] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40,

[0324] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41,

[0325] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and

[0326] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0327] In another aspect, the present invention provides antibodies that bind to latent TGF-β1 of humans, monkeys, mice and / or rats. In certain embodiments, the present invention provides antibodies that bind to latent TGF-β1 of humans, monkeys and mice. In certain embodiments, the present invention provides antibodies that form SLC binding with latent TGF-β1 of humans, monkeys and mice. In certain embodiments, the present invention provides antibodies that form LLC binding with latent TGF-β1 of humans, monkeys and mice. In certain embodiments, the present invention provides antibodies that form LLC binding with latent TGF-β1 of humans, monkeys and mice. In certain embodiments, the present invention provides antibodies that bind to the complex formed by latent TGF-β1 of humans, monkeys and mice and GARP or LRRC33. In certain embodiments, the present invention provides antibodies that bind to latent TGF-β1 on the cell surface of humans, monkeys and mice.

[0328] In another aspect, the invention provides antibodies that bind to the same epitope as any of the anti-latent TGF-β1 antibodies provided herein. The epitope may be present on TGF-β1 of humans, monkeys, mice and / or rats. For example, in certain embodiments, the invention provides antibodies that bind to the same epitope as a reference antibody, wherein the reference antibody is:

[0329] (1) An anti-latent TGF-β1 antibody comprising:

[0330] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20,

[0331] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21,

[0332] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22,

[0333] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23,

[0334] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and

[0335] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25;

[0336] (2) An anti-latent TGF-β1 antibody comprising:

[0337] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26,

[0338] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27,

[0339] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28,

[0340] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29,

[0341] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30, and

[0342] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31;

[0343] (3) An anti-latent TGF-β1 antibody comprising:

[0344] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32,

[0345] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33,

[0346] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34,

[0347] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35,

[0348] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, and

[0349] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37; or

[0350] (4) An anti-latent TGF-β1 antibody comprising:

[0351] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38,

[0352] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39,

[0353] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40,

[0354] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41,

[0355] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and

[0356] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0357] In another aspect, the present invention provides antibodies that compete with the anti-latent TGF-β1 antibodies provided herein for binding to human, monkey, mouse and / or rat TGF-β1. For example, in certain embodiments, the antibodies provided compete with the following antibodies for binding to human, monkey, mouse and / or rat TGF-β1:

[0358] (1) An anti-latent TGF-β1 antibody comprising:

[0359] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20,

[0360] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21,

[0361] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22,

[0362] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23,

[0363] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and

[0364] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25;

[0365] (2) An anti-latent TGF-β1 antibody comprising:

[0366] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26,

[0367] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27,

[0368] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28,

[0369] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29,

[0370] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30, and

[0371] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31;

[0372] (3) An anti-latent TGF-β1 antibody comprising:

[0373] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32,

[0374] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33,

[0375] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34,

[0376] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35,

[0377] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36, and

[0378] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37; or

[0379] (4) An anti-latent TGF-β1 antibody comprising:

[0380] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38,

[0381] (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39,

[0382] (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40,

[0383] (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41,

[0384] (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and

[0385] (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0386] In another aspect of the invention, the anti-latent TGF-β1 antibody according to any one of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody or a human antibody. In one embodiment, the anti-latent TGF-β1 antibody is an antibody fragment, for example, Fv, Fab, Fab', scFv, diabody or F(ab') 2In another embodiment, the antibody is a full-length antibody, for example, a complete IgG1, IgG2, IgG3 or IgG4 antibody or other antibody classes or isotypes defined herein. In another aspect, the anti-latent TGF-β1 antibody also includes any antigen binding molecule comprising an immunoglobulin variable heavy chain and / or variable light chain structure.

[0387] In yet another aspect, the anti-latent TGF-β1 antibody according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0388] 1. Antibody Binding Activity

[0389] 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 smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0390] In one embodiment, the binding activity of the antibody is measured by a radiolabeled antigen binding assay (RIA) and represented by KD. In one embodiment, the RIA is performed using a Fab form of the antibody of interest and its antigen. For example, the solution binding activity of the Fab for the antigen is measured by titrating the antibody with a minimum concentration of ( 125 I) Label the antigen to equilibrate the Fab, and then capture the bound antigen with an anti-Fab antibody-coated plate for measurement (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 (CappelLabs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 degrees Celsius (°C)). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I]-antigen is mixed with a serial dilution of the target Fab (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12, Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure that 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 discarded and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plate is dry, 150 microliters / well of scintillant (MICROSCINT-20 TM; Packard) is added, and the plate is counted for tens of minutes on a TOPCOUNTTM gamma counter (Packard). Concentrations of each Fab that produce less than or equal to 20% of maximum binding are selected for competitive binding assays.

[0391] In one embodiment, to measure the binding activity of the 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 surface plasmon resonance analysis as the measuring principle. BIACORE (registered trademark) control software is used for the operation of the device. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the manufacturer's instructions to immobilize molecules for ligand capture, for example, anti-tag antibodies, anti-IgG antibodies, protein A, etc. on a sensor chip coated with carboxymethyl dextran (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH value and injected at an appropriate flow rate and for an appropriate injection time. Binding activity measurement uses a buffer containing 0.05% polysorbate 20 (also known as Tween (registered trademark)-20) as a measurement buffer, the flow rate is 10-30 μl / min, and the measurement temperature is preferably at 25 degrees Celsius or 37 degrees Celsius. For measurements with antibodies captured by ligand capture molecules as ligands, antibodies are injected so that the target amount of antibodies is captured, and then serial dilutions of antigens and / or Fc receptors (analytes) prepared using measurement buffer are injected. For measurements with antigens and / or Fc receptors captured by ligand capture molecules as ligands, antigens and / or Fc receptors are injected so that the target amount of them is captured, and then serial dilutions of antibodies (analytes) prepared using measurement buffer are injected.

[0392] In one embodiment, the measurement results are analyzed using BIACORE (registered trademark) evaluation software. Kinetic parameter calculation is 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 where the binding activity is weak, especially when the dissociation is fast and the kinetic parameters are difficult to calculate, the equilibrium dissociation constant (KD) can be calculated using a steady-state model. As an additional parameter for binding activity, the "binding amount of the analyte per unit amount of ligand" can be calculated by dividing the binding amount of the analyte at a specific concentration (resonance unit: RU) by the amount of captured ligand.

[0393] 2. Antibody fragments

[0394] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fv and scFv fragments, as well as other fragments described below. For a review of 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. Pat. Nos. 5,571,894 and 5,587,458. For a review of Fab and F(ab') fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life 2 For a discussion of fragments, see U.S. Patent No. 5,869,046.

[0395] 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). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0396] A single domain antibody is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 Bl).

[0397] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0398] The present invention also relates to antigen binding molecules that bind to TGF-β1, including but not limited to, for example, miniantibodies (low molecular weight antibodies) and scaffold proteins. In the present invention, any scaffold protein is acceptable as long as it is a peptide having a stable three-dimensional structure and capable of binding at least to an antigen. Such peptides include, for example, antibody variable regions, fibronectin, protein A domains, LDL receptor A domains, lipocalin, and fragments of 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 referring 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".

[0399] 3. Chimeric and humanized antibodies

[0400] In certain embodiments, the antibodies provided herein are chimeric antibodies. 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 (e.g., 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. Chimeric antibodies include antigen-binding fragments thereof.

[0401] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and binding activity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs, such as CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or binding activity.

[0402] Humanized antibodies and methods of making the same are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in 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 specific determinant region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "guided selection" method of FR shuffling).

[0403] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); 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 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)).

[0404] 4. Human Antibodies

[0405] 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 routinely described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0406] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or is present outside the chromosome or randomly integrated into the animal chromosome. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describe XENOMOUSE TMTECHNOLOGY; U.S. Patent No. 5,770,429 describes the HUMAB (registered trademark) technology; U.S. Patent No. 7,041,870 describes the K-M MOUSE (registered trademark) technology, and U.S. Patent Application Publication No. US 2007 / 0061900 describes the VELOCIMOUSE (registered trademark) technology. The human variable regions from intact antibodies produced by such animals can be further modified, e.g., by combination with different human constant regions.

[0407] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell 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, e.g., the methods described in U.S. Patent No. 7,189,826 (describing the 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).

[0408] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0409] 5. Antibodies from the library

[0410] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Mol. Biol. 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and, for example, in McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Mol. Biol. 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).

[0411] In certain phage display methods, VH and VL gene libraries are cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments in the form of single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high binding activity antibodies against immunogens without the need to construct hybridomas. Alternatively, the initial library can be cloned (e.g., from humans) to provide a single source of antibodies against a variety of non-self and self antigens without any immunization, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, the initial library can also be prepared by cloning unrearranged V gene segments from stem cells, and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patents describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0412] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0413] 6. Multispecific Antibodies

[0414] In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites. In certain embodiments, one of the binding specificities is directed to TGF-β1 and the other is directed to any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of TGF-β1. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing TGF-β1. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0415] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305: 537 (1983)), WO 93 / 08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments (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., 152:5368-5476 (1994)). (1994)); and preparing trispecific antibodies, for example as described in Tutt et al. J. Immunol. 147: 60 (1991).

[0416] Engineered antibodies having three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576A1).

[0417] The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs," which comprise an antigen binding site that binds to TGF-β1 as well as another different antigen (eg, see, US 2008 / 0069820).

[0418] 7. Antibody variants

[0419] In certain embodiments, it is contemplated that the amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to improve the binding activity and / or other biological properties of the antibodies. The amino acid sequence variants of the antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibodies or by peptide synthesis. Such modifications include, for example, the deletion and / or insertion and / or substitution of residues in the antibody amino acid sequence. Any combination of deletion, insertion and substitution can be performed to reach the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0420] a) Substitution, insertion and deletion variants

[0421] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVR and FR. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1, and are further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product with the desired activity is screened, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0422] (Table 1)

[0423]

[0424] Amino acids can be grouped according to common side chain properties:

[0425] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0426] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0427] (3) Acidic: Asp, Glu;

[0428] (4) Basic: His, Lys, Arg;

[0429] (5) Residues that affect chain orientation: Gly, Pro;

[0430] (6) Aromaticity: Trp, Tyr, Phe.

[0431] Non-conservative substitutions will entail exchanging a member of one of these classes for another.

