Anti-TGF-beta antibodies and use thereof

Abstract

To provide a TGF-β antibody for treatment of conditions that are mediated by TGF-β.SOLUTION: The invention provides the use of an anti-TGF-β antibody that binds specifically to human TGF-β1, TGF-β2 and TGF-β3 in the production of a drug for treating an immune-mediated disease, fibrotic condition, or congenital or bone defect in a patient. The antibody comprises heavy chain complementarity-determining regions (CDR) 1-3 in a specific sequence and light chain CDR1-3 in another specific sequence. The antibody comprises a human IgG4 constant region having a proline at position 228 (EU numbering).SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 448,800 and European Application No. 17305061.8, both filed on January 20, 2017. The disclosures of the two priority applications are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application has been filed electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. Said ASCII copy, created on January 11, 2018, is named 022548_WO011_SL.txt and is 30,458 in size. It's a part-time job. [Background technology]

[0003] Transforming growth factor beta (TGF-β) is a cytokine that controls many important cellular functions, including proliferation, differentiation, survival, migration, and epithelial-mesenchymal transition. It regulates various biological processes such as extracellular matrix formation, wound healing, embryonic development, bone development, hematopoiesis, immune and inflammatory responses, and malignant transformation. Deregulation of TGF-β leads to pathological conditions, such as birth defects, cancer, chronic inflammation, and autoimmune and fibrotic diseases.

[0004] TGF-β has three known isoforms-TGF-β1, 2, and 3. All three isoforms are initially transcribed as precursor peptides. After cleavage, the mature C-terminus remains associated with the N-terminus (called latency-associated peptide or LAP) to form a small latent complex (SLC) that is secreted from the cell. The inability of SLC to bind to TGF-β receptor II (TGFβRII) prevents receptor engagement. Activation by dissociation of the N- and C-termini occurs by one of several mechanisms, including proteolytic cleavage, acidic pH, or integrin conformational changes (Non-Patent Document 1).

[0005] TGF-β1, 2, and 3 are pleiotropic in their functions and are expressed in different patterns across cell and tissue types. They are similar in activity in vitro, but individual knockouts in specific cell types suggest non-identical roles in vivo, even though they share the ability to bind to the same receptor (Non-Patent Document 2). Upon TGF-β binding to TGFβRII, the constitutive kinase activity of the receptor phosphorylates and activates TGF-βRI, phosphorylating SMAD2 / 3, allowing binding to SMAD4, localization to the nucleus, and transcription of TGF-β responsive genes. Ibid. In addition to this classical signaling cascade, non-classical pathways signal through other factors, including p38 MAPK, PI3K, AKT, JUN, JNK, and NF-κB. TGF-β signaling is also regulated by other pathways, including WNT, Hedgehog, Notch, INF, TNF, and RAS. Thus, the end result of TGF-β signaling is the crosstalk of all of these signaling pathways integrating the state and environment of the cell. Same document. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Connolly et al., Int J Biol Sci (2012) 8(7):964-78 [Non-Patent Document 2] Akhurst et al., Nat Rev Drug Discov (2012) 11(10):790-811 [Non-Patent Document 3] Bedinger et al., mAbs. (2016) 8(2):389-404 [Non-Patent Document 4] Larkin et al., N Engl J Med (2015) 373:23-34 [Non-Patent Document 5] Redman et al., BMC Med (2016) 14:20-30 Summary of the Invention [Problem to be solved by the invention]

[0007] Considering the diverse functions of TGF-β, there is a need for pan-TGF-β specific therapeutic antibodies that are safe for human patients (Non-Patent Document 3). However, TGF-β is highly conserved among species. As a result, the production of antibodies against human TGF-β in animals such as mice is a difficult task.

[0008] There is also a medical need for patients for whom there is currently no effective treatment. For example, in the Phase III Checkmate-067 study, more than 50% of patients with advanced melanoma treated with the anti-PD1 antibody nivolumab monotherapy did not show a complete or partial response to treatment (Non-Patent Document 4; Non-Patent Document 5). [Means for solving the problem]

[0009] The present invention provides improved monoclonal antibodies that specifically bind to human TGF-β1, TGF-β2, and TGF-β3 (i.e., pan-TGF-β specific). These antibodies are less likely to form half-antibodies (i.e., dimeric complexes with one heavy chain and one light chain) during production. They also exhibit superior pharmacokinetic profiles, such as extended half-lives, and thus can provide improved clinical benefit to patients. The inventors have also discovered that TGF-β inhibition, such as that caused by the antibodies and antigen-binding fragments of the present invention, alleviates the immune-suppressive microenvironment in tumors and enhances the efficacy of immunotherapies, such as those targeting programmed cell death protein 1 (PD-1), PD-1 ligand 1 (PD-L1) and 2 (PD-L2).

[0010] In one aspect, the invention provides an isolated monoclonal antibody that specifically binds human TGF-β1, TGF-β2, and TGF-β3, comprising heavy chain complementarity determining regions (CDRs) 1-3 in SEQ ID NO: 1 and light chain CDRs 1-3 in SEQ ID NO: 2, wherein the antibody comprises a human IgG4 constant region with a mutation at position 228 (EU numbering). In some embodiments, the mutation is a serine to proline mutation (S228P). In some embodiments, the antibody comprises a heavy chain variable domain (V H ) amino acid sequence and the light chain variable domain (V L ) amino acid sequence. In a further embodiment, the antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 1 (with or without the C-terminal lysine) and the light chain amino acid sequence set forth in SEQ ID NO: 2. The invention also features F(ab')2 antigen-binding fragments of the above antibodies.

[0011] In a preferred embodiment, the antibody or fragment of the present invention exhibits an increased half-life, an increased exposure, or both, compared to fresolimumab. For example, the increase is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. The exposure of a drug, such as the antibody or fragment of the present invention, is a function of the concentration of the drug in the body over time. The concentration of the drug in the body is often indicated by the level of the drug in the blood, plasma, or serum. The half-life and exposure (bio-exposure) of a drug are measured by well-known methods, as shown in Example 7 below.