[0432] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased binding activity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are antibodies that are matured in binding activity, which can be conveniently produced, for example, using phage display-based binding activity maturation techniques, such as those described herein. In brief, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding activity).

[0433] HVRs may be altered (e.g., substituted), for example, to improve antibody binding activity. Such changes may be made to HVR "hot spots", i.e., residues encoded by codons that undergo high frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigens, and the resulting variant VH or VL may be tested for binding activity. For example, Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)) have been described in the maturation of binding activity by construction and reselection from a secondary library. In some embodiments of binding activity maturation, diversity is introduced into the variable gene 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 constructed. The library is then screened to identify any antibody variant with the desired binding activity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0434] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not significantly reduce binding activity may be made to HVRs. For example, such changes may be outside of antigen contact residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two or three amino acid substitutions.

[0435] As described in Cunningham and Wells (1989) Science, 244: 1081-1085, a useful method for identifying residues or regions of antibodies that can be used as mutagenesis targets is called "alanine scanning mutagenesis". In this method, a residue or a group of target residues (e.g., charged residues, such as arg, asp, his, lys and glu) are identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex can be analyzed to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain desired properties.

[0436] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include enzymes that increase the plasma half-life of the antibody (e.g., for ADEPT) or fusions of polypeptides to the N-terminus or C-terminus of the antibody.

[0437] b) Glycosylation variants

[0438] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites in antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0439] In the case where the antibody comprises an Fc region, the carbohydrate attached thereto can be changed. The natural antibody produced by mammalian cells generally comprises a branched biantennary oligosaccharide, which is generally attached to the Asn297 of the CH2 domain of the Fc region by an N bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharide in the antibody of the present invention can be modified to produce antibody variants with some improved properties.

[0440] In one embodiment, an antibody variant having a carbohydrate structure is provided that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 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, due to minor sequence variations in antibodies, Asn297 may also be located about + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of disclosures relating to "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; WO2005 / 035778; WO2005 / 053742; WO2002 / 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 defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0441] Further provided are antibody variants with bisecting oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, 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 having at least one galactose residue in the oligosaccharides attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0442] c) Fc region variants

[0443] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variant can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. In another embodiment, the human Fc variant can include a chimeric human Fc region sequence (e.g., human IgG1 / 4 or human IgG2 / 4 Fc region), or a chimeric human Fc region sequence, which further includes an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0444] In certain embodiments, the present invention contemplates antibody variants with some but not all effector functions, making them ideal candidates for certain applications, where the antibody half-life in vivo is important but certain effector functions (e.g., complement and ADCC) are non-essential or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cell NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. 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 for evaluating ADCC activity of target molecules are described in U.S. Pat. Nos. 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 can be used (see, e.g., ACT1 by flow cytometry). TMNon-radioactive cytotoxicity assays (CellTechnology, 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 (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule can be assessed in vivo (e.g., 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, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC analysis 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)).

[0445] Antibodies with reduced effector function include antibodies with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acids 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. Pat. No. 7,332,581).

[0446] Certain antibody variants with increased or decreased binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0447] In certain embodiments, the antibody variant comprises one or more amino acid substitutions that improve ADCC, e.g., an Fc region having substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0448] In some embodiments, altering the Fc region results in altered (i.e., increased or decreased) Clq 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).

[0449] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for transferring 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 US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include variants 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, 3787 380, 382, 413, 424, or 434, e.g., a substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0450] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,82; and WO 94 / 29351.

[0451] d) Cysteine ​​engineered antibody variants

[0452] In certain embodiments, it may be desirable to produce cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In a particular embodiment, the substituted residues occur at accessible sites of the antibody. 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 further described herein, 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) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be produced as described in U.S. Patent No. 7,521,541.

[0453] e) Antibody derivatives

[0454] In certain embodiments, the antibodies provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization 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), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in production. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may be different, and if more than one polymer is attached, they may be the same or different molecules. Generally, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0455] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that do not harm ordinary cells, but heats the non-protein moiety to a temperature close to that at which cells of the antibody-non-protein moiety are killed.

[0456] B. Recombinant Methods and Compositions

[0457] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-latent TGF-β1 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of preparing 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, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0458] To recombinantly produce an anti-latent TGF-β1 antibody, nucleic acid encoding the antibody is isolated, e.g., as described above, and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0459] Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli). After expression, antibodies can be separated from bacterial cell paste as soluble fractions and can be further purified.

[0460] 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).

[0461] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0462] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0463] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Examples of other useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., 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 (MMT060562); TRI cells, e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 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 antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0464] C. Determination

[0465] 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.

[0466] 1. Binding assays and other assays

[0467] On the one hand, the antigen-binding activity of the antibody of the present invention is detected, for example, by known methods such as ELISA, Western blotting, surface plasmon resonance (eg, BIACORE (registered trademark)) or similar techniques (eg, KinExa or OCTET (registered trademark)).

[0468] In another aspect, competition assays can be used to identify antibodies that compete with any of the anti-latent TGF-β1 antibodies described herein (preferably hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) for binding to latent TGF-β1. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as any of the anti-latent TGF-β1 antibodies described herein (preferably hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). Detailed exemplary methods for epitope mapping of antibody binding are provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ). Methods for epitope mapping include, but are not limited to, X-ray crystallography and alanine scanning mutagenesis.

[0469] In certain embodiments, when such competing antibodies are present in excess, they block (e.g., reduce) the binding of the 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. 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., a linear epitope or a conformational epitope) as the anti-latent TGF-β1 antibodies described herein. In a further aspect, the reference antibody is hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191.

[0470] In an exemplary competition assay, immobilized latent TGF-β1 is incubated in a solution containing a first labeled antibody (reference antibody) (e.g., hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) that binds to latent TGF-β1 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to latent TGF-β1. The second antibody may be present in the hybridoma supernatant. As a control, immobilized latent TGF-β1 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to latent TGF-β1, excess unbound antibody is removed and the amount of label associated with immobilized latent TGF-β1 is measured. If the amount of label associated with immobilized latent TGF-β1 in the test sample is significantly reduced relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to latent TGF-β1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0471] In certain embodiments, the binding of anti-latent TGF-β1 antibodies to latent TGF-β1 on the cell surface can be detected by known methods such as ELISA, Western blot, BIAcore, flow cytometry, etc. For example, cells expressing latent TGF-β1 can be contacted with anti-latent TGF-β1 antibodies directly conjugated to PE- or APC, or with unconjugated anti-latent TGF-β1 antibodies, and then contacted with PE- or APC-conjugated secondary antibodies, and staining of latent TGF-β1 on the cell surface can be detected. See, for example, Oida et al., PLoS One. 2010 Nov 24; 5(11): e15523; Su et al., Hum Mol Genet. 2015 Jul 15; 24(14): 4024-36.

[0472] 2. Activity Assay

[0473] In one aspect, an assay for identifying an anti-latent TGF-β1 antibody having biological activity is provided. The biological activity may include, for example, inhibiting activation of latent TGF-β1, inhibiting release of mature TGF-β1 from latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 without blocking protease access to latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 while allowing protease to cleave the LAP region of latent TGF-β1, inhibiting protease-mediated release of mature TGF-β1 from latent TGF-β1 without inhibiting or partially inhibiting integrin-mediated activation of TGF-β1, etc. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0474] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0475] In some embodiments, whether the detection antibody inhibits activation of latent TGF-β1, i.e., inhibits the release of mature TGF-β1 from latent TGF-β1, is determined by contacting a latent TGF-β1 activator (e.g., a protease, an integrin, other non-protease activators, etc.) with latent TGF-β1 in the presence or absence of the detection antibody, and then 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 PBMC 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, after latent TGF-β1 is incubated with or without the detection antibody, mature TGF-β1 is detected using the above method to determine whether the detection antibody inhibits spontaneous activation of latent TGF-β1. In some embodiments, when the amount of mature TGF-β1 detected in the presence of the detection antibody (or after contact) is reduced compared to the amount detected in the absence of the detection antibody, the detection antibody is identified as an antibody that can inhibit activation of latent TGF-β1. In an example, the amount of mature TGF-β1, whether it is reduced or increased, can be measured according to 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 according to the optical density (OD) (e.g., wavelength in mm or nm, etc.) of a marker directly or indirectly associated with mature TGF-β.

[0476] In certain embodiments, inhibition of TGF-β1 activation in an assay comprises a reduction in the amount of mature TGF-β1 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 mature TGF-β1 release by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more.

[0477] In some embodiments, whether the detection 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 in cells expressing TGF-β1 receptors, for example, the activity of binding to TGF-β1 receptors, or the activity of mediating signal transduction, etc. In some embodiments, the binding of mature TGF-β1 to the TGF-β1 receptor 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 produced by transfecting cells with TGF-β1 receptor genes. For example, the HEK-Blue used in the working examples described herein can be used TM TGF-β1 cells, or those cells transiently or stably genetically modified to express a transgene encoding a TGF-β1 receptor. TGF-β1-mediated signaling can be detected at any level of the signaling pathway, for example, by examining phosphorylation of Smad polypeptides, examining expression of TGF-β1-regulated genes (including reporter genes), or measuring TGF-β1-dependent cell proliferation.

[0478] 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, for example, 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 "wounded" monolayer cultures of BAE cells, examining the ability of TGF-β to inhibit cell growth, examining the ability of TGF-β to inhibit the activity of plasminogen activator (PA), 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))

[0479] The inhibition of TGF-β1 activation can also be detected and / or measured using the method described and illustrated in the working examples. Using these or other suitable types of assays, it is possible to screen for antibodies that can inhibit TGF-β1 activation in the detection antibody. In certain embodiments, compared with the negative control under similar conditions, inhibition of TGF-β1 activation includes reducing TGF-β1 activation in the assay by at least 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% or more. In some embodiments, it refers to inhibiting TGF-β1 activation by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more. In certain embodiments, compared with the negative control under similar conditions, inhibition of TGF-β1 activation includes reducing the amount of mature TGF-β1 detected in the assay by at least 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% or more. In some embodiments, it refers to a decrease in the amount of mature TGF-β1 by at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more.

[0480] In some embodiments, whether the detection antibody inhibits the cleavage of the LAP portion of latent TGF-β1 is determined by contacting the protease with the latent TGF-β1 in the presence or absence of the detection antibody, and then detecting the cleavage product of the latent TGF-β1 and / or the 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., FLAG-tag, etc.) is added to the N-terminus of the LAP region of the latent TGF-β1, when the protease-mediated cleavage occurs, the portion to which the protein tag is added is removed. Therefore, the cleavage product of the latent TGF-β1 can be detected by detecting the latent TGF-β1 (or the LAP region of the latent TGF-β1) without the protein tag, and / or the uncleaved latent TGF-β1 can be detected by detecting the latent TGF-β1 with the protein tag.

[0481] For another example, where a protein tag (e.g., FLAG-tag, etc.) is added to the N-terminus of the LAP region of latent TGF-β1, and where the protease cleavage site is not located near the N-terminus of the LAP region of latent TGF-β1, when protease-mediated cleavage occurs, the LAP region with the protein tag becomes shorter. Therefore, by detecting the latent TGF-β1 with the shortened LAP region (or the shortened LAP region of latent TGF-β1) with the protein tag, the cleavage product of the latent TGF-β1 can be detected.

[0482] In some embodiments, when the amount of latent TGF-β1 cleavage product detected in the presence (or after contact) of the detection antibody is reduced compared to the amount detected in the absence of the detection antibody, the detection antibody is identified as an antibody that can inhibit the cleavage of latent TGF-β1. Conversely, when the amount of latent TGF-β1 cleavage product detected in the presence (or after contact) of the detection antibody is not significantly reduced compared to the amount detected in the absence of the detection antibody, the detection antibody is identified as an antibody that does not inhibit the cleavage of latent TGF-β1. In some embodiments, when an increased amount of uncleaved latent TGF-β1 is detected in the presence (or after contact) of the detection antibody compared to the amount detected in the absence of the detection antibody, the detection antibody is identified as an antibody that can inhibit the cleavage of latent TGF-β1. Conversely, when the amount of uncleaved latent TGF-β1 in the presence (or after contact) of the detection antibody is not significantly increased compared to the amount detected in the absence of the detection antibody, the detection antibody is identified as an antibody that does not inhibit the cleavage of latent TGF-β1. In certain embodiments, whether the detection antibody blocks the access of the protease to latent TGF-β1 is determined by a method that detects protein interactions between the protease and latent TGF-β1, for example, ELISA or surface plasmon resonance (e.g., BIACORE (registered trademark)) or similar techniques (e.g., KinExa or OCTET (registered trademark)). When a decrease in the interaction between the protease and latent TGF-β1 is detected in the presence of (or after contact with) the detection antibody compared to the interaction detected in the absence of the detection antibody, the detection antibody is identified as an antibody that can block the access of the protease to latent TGF-β1.

[0483] In certain embodiments, non-inhibition of cleavage of latent TGF-β1 comprises an increase in the amount of cleavage products of latent TGF-β1 in the 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, non-inhibition of cleavage of latent TGF-β1 comprises an increase in the amount of uncleaved latent TGF-β1 in the assay by 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.

[0484] In some embodiments, the anti-latent TGF-β1 antibody may be subjected to other biological activity assays, for example, to evaluate its effectiveness as a therapeutic agent. 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 blocking TGF-β1 by an anti-latent TGF-β1 antibody can be evaluated in a unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis model (e.g., as described in Chevalier RL et al., Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy. Kidney Int. 2009 Jun;75(11):1145-1152), a choline-deficient L-amino acid-defined high-fat diet (CDAHFD)-induced NASH / liver fibrosis mouse model, a bleomycin (BLM)-induced lung fibrosis mouse model, and / or a syngeneic tumor model (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, the anti-latent TGF-β1 antibodies may be subjected to the biological activity assays described herein.

[0485] 3. Screening methods

[0486] In one aspect, the method for screening antibodies of the invention comprises various assays described herein and known in the art. For example, the method for screening anti-latent TGF-β1 antibodies comprises:

[0487] (a) contacting a biological sample containing latent TGF-β1 and a protease with a detection antibody;

[0488] (b) testing (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

[0489] (c) Selection of detection antibodies that inhibit activation of latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP portion of latent TGF-β1.

[0490] Alternatively, in addition to the above steps (b) and (c), the method for screening anti-latent TGF-β1 antibodies comprises, for example, the following steps (b) and (c):

[0491] (b) measuring (i) the amount of uncleaved latent TGF-β1 and (ii) the amount of mature TGF-β1; and

[0492] (c) If the amount of uncleaved latent TGF-β1 is not significantly increased and the amount of mature TGF-β1 is decreased compared to the absence of the detection antibody, select a detection antibody that inhibits protease-mediated release of mature TGF-β1 from latent TGF-β1 but does not inhibit protease-mediated cleavage of the LAP region of latent TGF-β1.

[0493] Alternatively, in addition to the above steps (b) and (c), the method for screening anti-latent TGF-β1 antibodies comprises, for example, the following steps (b) and (c):

[0494] (b) measuring (i) the amount of latent TGF-β1 cleavage products and (ii) the level of mature TGF-β1 activity; and

[0495] (c) If the amount of cleavage product is not significantly reduced and the level of mature TGF-β1 activity is reduced compared to when the detection antibody is not present, a detection antibody is selected that inhibits protease-mediated activation of latent TGF-β1 but does not inhibit protease-mediated cleavage of the LAP region of latent TGF-β1.

[0496] In addition, the present invention provides a method for producing an anti-latent TGF-β1 antibody, which, in addition to the above steps (a) to (c), further comprises, for example, the following steps (d) and (e):

[0497] (d) obtaining the amino acid sequence information of the anti-latent TGF-β1 antibody selected in step (c); and

[0498] (e) Introducing a gene encoding an anti-latent TGF-β1 antibody into a host cell.

[0499] In this case, the term "not significantly increased / decreased", for example, in the phrases "the amount of uncleaved latent TGF-β1 is not significantly increased" and "the amount of the cleavage product (of latent TGF-β1) is not significantly decreased", means that the level / degree of increase / decrease may be zero, or may not be zero but close to zero, or may be so low that it can be technically ignored or actually / substantially considered to be zero by a person skilled in the art. For example, in immunoblot analysis, when researchers cannot 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 the cleavage product (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".

[0500] In some embodiments, whether a test antibody inhibits cleavage of the LAP region of latent TGF-β1, and whether a test antibody inhibits activation of latent TGF-β1 can be determined by various assays described herein and known in the art.

[0501] D. Immunoconjugates

[0502] The present invention also provides immunoconjugates comprising an anti-latent TGF-β1 antibody herein conjugated to one or more cytotoxic agents (e.g., 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).

[0503] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) wherein the antibody is conjugated to one or more drugs including, but not limited to, maytansine alkaloids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent EP 0 425 235 B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; 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)); anthracyclines 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; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; and CC1065.

[0504] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0505] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 212 When the radioconjugate is used for detection, it may contain a radioactive atom such as Tc-99m or 123 I, or a spin label for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0506] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

[0507] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinyl sulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0508] E. Methods and compositions for diagnosis and detection

[0509] In certain embodiments, any anti-latent TGF-β1 antibody provided herein can be used to detect the presence of TGF-β1 (e.g., latent TGF-β1) in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection / measurement. In certain embodiments, biological samples include cells or tissues, such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, peritoneal fluid, lens fluid and mucus.

[0510] In one embodiment, an anti-latent TGF-β1 antibody for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of TGF-β1 (e.g., latent TGF-β1) in a biological sample is provided. For example, the method for detecting the presence of latent TGF-β1 comprises:

[0511] (a) contacting the biological sample with an anti-latent TGF-β1 antibody of the invention as described herein under conditions that allow the anti-latent TGF-β1 antibody to bind to latent TGF-β1; and

[0512] (b) Detection of whether a complex is formed between the anti-latent TGF-β1 antibody and latent TGF-β1.

[0513] Such methods can be in vitro or in vivo methods. In one embodiment, the anti-latent TGF-β1 antibody is used to select subjects eligible for treatment with the anti-latent TGF-β1 antibody, for example, where TGF-β1, such as latent TGF-β1, is a biomarker for selecting patients. That is, the anti-latent TGF-β1 antibody can be used as a diagnostic agent targeting TGF-β1.

[0514] More specifically, the anti-latent TGF-β1 antibodies can be used to diagnose fibrosis, preferably myocardial fibrosis, pulmonary / lung fibrosis, liver fibrosis, kidney fibrosis, skin fibrosis, eye fibrosis and bone marrow fibrosis. The anti-latent TGF-β1 antibodies of the present invention can also be used to diagnose cancer.

[0515] In some embodiments, the present invention provides a method for inhibiting the release of mature TGF-β1 from latent TGF-β1 in a biological sample without inhibiting protease-mediated cleavage of the LAP region of latent TGF-β1, comprising contacting a biological sample containing latent TGF-β1 with an anti-latent TGF-β1 antibody of the present invention under conditions that allow the antibody to bind to latent TGF-β1.

[0516] In certain embodiments, for example, for detection / diagnosis purposes, labeled anti-latent TGF-β1 antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detectable moieties, such as enzymes or ligands, such as by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, which are coupled to enzymes that oxidize dye precursors such as HRP, lactoperoxidase or microperoxidase using hydrogen peroxide, biotin / avidin, spin labels, phage labels, stable free radicals, etc.

[0517] F. Pharmaceutical Preparations

[0518] Pharmaceutical formulations of anti-latent TGF-β1 antibodies as described herein are prepared by mixing such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally 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 (e.g., 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 dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; counterions to form salts, such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable 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, the sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0519] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter including a histidine-acetate buffer.

[0520] In one aspect, the present invention provides a pharmaceutical preparation comprising an anti-latent TGF-β1 antibody for treating fibrosis, preferably myocardial fibrosis, pulmonary fibrosis, liver fibrosis, kidney fibrosis, skin fibrosis, eye fibrosis and bone marrow fibrosis. The present invention also provides a pharmaceutical preparation comprising an anti-latent TGF-β1 antibody for treating cancer.

[0521] The formulations herein may also contain more than one active ingredient, preferably with complementary activities that do not adversely affect each other, as necessary for the particular indication being treated. For example, it may be desirable to further provide an immune checkpoint inhibitor, which is described below as III. Combination therapy ” is described in .

[0522] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980).

[0523] Sustained release preparations may 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, e.g. films, or microcapsules.