[0012] The present invention further provides a composition comprising an antibody of the present invention, wherein the composition comprises less than 1% half-antibody. Half-antibody formation is determined, for example, by purity analysis of a monoclonal antibody preparation using SDS-capillary electrophoresis under non-reducing conditions or non-reducing SDS-PAGE analysis followed by densitometry, or RP-HPLC. (Angal et al., Mol Immunol (1993) 30(1):105-8; Bloom et al., Protein Science (1997) 6:407-415; Schuurman et al., (2001) 38(1):1-8; and Solanos et al., Anal Chem (2006) 78:6583-94). In some embodiments, the composition is a pharmaceutical composition that also includes a pharma- ceutically acceptable excipient.

[0013] In another aspect, the invention provides a method of inhibiting TGF-β signaling in a patient (human) in need thereof, comprising administering to the patient a therapeutic amount of an antibody or fragment of the invention. In some embodiments, the patient has an immune-mediated disease (e.g., scleroderma), a fibrotic condition (e.g., a fibrotic condition of the kidney, such as focal segmental glomerulosclerosis (FSGS), or a fibrotic condition of the lung, such as idiopathic pulmonary fibrosis), or a congenital or bone defect (e.g., osteogenesis imperfecta). In some embodiments, the patient has cancer. In some embodiments, the antibody or fragment used in the method is an antibody or fragment that inhibits CD4 + The antibody or fragment inhibits the differentiation of T cells into inducible regulatory T cells (iTreg). The antibody or fragment may alleviate the immunosuppressive tumor microenvironment. This action of the antibody or fragment helps to activate the immune system and enhance the efficacy of immunotherapy. The efficacy of the treatment methods described herein is indicated, for example, by one or more of the following in a patient (e.g., in the patient's tumor tissue): (1) an increase in MIP2 and / or KC / GRO levels, (2) an increase in INF-γ-positive CD8 + CD8 like T cells + T cell activation or infiltration into tumor tissue, and (3) an increase in the clustering of natural killer (NK) cells.

[0014] The present invention further provides a method for treating cancer in a patient (human), comprising administering to the patient (1) a therapeutically effective amount of an antibody or fragment of the present invention, and (2) a therapeutically effective amount of an inhibitor of an immune checkpoint protein. The two agents are administered simultaneously (e.g., in a single composition or in separate compositions), or sequentially in any order. The two agents are administered, for example, on the same day. In some embodiments, therapeutic agent (1) is administered to the patient prior to therapeutic agent (2) (e.g., one or several days prior).

[0015] In some embodiments, the immune checkpoint protein is PD-1, PD-L1, or PD-L2. In further embodiments, the inhibitor of an immune checkpoint protein is an anti-PD-1 antibody. In further embodiments, the anti-PD-1 antibody comprises (1) a heavy chain CDR1-3 in SEQ ID NO:5 and a light chain CDR1-3 in SEQ ID NO:6, (2) a V corresponding to residues 1-117 of SEQ ID NO:5, and (3) a V corresponding to residues 2-31 of SEQ ID NO:5. H Amino acid sequence and V corresponding to residues 1 to 107 of SEQ ID NO:6 L or (3) a heavy chain amino acid sequence set forth in SEQ ID NO:5 (with or without a C-terminal lysine) and a light chain amino acid sequence set forth in SEQ ID NO:6. In one particular embodiment, the method comprises administering to a cancer patient an anti-TGF-β antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO:1 (with or without a C-terminal lysine) and a light chain amino acid sequence set forth in SEQ ID NO:2, and an anti-PD-1 antibody comprising a heavy chain amino acid sequence set forth in SEQ ID NO:5 (with or without a C-terminal lysine) and a light chain amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the patient is refractory to anti-PD-1 antibody monotherapy. The patient may have advanced or metastatic melanoma, or squamous cell carcinoma.

[0016] In some regimens, the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient every 2 weeks or every 3 weeks. In some regimens, the two agents are each administered at a dose of 0.01 to 40 (e.g., 0.02 to 20, 0.05 to 15, or 0.05 to 20) mg / kg body weight.

[0017] The present invention also provides a method of increasing an immune response in a patient in need thereof comprising administering to the patient an immune checkpoint inhibitor and an antibody or fragment of the present invention. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, such as one comprising: (1) HCDRs 1-3 in SEQ ID NO:5 and LCDRs 1-3 in SEQ ID NO:6; (2) a VH and VL corresponding to residues 1-117 of SEQ ID NO:5 and residues 1-107 of SEQ ID NO:6, respectively; or (3) a heavy chain having the amino acid sequence of SEQ ID NO:5 (with or without a C-terminal lysine) and a light chain having the amino acid sequence of SEQ ID NO:6.

[0018] The method of the present invention is used to treat various cancers, including, but not limited to, melanoma (e.g., metastatic or progressive), lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, fallopian tube cancer, uterine cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), urothelial cancer, and kidney cancer (e.g., renal cell carcinoma). In some embodiments, the patient has a mesenchymal tumor or a mesenchymal subtype of a solid tumor. Examples of such solid tumors include those in the colon (e.g., colorectal cancer), ovary, head and neck (e.g., head and neck squamous cell carcinoma), liver (e.g., hepatocellular carcinoma), and urothelial system.

[0019] In some embodiments, cancers, including mesenchymal tumors, are characterized by overexpression of one or more of ACTA2 (smooth muscle alpha 2 actin), VIM (vimentin), MGP (matrix Gla protein), ZWINT (ZW10 interacting centromere protein), and ZEB2 (zinc finger E-box binding homeobox 2). Expression levels of such biomarkers are determined at the mRNA or protein level in a biological sample from a patient, such as, for example, a tumor biopsy or circulating tumor cells.

[0020] The invention also provides the above-mentioned antibodies, fragments, or compositions for use in the treatment of the conditions described herein, as well as the use of the above-mentioned antibodies, fragments, or compositions in the manufacture of a medicament for the treatment of the conditions described herein.