[0524] Preparations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0525] G. Methods of treatment and compositions

[0526] Any anti-latent TGF-β1 antibody provided herein can be used in a method of treatment. On the one hand, an anti-latent TGF-β1 antibody for use as a drug is provided. In a further aspect, an anti-latent TGF-β1 antibody for treating cancer or fibrosis (e.g., liver fibrosis, kidney fibrosis, or lung fibrosis) and the like 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 invention provides an anti-latent TGF-β1 antibody for a method of treating an individual suffering from cancer or fibrosis (e.g., liver fibrosis, kidney fibrosis, or lung fibrosis) and the like, comprising administering an effective amount of an anti-latent TGF-β1 antibody to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, for example, as described below. In a further embodiment, the present invention provides an anti-latent TGF-β1 antibody for inhibiting protease-mediated activation of latent TGF-β1. In certain embodiments, the present invention provides an anti-latent TGF-β1 antibody for use in a method of inhibiting protease-mediated activation of latent TGF-β1 in an individual, the method comprising administering to the individual an effective anti-latent TGF-β1 antibody to inhibit protease-mediated activation of latent TGF-β1. The "individual" according to any of the above embodiments is preferably a human.

[0527] In another aspect, the present invention provides the use of anti-latent TGF-β1 antibodies in the production or preparation of drugs. In one embodiment, the drug is used to treat cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc. In another embodiment, the drug is used to treat a method of cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc., comprising administering an effective amount of the drug to an individual suffering from cancer or fibrosis (e.g., liver fibrosis, renal fibrosis, or pulmonary fibrosis), etc. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, for example, as described below. In a further embodiment, the drug is used to inhibit protease-mediated activation of latent TGF-β1. In a further embodiment, the drug is used to inhibit protease-mediated activation of latent TGF-β1 in an individual, comprising administering an effective amount of the drug to the individual to inhibit protease-mediated activation of latent TGF-β1. According to any of the above embodiments, the "individual" can be a human.

[0528] In another aspect, the present invention provides a method for treating cancer or fibrosis (e.g., liver fibrosis, kidney fibrosis, or lung fibrosis), etc. In one embodiment, the method comprises administering an effective amount of an anti-latent TGF-β1 antibody to an individual suffering from such cancer or fibrosis (e.g., liver fibrosis, kidney fibrosis, or lung fibrosis), etc. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, as described below. In some embodiments, the antibody and the agent are administered simultaneously. According to any of the above embodiments, the "individual" may be a human.

[0529] In another aspect, the present invention provides a method for inhibiting protease-mediated activation of latent TGF-β1 in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-latent TGF-β1 antibody to inhibit protease-mediated activation of latent TGF-β1. In one embodiment, the "individual" is a human.

[0530] In another aspect, the present invention provides a pharmaceutical preparation comprising any anti-latent TGF-β1 antibody provided herein, for example, for use in any of the above-mentioned treatment methods. In one embodiment, the pharmaceutical preparation comprises any anti-latent TGF-β1 antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical preparation comprises any anti-latent TGF-β1 antibody provided herein and at least one additional therapeutic agent, for example, as described below.

[0531] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intraluminally, and intranasally, and, if local treatment is required, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, such as by injection, for example, intravenous injection or subcutaneous injection, depending in part on whether the administration is short-term or long-term. A variety of dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0532] The antibodies of the present invention will be formulated, dosed, and administered in a manner that is in keeping with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammalian being treated, the clinical condition of the individual patient, the cause, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to the physician. The antibodies need not, but optionally can be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors mentioned above.

[0533] For the prevention or treatment of a disease, the appropriate dosage of the antibody of the present invention (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 antibody is being administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody will be administered to the patient either as a single dose or in a series of treatments.

[0534] It is to be understood that any article described herein can include the immunoconjugate of the present invention in place of the anti-latent TGF-β1 antibody or, in addition to the anti-latent TGF-β1 antibody, any article described herein can also include the immunoconjugate of the present invention.

[0535] III. Combination therapy

[0536] The anti-latent TGFβ-1 antibody of the present invention can be used alone or in combination with other agents for treatment, preferably for the treatment of cancer or fibrosis, more preferably for the treatment of cancer. For example, the antibody of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the antibody and the agent are administered simultaneously. In certain embodiments, the additional therapeutic agent is one or more immune checkpoint inhibitors, such as CTLA-4, PD-1, PD-L1, PD-L2, CD160, CD57, CD244, LAG-3, CD272, KLRG1, CD26, CD39, CD73, CD305, TIGIT, TIM-3 and / or VISTA inhibitors. In some embodiments, the immune checkpoint inhibitor is, for example, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CD160 antibody, an anti-CD57 antibody, an anti-CD244 antibody, an anti-LAG-3 antibody, an anti-CD272 antibody, an anti-KLRG1 antibody, an anti-CD26 antibody, an anti-CD39 antibody, an anti-CD73 antibody, an anti-CD305 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, and / or an anti-VISTA antibody. Preferably, the immune checkpoint inhibitor is a PD-1 axis binding antagonist. More preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody is Nivolumab, Pembrolizumab, or Cemiplimab. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab or durvalumab, preferably atezolizumab. Preferably, the combination therapy comprises the anti-latent TGFβ-1 antibody of the present invention and atezolizumab. In some embodiments, the combination therapy comprising the anti-latent TGFβ-1 antibody of the present invention and one or more immune checkpoint inhibitors has additive or synergistic therapeutic effects, for example, additive, combined or synergistic anti-tumor effects, compared with anti-TGFβ antibody monotherapy or immune checkpoint inhibitor monotherapy.

[0537] On the one hand, the combination therapy of the present invention is used to treat cancer or fibrosis, preferably cancer. In one embodiment, cancer is resistant to immune checkpoint inhibitors and / or has limited response to immune checkpoint inhibitors. Without being bound by any theory, some immune checkpoint resistant cancers lack response and / or show limited response to immune checkpoint inhibitors and are related to TGF-β signaling characteristics in fibroblasts, especially in patients in which CD8+ T cells are excluded from tumor parenchyma and found in the tumor peri-matrix rich in fibroblasts and collagen. Therefore, anti-latent TGF-β1 antibodies used in combination with immune checkpoint inhibitors can reduce TGF-β signaling in stromal cells, promote T cell infiltration into the tumor center, and can show enhanced anti-tumor activity.

[0538] Programmed cell death protein 1 (PD-1; also known as CD274 or B7-H1) is a type I membrane protein that belongs to the CD28 / CTLA-4 family of T-cell regulators. PD-1 has two ligands, PD-L1 and PD-L2, which belong to the B7 family. PD-1 and the ligands are thought to negatively regulate immune responses, such as T-cell responses. PD-L1 and PD-1 are highly expressed in several types of cancer and are thought to play a role in cancer immune escape. Inhibitors, such as "(immune) checkpoint inhibitors" that inhibit the interaction between PD-1 and PD-L1, can enhance T-cell responses and increase anti-tumor activity.

[0539] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby eliminating T cell dysfunction caused by signaling on the PD-1 signaling axis - the result is restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0540] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction generated by the interaction of PD-1 with one or more of its binding partners (e.g., PD-L1, PD-L2). In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-1 signaling, thereby reducing dysfunctional T cell dysfunction (e.g., enhancing effector responses to antigen recognition). In a specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab), MK-3475 (lambrolizumab), CT-011 (pidilizumab) or AMP-224 or AMP-514 (MEDI0680). In another specific aspect, the PD-1 antagonist is selected from the group consisting of PDR001, REGN2810, BGB A317 and SHR-1210.

[0541] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction caused by the interaction of PD-L1 with one or more of its binding partners (e.g., PD-1, B7-1). In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In specific aspects, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-L1 with one or more of its binding partners (e.g., PD-1, B7-1). In one embodiment, the PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-L1 signaling, thereby reducing the degree of dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In specific aspects, the anti-PD-L1 antibody is YW243.55.S70 (atezolizumab), MDX-1105, avelumab, MPDL3280A, or MEDI4736 (durvalumab).

[0542] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (e.g., PD-1). In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-L2 with one or more of its binding partners (e.g., PD-1). In one embodiment, a PD-L2 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-L1 signaling, thereby reducing dysfunctional T cell dysfunction (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0543] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case the antibodies of the invention may be administered before, simultaneously with, and / or after the administration of the additional therapeutic agent or agents. In one embodiment, the administration of the anti-latent TGF-β1 antibody and the administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The antibodies of the invention may also be used in combination with radiotherapy.

[0544] On the one hand, when the above-mentioned combination therapy covers joint administration and two or more therapeutic agents are included in the same pharmaceutical preparation, the pharmaceutical preparation herein comprises, for example, the anti-latent TGFβ-1 antibody of the present invention and one or more of the above-mentioned immune checkpoint inhibitors. Preferably, the pharmaceutical preparation herein comprises the anti-latent TGFβ-1 antibody of the present invention, a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab) and a pharmaceutically acceptable carrier.

[0545] On the one hand, the present invention provides an anti-latent TGF-β1 antibody used in combination with another therapeutic agent to treat one or more diseases. On the other hand, the present invention provides a pharmaceutical preparation comprising an anti-latent TGF-β1 antibody, which is used in combination with another therapeutic agent to treat one or more diseases. In one embodiment, the one or more diseases are cancer and / or fibrosis, preferably cancer. In one embodiment, the additional therapeutic agent is one or more of the above-mentioned one or more immune checkpoint inhibitors. Preferably, the present invention provides an anti-latent TGF-β1 antibody used in combination with a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab) for the treatment of cancer.

[0546] In one aspect, the present invention provides a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab) used in combination with an anti-latent TGF-β1 antibody for treating one or more diseases. In another aspect, the present invention provides a pharmaceutical preparation comprising a PD-1 axis binding antagonist (preferably an anti-PD-L1 antibody, more preferably atezolizumab) used in combination with an anti-latent TGF-β1 antibody for treating one or more diseases. In another aspect, the one or more diseases are cancer and / or fibrosis, preferably cancer.