[0021] Also included in the invention are nucleic acid expression vectors encoding the heavy or light chains, or both, of the antibodies of the invention; host cells comprising antibody heavy and light chain coding sequences; and methods of making antibodies using the host cells comprising culturing the host cells in an appropriate culture medium, expressing the antibody genes, and then recovering the antibody. [Brief description of the drawings]

[0022] [Figure 1A] Graphs showing the effect of Ab1, fresolimumab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with 1 ng / ml of human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E). Antibody concentrations are in μg / ml. [Figure 1B] Graphs showing the effect of Ab1, fresolimumab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with 1 ng / ml of human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E). Antibody concentrations are in μg / ml. [Figure 1C] Graphs showing the effect of Ab1, fresolimumab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with 1 ng / ml of human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E). Antibody concentrations are in μg / ml. [Figure 1D]Graphs showing the effect of Ab1, fresolimumab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with 1 ng / ml of human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E). Antibody concentrations are in μg / ml. [Figure 1E] Graphs showing the effect of Ab1, fresolimumab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with 1 ng / ml of human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E). Antibody concentrations are in μg / ml. [Diagram 2] 1 is a bar graph showing the effect of Ab1 50 μg / ml on human induced regulatory T cell (iTreg) differentiation. Stimuli provided to T cells were anti-CD3 and anti-CD28 antibodies plus IL-2. [Diagram 3] 1 is a bar graph showing the effect of Ab1 on human inducible regulatory T cell (iTreg) differentiation in human CD4+ T cell cultures treated with 2 ng / ml human TGF-β1. Stimuli provided to T cells were anti-CD3 and anti-CD28 antibodies plus IL-2. [Figure 4] 1 is a bar graph showing the effect of Ab1 (30 μg / ml) and human TGF-β1 (18 ng / ml) on NFATc-driven luciferase expression in Jurkat T cells following T cell stimulation and anti-PD-1 treatment. [Diagram 5] Graph showing median tumor volume by median absolute deviation (MAD) in the indicated treatment groups using the C57BL / 6 MC38 colon mouse model. Vehicle: PBS. "Anti-PD-1": x-anti-mPD-1 Mab (see detailed description below). "Isotype control for Ab1": anti-HEL hlgG4. [Figure 6] Scatter plot showing tumor volume change from baseline on day 27 of the indicated treatments using a C57BL / 6 MC38 colon mouse model. Control: PBS. "Anti-PD-1 RPM114mlgG1": x-anti-mPD-1 Mab. [Figure 7A] FIG. 1 is a graph showing tumor volume over time for each indicated treatment group using a C57BL / 6 MC38 colon mouse model. Each line in the graph represents one animal. "mpk": mg / kg. "Ab1 isotype Ctrl": anti-HEL hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7B] FIG. 1 is a graph showing tumor volume over time for each indicated treatment group using a C57BL / 6 MC38 colon mouse model. Each line in the graph represents one animal. "mpk": mg / kg. "Ab1 isotype Ctrl": anti-HEL hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7C] FIG. 1 is a graph showing tumor volume over time for each indicated treatment group using a C57BL / 6 MC38 colon mouse model. Each line in the graph represents one animal. "mpk": mg / kg. "Ab1 isotype Ctrl": anti-HEL hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7D] FIG. 1 is a graph showing tumor volume over time for each indicated treatment group using a C57BL / 6 MC38 colon mouse model. Each line in the graph represents one animal. "mpk": mg / kg. "Ab1 isotype Ctrl": anti-HEL hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7E] FIG. 1 is a graph showing tumor volume over time for each indicated treatment group using a C57BL / 6 MC38 colon mouse model. Each line in the graph represents one animal. "mpk": mg / kg. "Ab1 isotype Ctrl": anti-HEL hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7F] FIG. 1 is a graph showing tumor volume over time for each indicated treatment group using a C57BL / 6 MC38 colon mouse model. Each line in the graph represents one animal. "mpk": mg / kg. "Ab1 isotype Ctrl": anti-HEL hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 8] FIG. 13 is a graph showing the effect of Ab1 on active TGF-β1 concentrations in LoVo tumor lysates. [Figure 9A] FIG. 1 is a graph showing serum concentrations of Ab1 and fresolimumab over time in five groups of rats given a single dose of 5 mg / kg of either antibody. Groups (Gr.) 1-3 received three different batches of fresolimumab (B1, B2, and B3). Groups 4 and 5 received two different batches of Ab1 (B1 and B2). [Figure 9B] FIG. 1 is a graph showing serum concentrations of Ab1 and fresolimumab over time in monkeys given a single dose of 1 mg / kg of either antibody. [Figure 9C] Graph showing serum concentrations of Ab1 and fresolimumab over time in monkeys given five weekly doses of Ab1 at 1 mg / kg per dose or biweekly doses of fresolimumab at 1 mg / kg per dose for the study durations indicated. [Figure 9D] FIG. 1 is a graph showing serum concentrations of Ab1 and fresolimumab over time in monkeys given a single dose of 10 mg / kg of either antibody. [Figure 9E] 1 is a graph showing serum concentrations of Ab1 and fresolimumab over time in monkeys given five weekly doses of Ab1 at 10 mg / kg per dose or biweekly doses of fresolimumab at 10 mg / kg per dose for the study durations indicated. [Figure 10A] FIG. 1 is a graph showing changes in the levels of TGF-β1 in MC38 tumors following treatment with Ab1 (+ / - anti-PD1). [Figure 10B] FIG. 1 is a graph showing changes in MIP-2 levels in MC38 tumors following treatment with Ab1 (+ / - anti-PD1). [Figure 10C] FIG. 1 is a graph showing changes in KC / GRO levels in MC38 tumors after treatment with Ab1 (+ / - anti-PD1). [Figure 11A] 1 is a graph quantifying CellTrace Violet staining and IFN-γ staining of CD8 pos cells. [Figure 11B] FIG. 1 is a graph showing that Ab1 restored both proliferation and IFN-γ production in TGFβ-treated CD8+ T cells. [Figure 12A] Graph showing relative abundance (log2 transformed) of CD8+ T cells across a list of syngeneic mouse tumor models of colon cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma, and renal cancer. [Figure 12B] 1 is a graph showing TGFβ pathway activation across a list of syngeneic mouse tumor models of colon cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma, and renal cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention features improved pan-TGF-β specific monoclonal antibodies that are less likely to form half-antibodies than prior known antibodies and also have superior pharmacokinetic profiles such as higher exposure in vivo. The antibodies, collectively referred to as "Ab1 and related antibodies", share common structural features, including heavy chain CDRs (HCDRs) 1-3 in SEQ ID NO: 1 and light chain CDRs (LCDRs) 1-3 in SEQ ID NO: 2, and a human IgG4 constant region in which residue 228 (EU numbering) in the hinge region is mutated from serine to proline. P228 is boxed and bold in the sequence of SEQ ID NO: 1 shown below.