[0547] IV. Products, Kits

[0548] A. Products

[0549] In another aspect of the invention, an article containing materials useful for treating, preventing and / or diagnosing the above-mentioned conditions (e.g., fibrosis and cancer) is provided. The article comprises a container and a label on the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed of a variety of materials, such as glass or plastic. The container holds a composition, which is used alone or in combination with another composition that is effective for treating, preventing and / or diagnosing a condition (e.g., fibrosis and cancer) and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition is an antibody or immunoconjugate of the invention. The label or package insert indicates that the composition is used to treat a selected condition (e.g., fibrosis and cancer). In addition, the article can comprise (a) a first container containing a composition, wherein the composition comprises an antibody / immunoconjugate of the invention; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a specific condition (e.g., fibrosis and cancer). Alternatively, or in addition, the article of manufacture may 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. From a commercial and user perspective, it may further include other desirable materials, including other buffers, diluents, filters, needles, and syringes.

[0550] It is to be understood that any of the above-described articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-latent TGF-β1 antibody.

[0551] B. Kit

[0552] The present disclosure provides a kit for treating, preventing and / or diagnosing the conditions described herein, in particular, a method for treating an individual with fibrosis or cancer, the kit containing an anti-latent TGF-β1 antibody, an immunoconjugate containing an anti-latent TGF-β1 antibody, an isolated nucleic acid encoding an anti-latent TGF-β1 antibody, or a vector containing a nucleic acid of the present disclosure, or a vector produced by the method of the present disclosure. The kit may further contain any therapeutic agent exemplified in "III. Combination therapy" herein, such as an immune checkpoint inhibitor, including an anti-PD-L1 antibody. The kit may be packaged with additional pharmaceutically acceptable carriers or media disclosed herein, or instructions describing how to use the kit, etc. As with the articles described herein, the kit may contain materials for treating fibrosis or cancer; containers and labels on containers or package inserts associated with containers; compositions used alone or in combination with another composition effective for treating fibrosis or cancer; sterile access ports, etc. The kit may further include a label or package insert indicating that the composition can be used to treat fibrosis or cancer. Alternatively, or in addition, the kit may further 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. From a commercial and user perspective, the kit may further include other materials, including other buffers, diluents, filters, needles and syringes.

[0553] Example

[0554] The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, given the general description provided above.

[0555] Example 1: Expression and purification of antigens

[0556] (1-1) Expression and purification of latent TGF-β1

[0557] The sequences used for expression and purification are: human latent TGF-β1 with flag tag (SEQ ID NO: 1, 2), mouse latent TGF-β1 with flag tag (SEQ ID NO: 3, 4), and cynomolgus monkey latent TGF-β1 with flag tag (SEQ ID NO: 5, 6). Each of these latent TGF-β1 with flag tag has a signal sequence derived from rat serum albumin (SEQ ID NO: 7), a Flag tag, and a latent TGF-β1 sequence from its N-terminus to its C-terminus. The Cys residue at position 30 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)-beta1 association with latent TGF-beta binding protein yields inflammation and tumors. Proc Natl Acad Sci US A. 2008; 105(48): 18758-18763).

[0558] Human latent TGF-β1 with a Flag tag (hereinafter referred to as "human latent TGF-β1 (SLC)" or "human latent TGF-β1"), mouse latent TGF-β1 with a Flag tag (hereinafter referred to as "mouse latent TGF-β1 (SLC)" or "mouse latent TGF-β1"), or cynomolgus monkey latent TGF-β1 with a Flag tag (hereinafter referred to as "monkey latent TGF-β1 (SLC)" or "monkey latent TGF-β1") was transiently expressed using FreeStyle293-F or Expi293F cell lines (Thermo Fisher Scientific). Conditioned medium expressing human, mouse, or monkey latent TGF-β1 (SLC) was applied to a column loaded with anti-Flag M2 affinity resin (Sigma), and latent TGF-β1 (SLC) was eluted with Flag peptide (Sigma). The fractions containing human, mouse or monkey latent TGF-β1 (SLC) were collected and subsequently placed on a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1× PBS. The fractions containing human, mouse or monkey latent TGF-β1 (SLC) were then pooled and stored at -80°C.

[0559] (1-2) Expression and purification of mouse latency-associated peptide (LAP)

[0560] The sequence used for expression and purification is: mouse LAP with a flag tag (SEQ ID NO: 8, 9), which has a signal sequence derived from rat serum albumin (SEQ ID NO: 7), a Flag tag, and a sequence of latency-associated protein (LAP) from its N-terminus to its C-terminus. The Cys residue at the thirtieth position (30th) in the LAP with a flag tag is substituted with Ser, which corresponds to the "C33S mutation". The expression and purification of mouse LAP with a Flag tag (SEQ ID NO: 8, 9) (hereinafter referred to as "recombinant mouse latency-associated protein (LAP)") are exactly the same as described in Example (1-1).

[0561] Example 2. Humanization and optimization of anti-TGF-β1 antibodies

[0562] (2-1) Humanization

[0563] The parent anti-TGF-β antibody TBA0947 (chimeric antibody) was humanized as follows. First, the variable regions of the heavy and light chains of the humanized antibodies were designed using the variable regions of TBA0947 and the human germline framework. Then, the polynucleotides of each 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: 10) and the light chain constant region SK1 sequence (SEQ ID NO: 11), respectively. The humanized antibodies were transiently expressed in FreeStyle 293-F cells (Thermo Fisher Scientific) and subjected to Biacore analysis. Humanized antibodies that showed Biacore binding activity at least similar to that of the parent antibody were selected.

[0564] (2-2) Optimization

[0565] The humanized antibodies obtained in Example (2-1) were optimized to hT0947AE04, hT0947AE07, hT0947AE08 and hT0947AE09, which have improved binding activity to latent TGF-β1 (SLC). In brief, all residues in the complementary determining regions (CDRs) of the heavy and light chains were subjected to comprehensive mutagenesis. Each amino acid was replaced by 18 other naturally occurring amino acids, excluding the original amino acid and cysteine. The variants were transiently expressed in FreeStyle 293-F cells (Thermo Fisher Scientific) and purified from the culture supernatant for Biacore evaluation. Target variants with improved binding activity to human and mouse latent TGF-β1 (SLC) were selected. Antibodies with these mutation combinations in the CDRs were then generated.

[0566] (2-3) Optimizing the amino acid sequence of the antibody

[0567] The amino acid sequences of the variable regions of hT0947AE04, hT0947AE07, hT0947AE08 and hT0947AE09 are identified as follows:

[0568] - the heavy chain variable region of hT0947AE04 comprises the amino acid sequence of SEQ ID NO: 12 (hT0947AE04H), and the light chain variable region of hT0947AE04 comprises the amino acid sequence of SEQ ID NO: 13 (hT0947AE04L).

[0569] - The heavy chain variable region of hT0947AE07 comprises the amino acid sequence of SEQ ID NO: 14 (hT0947AE07H), and the light chain variable region of hT0947AE07 comprises the amino acid sequence of SEQ ID NO: 15 (hT0947AE07L).

[0570] - the heavy chain variable region of hT0947AE08 comprises the amino acid sequence of SEQ ID NO: 16 (hT0947AE08H), and the light chain variable region of hT0947AE08 comprises the amino acid sequence of SEQ ID NO: 17 (hT0947AE08L).

[0571] - The heavy chain variable region of hT0947AE09 comprises the amino acid sequence of SEQ ID NO: 18 (hT0947AE09H), and the light chain variable region of hT0947AE09 comprises the amino acid sequence of SEQ ID NO: 19 (hT0947AE09L).

[0572] The amino acid sequences of the CDRs (HVRs) of hT0947AE04, hT0947AE07, hT0947AE08, and hT0947AE09 were identified according to Kabat as follows:

[0573] - hT0947AE04 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 23, 24 and 25, respectively.

[0574] - hT0947AE07 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 31, respectively.

[0575] - hT0947AE08 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 33 and 34, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 35, 36 and 37, respectively.

[0576] - hT0947AE09 comprises heavy chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively, and light chain CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively.

[0577] (2-4) Construction of full-length heavy and light chains

[0578] Multiple amino acid substitutions were introduced into the heavy chain constant region SG1 (SEQ ID NO: 44). SG1 is a wild-type human IgG1 heavy chain constant region lacking the last two C-terminal amino acids Gly-Lys (GK). As a result, SG181 (SEQ ID NO: 10) and SG191 (SEQ ID NO: 45) were produced. According to the EU index, SG181 includes amino acid substitutions L235R / G236R (amino acid substitutions to reduce effector function) and K214R. According to the EU index, SG191 includes amino acid substitutions L235R / G236R (amino acid substitutions to reduce effector function), M428L / N434A (amino acid substitutions to enhance binding activity to FcRn), Q438R / S440E (amino acid substitutions to reduce binding to rheumatoid factor) and K214R. In addition, mF18 (SEQ ID NO: 46), a mouse IgG heavy chain constant region, was generated that includes P235K / S239K (amino acid substitutions to reduce effector function).

[0579] Each heavy chain variable region was combined with a heavy chain constant region SG181 (SEQ ID NO: 10), SG191 (SEQ ID NO: 45) or mF18 (SEQ ID NO: 46). Thus, a full-length heavy chain sequence having the following amino acid sequence was constructed:

[0580] (a1) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 47, which comprises hT0947AE04H (heavy chain variable region) and SG181 (heavy chain constant region)

[0581] (a2) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 48, which comprises hT0947AE07H (heavy chain variable region) and SG181 (heavy chain constant region)

[0582] (a3) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 49, which comprises hT0947AE08H (heavy chain variable region) and SG181 (heavy chain constant region)

[0583] (a4) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 50, which comprises hT0947AE09H (heavy chain variable region) and SG181 (heavy chain constant region)

[0584] (b1) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 51, which comprises hT0947AE04H (heavy chain variable region) and SG191 (heavy chain constant region)

[0585] (b2) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 52, which comprises hT0947AE07H (heavy chain variable region) and SG191 (heavy chain constant region)

[0586] (b3) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 53, which comprises hT0947AE08H (heavy chain variable region) and SG191 (heavy chain constant region)

[0587] (b4) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 54, comprising hT0947AE09H (heavy chain variable region) and SG191 (heavy chain constant region)

[0588] (c1) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 55, which comprises hT0947AE04H (heavy chain variable region) and mF18 (heavy chain constant region)

[0589] (c2) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 56, which comprises hT0947AE07H (heavy chain variable region) and mF18 (heavy chain constant region)

[0590] (c3) a full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 57, which comprises hT0947AE08H (heavy chain variable region) and mF18 (heavy chain constant region)

[0591] (c4) A full-length heavy chain comprising the amino acid sequence of SEQ ID NO: 58, which comprises hT0947AE09H (heavy chain variable region) and mF18 (heavy chain constant region).