[0024] Antibody Ab1 has a predicted molecular weight of 144 KD when unglycosylated. Its heavy and light chain amino acid sequences are SEQ ID NOs: 1 and 2, respectively. The two sequences are shown below. The variable domains are in italics. The CDRs are shown in boxes. The glycosylation site in the constant domain of the heavy chain is in bold (N297).

[0025] [ka]

[0026] In some embodiments, antibodies of the invention, such as anti-TGF-β antibodies, do not have a C-terminal lysine in their heavy chains. The C-terminal lysine is removed during production or by recombinant techniques (i.e., the coding sequence for the heavy chain does not contain a codon for the C-terminal lysine). Thus, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO: 1 without the C-terminal lysine are also contemplated within the invention.

[0027] Ab1 and related antibodies specifically bind to human TGF-β1, -β2, and -β3. By "specifically," we mean that binding occurs over a 10-fold increase in specificity as determined, for example, by surface plasmon resonance (see, e.g., Example 1 below) or Bio-Layer interferometry. -8 M (e.g., 1 to 5 nM) or less, such as 10 -7 K is smaller than M D Ab1 and related antibodies may also have strong TGF-β neutralizing potency when assayed in a mink lung epithelial cell assay (see, e.g., Example 2 below), or an EC50 of about 0.05 to 1 μg / ml as determined in an A549 cell IL-11 induction assay (see, e.g., Example 6 of PCT Publication WO 2006 / 086469, the disclosure of which is incorporated herein by reference in its entirety).

[0028] These antigen-binding and neutralizing properties of Ab1 and related antibodies are comparable to the earlier anti-TGF-β antibody fresolimumab (germline IgG4 PET1073G12 antibody described in WO 2006 / 086469). The heavy and light chain sequences of fresolimumab, including the leader sequence, are shown in SEQ ID NOs: 3 and 4, respectively. As shown in SEQ ID NO: 3, fresolimumab does not have a proline at position 228 (EU numbering, which corresponds to actual position 247 in SEQ ID NO: 3). Ab1 and related antibodies have several improved properties over fresolimumab.

[0029] During manufacturing, fresolimumab can form at most 6-18% half antibodies (i.e., dimers with one heavy chain and one light chain rather than tetramers with two heavy chains complexed with two light chains) under non-reducing denaturing conditions. In contrast, Ab1 produces substantially fewer half antibodies (<1%). Thus, Ab1 and related antibodies yield a purer drug product during manufacturing.

[0030] Furthermore, Ab1 and related antibodies can have improved pharmacokinetic (PK) profiles than fresolimumab. They can have linear PK behavior with a much longer half-life and lower elimination rate than fresolimumab, resulting in approximately 1.7-fold higher exposure in vivo than fresolimumab. For example, in rats, Ab1 has been shown to have an average half-life of 7.1 days compared to 4.3 days for fresolimumab, and an elimination rate (CL) of 0.30 ml / hr / kg compared to 0.51 ml / hr / kg for fresolimumab (Example 7, below). In cynomolgus monkeys, Ab1 has been shown to have a half-life of 7.1 days compared to 4.3 days for fresolimumab, and an elimination rate (CL) of 0.30 ml / hr / kg compared to 0.51 ml / hr / kg for fresolimumab (Example 7, below). It has been shown to have an average half-life of 13 days compared to 4.5 days for fresolimumab, and an elimination rate (CL) of 0.40 ml / hr / kg compared to 0.66 ml / hr / kg for fresolimumab. Id. These improved PK properties indicate that Ab1 and related antibodies can be given to patients in lower doses and / or less frequently than fresolimumab to achieve the same or better clinical efficacy, with fewer adverse side effects and less anti-drug antibody response, thus allowing for longer periods of treatment if necessary.

[0031] Furthermore, during toxicity studies of fresolimumab in non-human primates, a correlation between drug exposure and adverse events such as anemia was observed, although no such events were observed in similar studies performed with Ab1, even at equal or higher exposures.

[0032] Without wishing to be bound by theory, the inventors hypothesize that mutation of residue 228 in the heavy chain of Ab1 and related antibodies results in increased stability and improved PK and toxicity profiles.

[0033] The constant domains of Ab1 and related antibodies are optionally further modified, for example at Kabat residue L248 (eg, by introducing an L248E mutation) to reduce any undesirable effector functions of the molecule.

[0034] As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetrameric protein containing two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (V H ) and the heavy chain constant region (C H Each light chain contains a light chain variable domain (V L ) and the light chain constant region (C L ) is composed of V H and V L The domains are further subdivided into regions of hypervariability, called "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, called "framework regions" (FRs). H or V L consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each region are based on the IMGT® definition (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or the Kabat definition, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989).

[0035] The term "human antibody" refers to an antibody in which the variable domain and constant region sequences are derived from human sequences. The term includes antibodies with sequences derived from human genes, but whose sequences have been modified, for example to reduce immunogenicity, increase affinity, and increase stability. The term includes antibodies produced recombinantly in non-human cells, which may be conferred glycosylation that is not typical in human cells.

[0036] The term "chimeric antibody" refers to an antibody that contains sequences from two different animal species. For example, a chimeric antibody is one that combines the V sequences of a mouse antibody (i.e., an antibody encoded by mouse antibody genes, such as an antibody obtained from an immunized mouse using hybridoma technology) with the constant region of an antibody from another species (e.g., human, rabbit, or rat). H and V L may include.

[0037] The term "antigen-binding fragment" of an antibody refers to a fragment of an antibody that retains the ability to specifically bind to an antigen. In some embodiments, an antigen-binding fragment of the invention is a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region (Fab is a V L , V H , C L and C H1 A monovalent antibody fragment consisting of a domain In some embodiments, the antigen-binding fragments of the invention also comprise H2 or C H3 It may also include a domain.