[0592] Each light chain variable region is combined with the human IgG light chain constant region (κ) SK1 (SEQ ID NO: 11) or the mouse IgG light chain constant region (κ) mk1 (SEQ ID NO: 59). Thus, full-length light chain sequences with the following amino acid sequences were constructed:

[0593] (d1) A full-length light chain containing the amino acid sequence of SEQ ID NO: 60, which contains hT0947AE04L (light chain variable region) and SK1 (light chain constant region)

[0594] (d2) A full-length light chain containing the amino acid sequence of SEQ ID NO: 61, which contains hT0947AE07L (light chain variable region) and SK1 (light chain constant region)

[0595] (d3) A full-length light chain containing the amino acid sequence of SEQ ID NO: 62, which contains hT0947AE08L (light chain variable region) and SK1 (light chain constant region)

[0596] (d4) A full-length light chain containing the amino acid sequence of SEQ ID NO: 63, which contains hT0947AE09L (light chain variable region) and SK1 (light chain constant region)

[0597] (e1) A full-length light chain containing the amino acid sequence of SEQ ID NO: 64, which contains hT0947AE04L (light chain variable region) and mk1 (light chain constant region)

[0598] (e2) A full-length light chain containing the amino acid sequence of SEQ ID NO: 65, which contains hT0947AE07L (light chain variable region) and mk1 (light chain constant region)

[0599] (e3) A full-length light chain containing the amino acid sequence of SEQ ID NO: 66, which contains hT0947AE08L (light chain variable region) and mk1 (light chain constant region)

[0600] (e4) A full-length light chain containing the amino acid sequence of SEQ ID NO: 67, which contains hT0947AE09L (light chain variable region) and mk1 (light chain constant region).

[0601] Then, the respective full-length heavy chains and light chains were combined, and the antibodies shown in Table 2 were constructed. The constructed antibodies are named as shown in Table 2 and are referred to by their respective names in this specification.

[0602] (Table 2)

[0603]

[0604] Example 3. Biacore analysis for evaluating the binding activity of anti-latent TGF-β1 antibodies

[0605] The binding activity of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, and hT0947AE09-SG191) to latent TGF-β1 (SLC) of human, cynomolgus monkey, or mouse was measured using a Biacore 8k instrument (GE Healthcare). Mouse anti-human Igκ light chain antibody (BD Pharmingen) was immobilized on all flow cells of a CM5 sensor chip using an amine coupling kit (GE Healthcare). The antibody was captured onto the anti-κ sensor surface at a capture level of approximately 20 RU (resonance unit), and then latent TGF-β1 (SLC) of human, cynomolgus monkey, or mouse prepared in Example (1-1) was injected over the flow cell. All antibodies and analytes were measured in a 5% PBS containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN 3 ACES pH 7.4 was prepared. The assay temperature was set at 37 degrees Celsius. The sensor surface was regenerated with 10 mM glycine-HCl, pH 2.1 in each cycle. The data were processed and fitted to a 1:1 binding model using Biacore Insight software, version 1.1.1.7442 (GE Healthcare) to determine binding activity.

[0606] Table 3 shows the binding activity (ka, kd and KD) of the anti-latent TGF-β1 antibodies to latent TGF-β1 of human, cynomolgus monkey or mouse.

[0607] (Table 3)

[0608]

[0609] Example 4. Characterization of Anti-Latent TGF-β1 Antibodies

[0610] (4-1) Anti-latent TGF-β1 antibody that binds to latent TGF-β1 on the cell surface

[0611] The binding activity of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) to cell surface latent TGF-β1 was determined by FACS using Ba / F3 cells expressing mouse latent TGF-β1 or FreeStyle expressing human latent TGF-β1. TM293-F cells (ThermoFisher) were used for detection. 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% FBS, 2 mM EDTA in PBS). Anti-KLH antibody with human IgG1 Fc region (IC17-hIgG1) that does not bind to mouse latent TGF-β1 and human latent TGF-β1 was used as a negative control antibody. Goat F(ab')2 anti-human IgG, mouse ads-PE (Southern Biotech, Cat. 2043-09) was then added and incubated at 4°C for 30 minutes and washed with FACS buffer. Data acquisition was performed on a FACS Verse (Becton Dickinson), and the mean fluorescence intensity (MFI) was analyzed and calculated using FlowJo software (Tree Star) and GraphPad Prism software (GraphPad). As shown in Figure 1, all anti-latent TGF-β1 antibodies bound to mouse cell surface latent TGF-β1 expressed on Ba / F3 cells and FreeStyle TM Binding of latent human cell surface TGF-β1 expressed on 293-F cells.

[0612] (4-2) Anti-latent TGF-β1 antibody does not bind to mature TGF-β1 but binds to mouse LAP

[0613] The binding activity of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) to mature TGF-β1 was detected by ELISA. 384-well plates were coated with mouse or human mature TGF-β1 overnight at 4 degrees Celsius and then washed four times with PBS-T. After washing, the plates were blocked with blocking buffer (1×TBS / Tween-20 + 0.5% BSA + 1×Block ace) at room temperature for at least 1 hour and then washed four times with PBS-T. After washing, the antibody solution was added to the plates and incubated at room temperature for 2 hours and then washed four times with PBS-T. After washing, the diluted secondary antibody (goat anti-human IgG-HRP, Abcam, Cat.ab98624) was added to the plates and incubated at room temperature for 1 hour and then washed four times with PBS-T. After washing, TMB solution was added to the plate and incubated at room temperature for 15 minutes, and then 1N sulfuric acid was added to stop the reaction. The absorbance (optical density; OD) was measured at 450 nm / 570 nm. Anti-KLH antibody (IC17-IgG1) was used as a negative control antibody, and anti-mature TGF-β antibody GC1008 with a human IgG1 Fc region (GC1008-F1332m) (as described in U.S. Patent No. US8,383,780) was used as a positive control antibody. Figure 2A and 2B As shown, the anti-latent TGF-β1 antibody did not bind to either mouse mature TGF-β1 or human mature TGF-β1.

[0614] In addition, the binding activity of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947A09-SG191) to mouse latency-associated protein (LAP) was tested by ELISA as described above. Figure 2C As shown, anti-latent TGF-β1 antibody binds to mouse LAP.

[0615] (4-3) Anti-latent TGF-β1 antibodies inhibit spontaneous latent TGF-β1 activation

[0616] Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were incubated at 37 degrees Celsius for 1 hour each in the presence or absence of anti-latent TGF-β1 antibody (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191 or hT0947AE09-SG191). Anti-KLH antibody (IC17-IgG1) was used as a negative control. Spontaneous latent TGF-β1 activation and antibody-mediated inhibition of spontaneous latent TGF-β1 activation were analyzed by mature TGF-β1 ELISA (Human TGF-β1 Quantikine ELISA Kit, R&D systems) according to the manufacturer's procedure.

[0617] As shown in Figure 3, anti-latent TGF-β1 antibody inhibited the spontaneous activation of latent TGF-β1.

[0618] (4-4) Anti-latent TGF-β1 antibody inhibits plasmin (PLN)-mediated activation of latent TGF-β1

[0619] Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were each incubated with human plasmin (Calbiochem) at 37 degrees Celsius for 1 hour in the presence or absence of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). Prior to incubation with plasmin, the antibody was pre-incubated with mouse or human latent TGF-β1 (SLC) at room temperature for 30 minutes. Anti-KLH antibody (IC17-hIgG1) was used as a negative control. Plasmin-mediated activation of latent TGF-β1 and antibody-mediated inhibition were analyzed by mature TGF-β1 ELISA (human TGF-β1 Quantikine ELISA kit, R&D systems) according to the manufacturer's procedure. As shown in FIG4 , anti-latent TGF-β1 antibody inhibited plasmin-mediated activation of latent TGF-β1.

[0620] (4-5) Anti-latent TGF-β1 antibodies inhibit plasma kallikrein (PLK)-mediated activation of latent TGF-β1

[0621] Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were each incubated with human kallikrein (Enzyme Research Laboratories) at 37 degrees Celsius for 2 hours in the presence or absence of anti-latent TGF-β1 antibody (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). Prior to incubation with kallikrein, the antibody was pre-incubated with mouse or human latent TGF-β1 (SLC) at room temperature for 30 minutes. Anti-KLH antibody (IC17-hIgG1) was used as a negative control. Kallikrein-mediated activation of latent TGF-β1 and antibody-mediated inhibition were analyzed by mature TGF-β1 ELISA (Human TGF-β1 Quantikine ELISA Kit, R&D systems) according to the manufacturer's procedure. As shown in Figure 5, anti-latent TGF-β1 antibody inhibited kallikrein-mediated activation of latent TGF-β1.

[0622] (4-6) Anti-latent TGF-β1 antibodies inhibit MMP2- and MMP9-mediated activation of latent human TGF-β1

[0623] The human latent TGF-β1 (SLC) prepared in Example (1-1) was incubated with activated metalloproteinase 2 (MMP2) or MMP9 (R&D systems) at 37 degrees Celsius for 2 hours in the presence or absence of anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191 or hT0947AE09-SG191). Prior to incubation with MMP2 or MMP9, the antibody was pre-incubated with human latent TGF-β1 (SLC) at room temperature for 30 minutes. Anti-KLH antibody (IC17-hIgG1) was used as a negative control. MMP2- and MMP9-mediated activation of human latent TGF-β1 and antibody-mediated inhibition were analyzed by mature TGF-β1 ELISA (Human TGF-β1 Quantikine ELISA Kit, R&D systems) according to the manufacturer's procedure. As shown in FIG6 , the anti-latent TGF-β1 antibody inhibited both MMP2-mediated activation of human latent TGF-β1 and MMP9-mediated activation of human latent TGF-β1.