[0038] The antibodies and antigen-binding fragments described herein are isolated. The term "isolated protein", "isolated polypeptide" or "isolated antibody" refers to a protein, polypeptide or antibody that, depending on its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its natural state, (2) is substantially free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell in which it naturally occurs will be "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

[0039] I. Use of Ab1 and Related Antibodies TGF-β receptors are widely expressed on immune cells, resulting in the broad effects of TGF-β in both the innate and adaptive immune systems. TGF-β is associated with many disease states, such as birth defects, cancer, chronic inflammation, autoimmunity, and fibrotic diseases. Therapeutic amounts of Ab1 or related antibodies are used to treat these conditions. A "therapeutically effective" amount refers to an amount of Ab1, related antibodies, or another therapeutic agent referred to herein that alleviates one or more symptoms of the treated condition. This amount will vary based on the condition or patient being treated and will be determined by a medical professional using well-established principles.

[0040] In some embodiments, Ab1 or related antibodies are administered at 40, 20, or 15 mg / kg or less (such as 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / kg). In some further embodiments, the dose may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 mg / kg. The frequency of administration may be, for example, daily, every 2, 3, 4, or 5 days, weekly, biweekly, or 3 weeks, monthly, or bimonthly. The antibody is administered intravenously (e.g., by intravenous infusion over 0.5 to 8 hours), subcutaneously, topically, or by any other route of administration appropriate to the condition and formulation.

[0041] Ab1 and related antibodies are derived from human antibody genes and therefore have low immunogenicity in humans. The toxicity study of Ab1 is detailed in Example 8 below. In rats, certain cardiac and pulmonary side effects have been observed. Therefore, when patients are treated with Ab1 or related antibodies, they are monitored for adverse events.

[0042] In some embodiments, efficacy of the antibodies of the invention is demonstrated in a patient (e.g., in a diseased tissue, such as a tumor tissue, of a patient) by one or more of the following: (1) a decrease in the level or activity of TGF-β, (2) an increase in MIP2 and / or KC / GRO levels, (3) an increase in INF-γ-positive CD8 + CD8 like T cells + T cell activation or infiltration into tumor tissue, and (4) increased clustering of natural killer (NK) cells.

[0043] A. Non-oncological disease conditions Conditions treated by Ab1 and related antibodies include, but are not limited to, bone defects (e.g., osteogenesis imperfecta), glomerulonephritis, nerve or skin injuries, lung or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced fibrosis, liver fibrosis, myelofibrosis, scleroderma, immune-mediated diseases (rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Berger's disease, and transplant rejection), and Dupuytren's contracture.

[0044] They are also useful for treating, preventing and reducing the risk of developing renal failure, including, but not limited to, focal segmental glomerulosclerosis (FSGS), diabetic (type I and type II) nephropathy, radiation nephropathy, obstructive nephropathy, diffuse systemic sclerosis, congenital kidney disease (e.g., polycystic kidney disease, sponge kidney, horseshoe kidney), glomerulonephritis, nephrosclerosis, nephrocalcinosis, systemic or glomerular hypertension, tubulointerstitial nephropathy, renal tubular acidosis, renal tuberculosis, and renal infarction. In particular, they are useful when combined with antagonists of the renin-angiotensin-aldosterone system, including, but not limited to: renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, AngII receptor antagonists (also known as "AngII receptor blockers"), and aldosterone antagonists. See, for example, WO 2004 / 098637, the disclosure of which is incorporated herein by reference in its entirety.

[0045] Ab1 and related antibodies are useful in treating diseases and conditions associated with ECM deposition, such as systemic sclerosis, post-surgical adhesions, keloids and hypertrophic scars, proliferative vitreoretinopathy, glaucoma surgery, corneal injuries, cataracts, Peyronie's disease, adult tachycardia syndrome, liver cirrhosis, post-myocardial infarction scarring, restenosis after angioplasty, scarring after subarachnoid hemorrhage, post-laminectomy fibrosis, fibrosis after tendon and other repairs, biliary cirrhosis (including sclerosing cholangitis), pericarditis, pleurisy, tracheotomy, penetrating CNS injury, eosinophilic myalgia syndrome, vascular restenosis, venous occlusive disease, pancreatitis, and psoriatic arthropathy.

[0046] Ab1 and related antibodies are further useful in conditions where promotion of re-epithelialization is beneficial, including, but not limited to, skin conditions such as venous ulcers, ischemic ulcers (pressure ulcers), diabetic ulcers, transplant sites, transplant donor sites, abrasions and burns, conditions of the bronchial epithelium such as asthma, ARDS, mucositis associated with cytotoxic treatments, conditions of the intestinal epithelium such as esophageal ulcers (reflex disease), gastroesophageal reflux disease, gastric ulcers, small and large intestinal lesions (inflammatory bowel disease).

[0047] Further uses of Ab1 and related antibodies are in conditions where endothelial cell proliferation is desirable, for example, in stabilizing atherosclerosis, promoting healing of vascular anastomoses, or in conditions where inhibition of smooth muscle cell proliferation is desirable, such as in arterial disease, restenosis, and asthma.

[0048] Ab1 and related antibodies are also useful in enhancing immune responses to macrophage-mediated infections such as those caused by Leishmania spp., Trypanosoma cruzi, Mycobacterium tuberculosis and Mycobacterium leprae, as well as Toxoplasma gondii, Histoplasma capsulatum, Candida albicans, Candida parapsilosis, and Cryptococcus neoformans. They are also useful in reducing immunosuppression caused, for example, by tumors, AIDS, or granulomatous diseases.

[0049] Ab1 and related antibodies are also useful in the prevention and / or treatment of ophthalmic conditions such as glaucoma and post-fibrous zonectomy scarring.

[0050] B. Oncological Disease Conditions TGF-β regulates several biological processes, including cell proliferation, epithelial-mesenchymal transition (EMT), matrix remodeling, angiogenesis, and immune function. Each of these processes contributes to tumor progression. A broad detrimental role for TGF-β in cancer patients across indications has also been suggested by its elevation within the tumor microenvironment as well as systemically. See, for example, Kadam et al., Mol. Biomark. Diagn. (2013) 4(3). Studies have shown that in malignant conditions, TGF-β can induce EMT and the resulting mesenchymal phenotype leads to increased cell migration and invasion.

[0051] Ab1 and related antibodies may be used in, but are not limited to, the treatment of skin cancer (e.g., unresectable or metastatic The present invention is useful for treating hyperproliferative diseases such as cancers including melanoma, including metastatic melanoma, squamous cell carcinoma and keratoacanthoma, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), primary peritoneal cancer, bladder cancer, kidney or renal cancer (e.g., renal cell carcinoma), urothelial carcinoma, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), brain cancer, glioblastoma, glioma, mesothelioma, leukemia and lymphoma.

[0052] In some embodiments, Ab1 and related antibodies are useful for treating cancer in patients who have failed or are expected to fail a prior therapy based on anti-PD-1, anti-PD-L1, or anti-PD-L2 therapeutic agents, i.e., patients who are non-responders or are expected to be non-responders to anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy. In some embodiments, Ab1 and related antibodies are useful for treating cancer in patients who have relapsed from a prior anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy. As used herein, the term "predicted" means that a person skilled in the medical arts can predict, based on his / her general medical knowledge and the particular condition of the patient, without administering the treatment, whether the patient will be a responder or non-responder, and whether the treatment will fail or be ineffective.

[0053] In some embodiments, the cancer is a mesenchymal subtype of solid tumors, including but not limited to mesenchymal colorectal cancer, mesenchymal ovarian cancer, mesenchymal lung cancer, mesenchymal head cancer, and mesenchymal neck cancer. Epithelial-mesenchymal transition (EMT) promotes cell migration and invasion properties by downregulating epithelial cell genes and enhancing mesenchymal gene expression. EMT is a hallmark of tumor progression and invasion. Up to a quarter of colorectal and ovarian cancers are mesenchymal. Thus, by inhibiting TGF-β and its induction of EMT, Ab1 or related antibodies are used to treat mesenchymal solid tumors. Several genetic markers and pathological tests identify mesenchymal subtypes of solid tumors. Markers include ACAT2, VIM, MGP, ZEB2, and ZWINT, which are detected by qRT-PCR or immunohistochemistry. Such markers are used to select patients for anti-TGF-β monotherapy or combination therapy of the present invention.

[0054] In some embodiments, Ab1 and related antibodies are useful for treating patients with advanced solid tumors.

[0055] Ab1 and related antibodies are also used in the treatment of hematopoietic disorders or malignancies such as multiple myeloma, myelodysplastic syndromes (MDS), Hodgkin's lymphoma, non-Hodgkin's lymphoma, and leukemia, as well as various sarcomas such as Kaposi's sarcoma.

[0056] Ab1 and related antibodies are also useful for inhibiting cyclosporine-mediated malignancy or cancer progression (eg, metastasis).

[0057] In the context of cancer therapy, it will be understood that "treatment" includes any medical procedure that results in partial remission of cancer, slowing the growth of cancer, delaying the progression or recurrence of cancer, or reducing cancer metastasis, as well as extending the patient's life expectancy.

[0058] C. Combination Therapies in Oncology It has been observed that the level of cytotoxic T cell infiltration in cancer is associated with favorable clinical outcome (Fridman et al., Nat Rev Cancer (2012) 12(4):298-306; and Galon et al., Immunity (2013) 39(1):11-26). Furthermore, the level of cytotoxic T cells (CD4 + T H1 Helper T cells, which help immune cells (such as T cells) and the cytokines they produce (e.g., IFN-γ) are also often associated with positive patient outcomes. In contrast, the presence of Treg cells has been shown to be associated with poor patient prognosis (Fridman, supra).

[0059] TGF-β suppresses nearly all aspects of the antitumor immune response. The cytokine promotes iTreg differentiation and inhibits cytotoxicity (CD8 + ) reduces cell proliferation and invasion. Inhibition of TGF-β by Ab1 or related antibodies, as described above, alleviates the immunosuppressive tumor microenvironment and provides positive outcomes for cancer patients.

[0060] Furthermore, the inventors have discovered that by alleviating the immunosuppressive tumor microenvironment, Ab1 and related antibodies can enable checkpoint modifiers, such as anti-PD-1 antibodies, to better induce an immune response, and as a result, more patients will benefit from immunotherapies, such as anti-PD-1, anti-PD-L1, or anti-PD-L2 treatments.

[0061] Ab1 and related antibodies, with or without therapeutic agents targeting immune checkpoint molecules, are also used in conjunction with other cancer therapies, such as chemotherapy (e.g., platinum or taxoid-based therapies), radiation therapy, and therapies targeting cancer antigens or oncogenic drivers.

[0062] Cancers that may be treated with a combination comprising Ab1 or a related antibody and an immune checkpoint inhibitor, such as an anti-PD-1 antibody, include those cancers listed in the subsections above.

[0063] In some embodiments, the cancer is refractory to prior anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy, such as advanced or metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and Hodgkin's lymphoma. Refractory patients are those who experience disease progression without any evidence of response, e.g., radiologically confirmed, within 12 weeks of initiating treatment.

[0064] In some embodiments, Ab1 or a related antibody is used in conjunction with another cancer therapy, such as anti-PD-1 therapy, to treat mesenchymal cancers such as colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, head and neck squamous cell carcinoma, renal cell carcinoma, hepatocellular carcinoma, and squamous cell carcinoma. See also discussion above.

[0065] Examples of anti-PD-1 antibodies are nivolumab, pembrolizumab, pidilizumab, MEDI0608 (formerly AMP-514; see, e.g., WO 2012 / 145493 and U.S. Pat. No. 9,205,148), PDR001 (see, e.g., WO 2015 / 112900), PF-06801591 (see, e.g., WO 2016 / 092419), and BGB-A317 (see, e.g., WO 2015 / 035606). In some embodiments, the anti-PD-1 antibodies include those disclosed in WO 2015 / 112800 (H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2 in Table 1 of the PCT publication). , H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P and H4xH9008P, and those referred to as H4H7798N, H4H7795N2, H4H9008P and H4H9048P2 in Table 3 of the PCT Publication. The disclosure of WO 2015 / 112800 is incorporated herein by reference in its entirety.

[0066] For example, CDRs, V H and V L The sequences, or heavy and light chain sequences, and antibodies that bind to the same PD-1 epitope as the antibodies disclosed in the PCT publication, The antibodies and related antibodies disclosed in WO 2015 / 112800, including antibodies and antigen-binding fragments thereof, are used in conjunction with the Ab1 or related antibodies of the invention to treat cancer. In a related embodiment, useful anti-PD-1 antibodies have the heavy and light chain amino acid sequences shown below as SEQ ID NOs: 5 and 6, respectively; the V and V of SEQ ID NOs: 5 and 6, respectively; H and V Lor one or more (eg all six) CDRs of SEQ ID NOs:5 and 6 (shown in boxes).