[0624] (4-7) Anti-latent TGF-β1 antibodies inhibit the activation of latent TGF-β1 without preventing latent TGF-β1 propeptide from acting via fibrinolysis Cleavage of enzyme (PLN)

[0625] Mouse latent TGF-β1 (mSLC) and human latent TGF-β1 (hSLC) prepared in Example (1-1) were each incubated with human plasmin (Calbiochem) at 37 degrees Celsius for 1 hour in the presence or absence of anti-latent TGF-β1 antibody (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191). Prior to incubation with plasmin, the antibody was pre-incubated with mouse or human latent TGF-β1 (SLC) at room temperature for 30 minutes. Camostat mesylate (TOCRIS), a serine protease inhibitor known to inhibit the activity of plasmin, was used as a control. The sample was mixed with 4× SDS-PAGE sample buffer (Wako), then heated at 95 degrees Celsius for 5 minutes, and then loaded for SDS gel electrophoresis. By Trans-Blot (registered trademark) Turbo TM Transfer System (Bio-rad) was used to transfer proteins to the membrane. Mouse anti-FLAG, M2-HRP antibody (Sigma-Aldrich) was used to detect latent TGF-β1 propeptide. The membrane was incubated with ECL substrate and images were acquired by ImageQuant LAS 4000 (GE Healthcare).

[0626] As shown in Figure 7, anti-latent TGF-β1 antibodies did not inhibit the cleavage of latent TGF-β1 propeptide by plasmin.

[0627] (4-8) Anti-latent TGF-β1 antibody did not significantly inhibit integrin-mediated activation of latent TGF-β1 in mouse PBMCs. live

[0628] Mouse PBMC and HEK-Blue TM TGF-β cell co-culture assay to detect integrin-mediated activation of latent TGF-β1. Mouse PBMCs were isolated from mouse blood by using Histopaque-1083 density gradient medium (Sigma-Aldrich). HEK-Blue TM TGF-β cells (Invivogen), which express a Smad3 / 4 binding element (SBE)-inducible SEAP reporter gene, allow the detection of biologically active TGF-β1 (mouse TGF-β1 and human TGF-β1) by monitoring the activation of Smad3 / 4. Active TGF-β1 stimulates SEAP production and its secretion into the cell supernatant. The amount of secreted SEAP is measured using QUANTI-Blue TM Reagent (Invivogen) evaluation.

[0629] HEK-Blue TMTGF-β cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 50 U / mL streptomycin, 50 μg / mL penicillin, 100 μg / mL Normocin, 30 μg / mL blasticidin, 200 μg / mL HygroGold, and 100 μg / mL Zeocin. During the functional assay, the cell culture medium was replaced with assay medium (RPMI1640 with 10% FBS) and seeded into 96-well plates. Anti-latent TGF-β1 antibodies (hT0947AE04-SG191, hT0947AE07-SG191, hT0947AE08-SG191, or hT0947AE09-SG191) and mouse PBMCs were then applied to the wells and incubated with HEK-Blue TM TGF-β cells were incubated overnight. The cell supernatant was then mixed with QUANTI-Blue TM Mix, and measure the optical density (OD) at 620 nm in a colorimetric plate reader. RGD peptide (GRRGDLATIH, GenScript) is known to bind to integrins and inhibit integrin-mediated TGF-β1 activation as a decoy integrin ligand. Therefore, RGD peptide was used as a positive control. In addition, RGE control peptide (GRRGELATIH, GenScript), which is known not to act as a decoy integrin ligand, was used as a negative control. Anti-KLH antibody (IC17-hIgG1) was used as a negative control. Anti-mature TGF-β1 antibody (GC1008-F1332m) was used as a positive control. F1332m is a human IgG1 heavy chain constant region that includes amino acid substitutions to reduce effector function.

[0630] like Figure 8 As shown, anti-latent TGF-β1 antibody did not significantly inhibit integrin-mediated TGF-β1 activation in mouse PBMCs.

[0631] Example 5. Antitumor activity of anti-latent TGF-β1 antibody (1)

[0632] The in vivo efficacy of the anti-latent TGF-β1 monoclonal antibody hT0947AE04-mF18 alone or in combination with an anti-PD-L1 antibody was evaluated in a mouse syngeneic model using EMT6 murine breast cancer cells and Balb / c mice, in which immune checkpoint inhibitor treatment alone had limited effects on tumor growth and survival (see Nature. 2018 Feb 22;554(7693):544-548.).

[0633] (5-1) Establishment of a syngeneic mouse model

[0634] The EMT6 murine breast cancer cell line was obtained from the American Type Culture Collection (ATCC CRL-2755). Cells were cultured in RPMI-1640 medium (SIGMA) supplemented with 2 mM L-glutamine (SIGMA) and 10% fetal bovine serum (FBS; SIGMA). Six-week-old specific pathogen-free Balb / c female mice were purchased from Charles River Inc., Japan, and acclimated for 2 weeks before inoculation. EMT6 cells growing in the logarithmic phase were harvested and washed with Hank's balanced salt solution (HBSS; SIGMA) and incubated at 1 × 10 6 Resuspended in 50% HBSS and 50% Matrigel (CORNING) at a concentration of 1×10 cells / mL. 1×10 cells / mL in 100 μL HBSS:Matrigel (1:1) were inoculated in the left mammary fat pad #5 of the mouse. 5 EMT6 cells.

[0635] When the average tumor volume reaches about 100-300 mm 3 At 7 days post-inoculation, mice were randomly divided into groups based on tumor volume and body weight. Tumor volume was measured with a caliper and calculated as follows:

[0636] Tumor volume (mm 3 ) = (1 / 2) × length (mm) × width (mm) 2

[0637] (5-2) Evaluation of antitumor activity

[0638] After the mouse model in Example (5-1) was established, the mice were treated with isotype control antibody (mouse IgG1 antibody combined with rat IgG2b antibody, purchased from Bio X Cell), anti-mouse PD-L1 antibody (rat IgG2b clone 10F.9G2, purchased from Bio XCell), hT0947AE04-mF18, or hT0947AE04-mF18 combined with anti-mouse PD-L1 antibody, as shown in Table 4. The antibodies were administered 3 times per week for 3 weeks. The first dose was administered intravenously, and the second and subsequent doses were administered intraperitoneally.

[0639] (Table 4)

[0640]

[0641] Tumor volume was measured twice a week. Fig. 9 shown.

[0642] Antitumor activity was also evaluated by tumor growth inhibition (TGI [%]). The TGI [%] for a particular group on a particular day was calculated as follows:

[0643] TGI[%] = {1-(T-T0) / (C-C0)}×100

[0644] Where "T" is the mean tumor volume of the group on the day of measurement, "T0" is the mean tumor volume of the group on the day of randomization, "C" is the mean tumor volume of Group 1 (isotype control) on the day of measurement, and "C0" is the mean tumor volume of Group 1 (isotype control) on the day of randomization.

[0645] As a result, the TGI [%] values ​​of anti-mouse PD-L1 antibody (Group 2), hT0947AE04-mF18 (Group 3), and hT0947AE04-mF18 combined with anti-mouse PD-L1 antibody (Group 4) on day 14 after the first dose were 51, 12, and 83, respectively. Therefore, a synergistic antitumor effect between anti-latent TGF-β1 (hT0947AE04-mF18) and anti-PD-L1 antibody was observed.

[0646] Survival curves were also plotted to evaluate the survival rate of each group. "Surviving" mice were defined as those whose tumor volume did not exceed 1955 mm 3 .like Fig.10 As shown, the combined treatment of hT0947AE04-mF18 and anti-mouse PD-L1 antibody (Group 4) significantly increased the survival rate of mice compared with the anti-mouse PD-L1 antibody-treated mice (Group 2) and hT0947AE04-mF18-treated mice (Group 3).

[0647] Example 6. Anti-tumor activity of anti-latent TGF-β1 antibody (2)

[0648] The in vivo efficacy of the anti-latent TGF-β1 monoclonal antibodies hT0947AE04-mF18, hT0947AE07-SG181, or hT0947AE08-SG181 combined with anti-PD-L1 antibodies was evaluated in a mouse syngeneic model using EMT6 murine breast cancer cells and Balb / c mice.

[0649] (6-1) Establishment of a mouse syngeneic model

[0650] The EMT6 murine breast cancer cell line was obtained from the American Type Culture Collection (ATCC CRL-2755). Cells were cultured in RPMI-1640 medium (SIGMA) supplemented with 2 mM L-glutamine (SIGMA) and 10% fetal bovine serum (FBS; SIGMA). 7-week-old specific pathogen-free Balb / c female mice were purchased from Charles River Inc., Japan, and acclimated for 1 week before inoculation. EMT6 cells growing in the logarithmic phase were harvested and washed with Hank's balanced salt solution (HBSS; SIGMA) and incubated at 1 × 10 6Resuspended in 50% HBSS and 50% Matrigel (CORNING) at a concentration of 1×10 cells / mL. 1×10 cells / mL in 100 μL HBSS:Matrigel (1:1) were inoculated in the left mammary fat pad #5 of the mouse. 5 EMT6 cells.

[0651] When the average tumor volume reaches about 100-300 mm 3 At 7 days post-inoculation, mice were randomly divided into groups based on tumor volume and body weight. Tumor volume was measured with a caliper and calculated as follows:

[0652] Tumor volume (mm 3 ) = (1 / 2) × length (mm) × width (mm) 2

[0653] (6-2) Evaluation of anti-tumor activity

[0654] After the mouse model in Example (6-1) was established, the mice were treated with vehicle (150 mM NaCl / 20 mM His-HCl buffer pH 6.0), anti-mouse PD-L1 antibody (rat IgG2b clone 10F.9G2, purchased from Bio X Cell), hT0947AE04-mF18 combined with anti-mouse PD-L1 antibody, hT0947AE07-SG181 combined with anti-mouse PD-L1 antibody, or hT0947AE08-SG181 combined with anti-mouse PD-L1 antibody, as shown in Table 5. The antibody was administered 3 times a week for 3 weeks. The first dose was administered intravenously, and the second and subsequent doses were administered intraperitoneally.

[0655] (Table 5)

[0656]

[0657] Tumor volume was measured twice a week. Fig.11 shown.

[0658] The antitumor activity was also evaluated by tumor growth inhibition (TGI [%]). (TGI [%]) was calculated as {1-(T-T0) / (C-C0)}×100, which is the same as in Example (5-2).

[0659] The TGI [%] of anti-mouse PD-L1 antibody alone (Group 2), hT0947AE04-mF18 combined with anti-mouse PD-L1 antibody (Group 3), hT0947AE07-SG181 combined with anti-mouse PD-L1 antibody (Group 4), and hT0947AE08-SG181 combined with anti-mouse PD-L1 antibody (Group 5) on day 14 after the first dose were 64, 89, 86, and 76, respectively. Therefore, anti-latent TGF-β1 antibody showed combined efficacy with anti-PD-L1 antibody.