[0067] [ka]

[0068] In some embodiments, antibodies of the invention, such as anti-PD-1 antibodies, do not have a C-terminal lysine in their heavy chains. The C-terminal lysine is removed during production or by recombinant techniques (i.e., the coding sequence for the heavy chain does not contain a codon for the C-terminal lysine). Thus, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO:5 without the C-terminal lysine are also contemplated within the invention.

[0069] In some embodiments, the anti-TGF-β antibodies or fragments of the present invention are also used in conjunction with antibodies against immune-regulatory antigens such as PD-L1 and CTLA-4. Exemplary anti-PD-L1 antibodies are atezolizumab, avelumab, durvalumab, LY3300054 and BMS-936559. Exemplary anti-CTLA-4 antibodies are ipilimumab or tremelimumab.

[0070] D. Biomarkers of Treatment Efficacy The efficacy of Ab1 and related antibodies is determined by biomarkers or target occupancy. For example, in tumor tissue, target occupancy is assayed by assessing the levels of active TGF-β in biopsies using the Meso Scale Discovery (MSD) assay. In blood, target engagement is assayed by assessing the effect of reduction of circulating TGF-β on peripheral blood mononuclear cells such as lymphocytes (T cells, B cells, NK cells) and monocytes. For example, circulating CD8 + Increased T cell proliferation was observed by expressing CD45 as a marker in flow cytometry + RO + CCR7 + CD28 + Ki67 +Activation of circulating NK cells is assessed using CD3 as a marker for flow cytometry. - CD56 high / dim CD16 + or CD137 + In addition, Ki-67, PD-1, and ICOS are used as PD markers associated with T cell activation.

[0071] Immune modulation upon treatment with Ab1 or related antibodies is assayed, for example, by assessing changes in infiltrating immune cells and immune markers by multiplex immunohistochemistry (IHC) assays using the NeoGenomics platform. In particular, NeoGenomic's MultiOmyx TIL Panel staining of a panel of immune markers allows for quantitative determination of density and localization of various immune cells. Immune markers include iTreg differentiation; CD8 + T cell infiltration and proliferation; and CD8 + Ab1 can indicate the production of IFNγ by CD4 T cells. + Inhibits differentiation of T cells into iTregs (see, e.g., Example 3 below) and inhibits CD8 + It was shown to increase T cell proliferation and their production of IFNγ (as shown in a mixed lymphocyte reaction assay; data not shown). Thus, the efficacy of treatment with Ab1 or related antibodies was confirmed by inhibition of iTregs, CD8 + Induction of T cell proliferation and infiltration into tumors or other diseased tissues, increased IFNγ production, and / or CD8 + Immunomodulation upon treatment with Ab1 or related antibodies also increased the ratio of CD8 T cells to Treg cells. + T cells, Treg cells, NK cells, and other immune cells are assayed in peripheral blood by methylation PCR-based quantitative immune cell counting. The efficacy of treatment can be demonstrated clinically as a delay or amelioration in disease progression, such as tumor progression.

[0072] II. Antibody Production Methods Ab1 and related antibodies, as well as antibodies targeting other co-targets such as PD-1, PD-L1 or PD-L2, are produced by methods well established in the art. The DNA sequences encoding the heavy and light chains of the antibody are inserted into an expression vector such that the genes are operably linked to the necessary expression control sequences, such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody light chain coding sequence and the antibody heavy chain coding sequence are inserted into separate vectors and operably linked to the same or different expression control sequences (e.g., promoters). In one embodiment, both coding sequences are inserted into the same expression vector and operably linked to the same expression control sequence (e.g., a common promoter), to separate same expression control sequences (e.g., promoters), or to different expression control sequences (e.g., promoters). The antibody coding sequence is inserted into the expression vector by standard methods (eg, ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0073] In addition to the antibody chain genes, the recombinant expression vector may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters such as the native immunoglobulin and actin promoters.

[0074] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may have additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. For example, the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes may include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and glutamate synthesis genes.

[0075] An expression vector encoding an antibody of the invention is introduced into a host cell for expression. The host cell is cultured under conditions suitable for expression of the antibody, and then harvested and isolated. Host cells include mammalian, plant, bacterial or yeast host cells. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, in particular, Chinese Hamster Ovary ( CHO cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. Cell lines are selected based on their expression levels. Other cell lines that may be used are insect cell lines such as Sf9 or Sf21 cells.

[0076] Furthermore, expression of the antibody can be enhanced using a number of known techniques, for example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions.

[0077] The tissue culture medium for the host cells may or may not contain animal derived components (ADCs) such as bovine serum albumin. In some embodiments, ADC-free culture medium is preferred for human safety. Tissue culture is performed using fed-batch, continuous perfusion, or any other method appropriate for the host cells and the desired yield.

[0078] III. Pharmaceutical Compositions The antibody of the present invention is formulated for suitable storage stability.For example, the antibody is lyophilized or stored or reconstituted for use using a pharma-ceutically acceptable excipient.For combination therapy, two or more therapeutic agents such as antibodies are formulated together, for example, mixed and provided in a single composition.

[0079] The term "excipient" or "carrier" is used herein to describe any ingredient other than the compound of the present invention. The choice of excipient largely depends on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form to be administered. "Pharmaceutically acceptable excipient" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonicity and absorption delaying agents, etc. that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffer, dextrose, glycerin, ethanol, etc., and combinations thereof. In some cases, isotonicity agents, such as sugars, mannitol, sorbitol, or polyalcohols such as sodium chloride, are included in the composition. Further examples of pharmaceutically acceptable substances are auxiliary agents, such as wetting agents or minor amounts of wetting agents or emulsifying agents, preservatives, or buffers that enhance the shelf life or effectiveness of the antibody.

[0080] The pharmaceutical composition of the present invention is prepared, packaged, or sold in bulk as a single unit dose or as a plurality of single unit doses.As used herein, "unit dose" is an individual amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is usually equal to the dosage of active ingredient that is administered to subject, or a convenient fraction of such dosage, such as, for example, one-half or one-third of such dosage.