[0660] Example 7. In vivo efficacy of anti-latent TGF-β1 antibody in a mouse renal fibrosis model induced by UUO

[0661] The in vivo efficacy of monoclonal antibodies hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191 was evaluated in a unilateral ureteral obstruction (UUO) mouse model, which is known to induce progressive renal fibrosis.

[0662] (7-1) Establishment of UUO-induced renal fibrosis model in mice

[0663] The in vivo efficacy of monoclonal antibodies hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191 was evaluated in a unilateral ureteral obstruction (UUO) mouse model that induces progressive renal fibrosis.

[0664] Specific pathogen-free C57BL / 6NTac male mice, 6 weeks old, were purchased from Invivos Pte Ltd (Singapore) and acclimatized for 1 week before treatment. The animals were maintained at 20 to 26°C with a 12:12 h light / dark cycle and fed a commercial standard chow diet (5P75; PMI Nutrition INT'L (LabDiet), Missouri, United States) and had free access to tap water.

[0665] 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 stretched to expose the kidney. Using forceps, the kidney was pulled to the surface and the left ureter was tied twice with surgical thread under the kidney. The ligated kidney was gently placed back in its correct anatomical position, and the peritoneum and skin were then sutured. Analgesics were given to reduce the pain of the animals. In the sham group, only the peritoneum and skin were incised and sutured.

[0666] (7-2) In vivo efficacy evaluation

[0667] All monoclonal antibodies were administered at 15 mg / kg intravenously three times a week starting from the day before surgery. Anti-KLH antibody (IC17dk-SG181) was used as a negative control in this study. The sham group was administered with anti-KLH antibody (IC17dk-SG181). Animals were weighed and exsanguinated under isoflurane anesthesia 7 days after surgery. Blood samples were collected from the cardiac cavity or the posterior vena cava and kept at -80 degrees Celsius until assayed. Kidneys were removed rapidly. Parts of renal tissue were snap-frozen in liquid nitrogen or dry ice for molecular analysis.

[0668] The content of hydroxyproline in the kidney, which is one of the amino acids contained in collagen, was measured to evaluate the extramatrix deposition of the tissue. The wet kidney tissue was dried at 95°C for 3 hours and weighed. Then, 6N HCl (100 μl / 1 mg dry tissue) was added to the dry tissue and boiled overnight. The samples were purified through a filter, and 10 μl of each sample was inoculated into a 96-well plate. The plates containing the samples were dried at 60 degrees Celsius, and hydroxyproline was measured using a hydroxyproline assay kit (BioVision). The results of this experiment are shown in Fig.12 As shown. A significant increase in hydroxyproline content was observed in disease-induced kidneys, and all antibodies (hT0947AE04-SG191, hT0947AE07-SG191, and hT0947AE08-SG191) inhibited renal fibrosis. Data are expressed as mean + / - standard error of the mean (SEM). Statistical analysis was performed using Student's t-test analysis. Differences were considered significant when P value < 0.05.

[0669] Example 8. Toxicity evaluation of anti-latent TGF-β1 antibodies

[0670] The potential toxicity of the anti-latent TGF-β1 antibody was evaluated in a repeat-dose toxicity study in normal mice and cynomolgus monkeys compared to the anti-mature TGF-β antibody GC1008-mF18 (anti-mature TGF-β antibody GC1008 with mouse IgG Fc region mF18 (as described in U.S. Patent No. US 8,383,780)). Since the anti-latent TGF-β1 antibody cross-reacts in mice and cynomolgus monkeys, mice and cynomolgus monkeys were selected as the animal species to be evaluated in the in vivo toxicology studies. See Table 6 for a summary of all toxicology studies.

[0671] (Table 6) Summary of toxicology studies

[0672]

[0673] In the 3-month study in mice (IV; 5 or 20 mg / kg, Q2D, 46 doses total), anemic changes (20 mg / kg in the hT0947AE04-mF18 group; 5 and 20 mg / kg in the GC1008-mF18 group) and cardiac injury (5 and 20 mg / kg in the GC1008-mF18 group; see Table 7) were observed. These findings are believed to be caused by on-target toxicity of TGF-β inhibition. Considering the on-target toxicity in the 3-month study in mice, the NOAEL for hT0947AE04-mF18 is 5 mg / kg IV Q2D. In addition, due to adverse reactions in the GC1008-mF18 5 mg / kg group, the NOAEL for GC1008-mF18 was not determined under the conditions of the 3-month study in mice.

[0674] (Table 7) Main findings of histopathology in the 3-month toxicity study in mice

[0675]

[0676] In a 6-week study in monkeys (IV; 10, 30, or 100 mg / kg, Q2W, 4 doses total), no toxicologically relevant changes due to intravenous administration of hT0947AE07-SG191 were observed, and the NOAEL was at the highest dose tested, 100 mg / kg Q2W.

[0677] Example 9. Anti-tumor activity of anti-latent TGF-β1 antibody (3)

[0678] The in vivo efficacy of the anti-latent TGF-β1 monoclonal antibody hT0947AE07-SG191 combined with anti-mouse PD-L1 antibody was evaluated in the Balb / c mouse syngeneic model of EMT6 murine breast cancer.

[0679] The EMT6 murine breast cancer cell line was obtained from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (SIGMA) containing 10% fetal bovine serum (FBS; Nichirei Biosciences Inc). Six-week-old specific pathogen-free Balb / c female mice were purchased from Charles River Inc., Japan, and acclimated for 1 week before inoculation. EMT6 cells growing in the logarithmic phase were harvested and washed with Hank's balanced salt solution (HBSS; SIGMA) and incubated at 1 × 10 6 Resuspended in 50% HBSS and 50% Matrigel (CORNING) at a concentration of 1×10 cells / mL. 1×10 cells / mL in 100 μL HBSS:Matrigel (1:1) were inoculated in the left mammary fat pad #5 of the mouse. 5 EMT6 cells.

[0680] When the average tumor volume reaches about 100-300 mm 3 At 7 days after inoculation, mice were randomly divided into groups according to tumor volume and body weight. Tumor volume was measured with a caliper and calculated as 1 / 2 × l × w 2 , l=length, w=width.

[0681] Mice were treated with vehicle (150 mM NaCl / 20 mM His-HCl buffer pH 6.0), anti-mouse PD-L1 antibody (rat IgG2b clone 10F.9G2, purchased from Bio X cell, 10 mg / kg first dose, followed by 5 mg / kg), hT0947AE07-SG191 (10 mg / kg) combined with anti-mouse PD-L1 antibody, or hT0947AE07-SG191 (30 mg / kg) combined with anti-mouse PD-L1 antibody. The antibodies were administered 3 times a week for 2 weeks, with the first dose administered intravenously and subsequent doses administered intraperitoneally.

[0682] Tumor volume was measured twice a week. Antitumor activity was evaluated by tumor growth inhibition (TGI [%]) calculated as {1-(T-T0) / (C-C0)}×100, where the mean tumor volume of each group on the measurement day was T and on the randomization day was T0, and the vehicle control group was represented by C and C0.

[0683] The results of this experiment are as follows Fig.13 shown.

[0684] The TGI [%] 14 days after the first dose of anti-mouse PD-L1 antibody alone, hT0947AE07-SG191 (10 mg / kg) in combination with anti-mouse PD-L1 antibody, and hT0947AE07-SG191 (30 mg / kg) in combination with anti-mouse PD-L1 antibody were 60, 77, and 80, respectively. hT0947AE07-SG191 showed combined efficacy with anti-mouse PD-L1 antibody.

[0685] Although the present invention has been described in some detail herein by way of illustration and example for the purpose of clear understanding, these descriptions and examples should not be construed as limiting the scope of the present invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

[0686] Industrial Applicability

[0687] The present invention provides cross-species anti-latent TGF-β1 antibodies that inhibit protease-mediated activation of latent TGF-β1 without inhibiting integrin-mediated activation of latent TGF-β1. The present invention also provides a combination therapy comprising an anti-latent TGF-β1 antibody and a checkpoint inhibitor. The anti-latent TGF-β1 antibody of the present invention (which can be administered in combination with a checkpoint inhibitor) is expected to be useful for treating TGF-β1-related diseases, such as fibrosis and cancer.

Claims

1. An anti-latent TGF-β1 antibody, comprising: (a) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 23, 24 and 25, respectively; (b) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 31, respectively; (c) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33 and 34, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 36 and 37, respectively; or (d) HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively, and HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively.

2. The anti-latent TGF-β1 antibody according to claim 1, comprising: (a) (i) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13, or (iii) 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: 14, (ii) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15, 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: 16, (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 with the amino acid sequence of SEQ ID NO: 18, (ii) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 19, or (iii) a VH sequence as in (i) and a VL sequence as in (ii).

3. Anti-latent TGF-β1 antibody, which is 10 -8 nM or less, 10 -9 nM or less or 10 -10 Binds to latent TGF-β1 with a dissociation constant (KD) of nM or less.

4. An anti-latent TGF-β1 antibody that inhibits the activation of latent TGF-β1.

5. An anti-latent TGF-β1 antibody which inhibits protease-mediated and / or integrin-mediated release of mature TGF-β1 from latent TGF-β1.

6. An anti-latent TGF-β1 antibody which results in reduced or diminished toxicity and / or side effects associated with anti-TGF-β antagonists.

7. A method for producing an anti-latent TGF-β1 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell or host cell culture medium.

8. A method for screening anti-latent TGF-β1 antibodies, wherein include: (a) contacting a biological sample containing latent TGF-β1 and a protease with a detection antibody; (b) testing (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 (c) Selection of detection antibodies that inhibit activation of latent TGF-β1 without inhibiting protease-mediated cleavage of the LAP portion of latent TGF-β1.

9. A method for detecting the presence of latent TGF-β1, wherein include: (a) contacting the biological sample with an anti-latent TGF-β1 antibody under conditions that allow the anti-latent TGF-β1 antibody to bind to latent TGF-β1; and (b) Detection of whether a complex is formed between the anti-latent TGF-β1 antibody and latent TGF-β1.

10. A pharmaceutical preparation comprising an anti-latent TGF-β1 antibody for use in treating fibrosis or cancer.

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