[0081] The pharmaceutical composition of the present invention is typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by the administration of the pharmaceutical composition through physical bleaching and tissue bleaching of the subject's tissue, thus usually resulting in direct administration into the bloodstream, into muscle, or into an internal organ. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is considered to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion; as well as kidney dialysis drip techniques. Local perfusion is also considered. Preferred embodiments include intravenous and subcutaneous routes.

[0082] A formulation of a pharmaceutical composition suitable for parenteral administration typically comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations are prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations are prepared, packaged, or sold in unit dosage form, such as ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional components, including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (e.g., pH 3 to 9), although in some applications they are more suitably formulated as sterile nonaqueous solutions used in conjunction with a suitable vehicle such as sterile, pyrogen-free water, or in dry form. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextrose. Such dosage forms are suitably buffered if desired. Other orally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form, or in a liposomal formulation. Formulations for parenteral administration are formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed release.

[0083] IV. Illustrative Embodiments More specific embodiments of the present invention are described as follows: 1. An isolated monoclonal antibody that specifically binds to human TGF-β1, TGF-β2, and TGF-β3, comprising heavy chain complementarity determining regions (CDRs) 1 to 3 in SEQ ID NO: 1 and light chain CDRs 1 to 3 in SEQ ID NO: 2, said antibody comprising a human IgG4 constant region having a proline at position 228 (EU numbering). 2. A heavy chain variable domain (V H ) amino acid sequence and the light chain variable domain (V L 2. The antibody of embodiment 1, comprising the amino acid sequence: 3. The antibody of embodiment 2, comprising a heavy chain amino acid sequence as set forth in SEQ ID NO:1 (with or without a C-terminal lysine) and a light chain amino acid sequence as set forth in SEQ ID NO:2. 4. An antigen-binding fragment of the antibody described in embodiment 3, which is F(ab')2. 5. The antibody or fragment according to any one of embodiments 1 to 4, which exhibits an extended half-life or increased exposure compared to fresolimumab. 6. The following: a) CD4 + Inhibits differentiation of T cells into induced regulatory T cells (iTreg) b) CD8 + Increase T-cell proliferation; c) increasing clustering of natural killer (NK) cells; d) increasing the levels of MIP-2; and e) Increases KC / GRO levels 6. The antibody or fragment of any one of embodiments 1 to 5, having one or more of the following properties: A composition comprising the antibody or fragment of any one of embodiments 1 to 6, comprising less than 7.1% half antibodies. 8. The antibody or fragment according to any one of embodiments 1 to 6 as a medicament. 9. A method for inhibiting TGF-β signaling in a patient in need thereof, comprising administering to the patient a therapeutic amount of an antibody or fragment according to any one of embodiments 1 to 6. 10. The method of embodiment 9, wherein the patient has cancer. 11. Cancers include melanoma, lung cancer, squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, 11. The method of embodiment 10, wherein the cancer is selected from the group consisting of head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma. 12. The method of embodiment 10 or 11, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT. 13. The method of any one of embodiments 10-12, wherein the cancer is a mesenchymal tumor. 14. The method of any one of embodiments 10 to 13, wherein the antibody or fragment alleviates an immunosuppressive tumor microenvironment. 15. A method of treating cancer in a patient, comprising: (1) An antibody or fragment thereof according to any one of embodiments 1 to 6, and (2) Immune checkpoint protein inhibitors to the patient. 16. The method of embodiment 15, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2. 17. The method of embodiment 16, wherein the inhibitor of an immune checkpoint protein is an anti-PD-1 antibody. 18. The method of embodiment 17, wherein the anti-PD-1 antibody comprises heavy chain CDRs 1-3 in SEQ ID NO:5 and light chain CDRs 1-3 in SEQ ID NO:6. 19. The anti-PD-1 antibody comprises a V corresponding to residues 1 to 117 of SEQ ID NO:5. H Amino acid sequence and V corresponding to residues 1 to 107 of SEQ ID NO:6 L 18. The method of embodiment 17, comprising an amino acid sequence. 20. The method of embodiment 17, wherein the anti-PD-1 antibody comprises a heavy chain amino acid sequence set forth in SEQ ID NO:5 (with or without the C-terminal lysine) and a light chain amino acid sequence set forth in SEQ ID NO:6. 21. The method according to any one of embodiments 15 to 20, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO:1 (with or without a C-terminal lysine) and the light chain amino acid sequence set forth in SEQ ID NO:2. 22. The method of any one of embodiments 15-21, wherein the cancer is refractory to anti-PD-1 antibody treatment. 23. The method of any one of embodiments 15 to 22, wherein the cancer is progressive or metastatic melanoma, or squamous cell carcinoma. 24. The method of any one of embodiments 15 to 23, wherein the cancer is a mesenchymal subtype of a solid tumor. 25. The method of any one of embodiments 15 to 24, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. 26. The method of any one of embodiments 15 to 25, wherein the cancer is selected from the group consisting of melanoma, lung cancer, squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma. 27. The method of any one of embodiments 15 to 26, wherein the antibody or fragment alleviates an immunosuppressive tumor microenvironment. 28. The method of any one of embodiments 15-27, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient on the same day. 29. The method of any one of embodiments 15-28, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient every two weeks. 30. The method of any one of embodiments 15-29, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are each administered at a dose of 0.05 to 20 mg / kg body weight. 31. A method for increasing an immune response in a patient in need thereof, comprising administering to the patient an immune checkpoint inhibitor and an antibody or fragment thereof according to any one of embodiments 1 to 6. 32. The method of embodiment 31, wherein the checkpoint inhibitor is an anti-PD-1 antibody. 33. Anti-PD-1 antibodies: a) HCDRs 1 to 3 of SEQ ID NO: 5 and LCDRs 1 to 3 of SEQ ID NO: 6; b) V corresponding to residues 1 to 117 of SEQ ID NO:5 and residues 1 to 107 of SEQ ID NO:6, respectively H and V L ;or c) a heavy chain having the amino acid sequence set forth in SEQ ID NO:5 (with or without the C-terminal lysine) and a light chain having the amino acid sequence set forth in SEQ ID NO:6 33. The method of embodiment 32, comprising: 34. The method of any one of embodiments 31 to 33, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO:1 (with or without the C-terminal lysine) and the light chain amino acid sequence set forth in SEQ ID NO:2. 35. The method of any one of embodiments 31-34, wherein the patient has cancer. 36. The method of embodiment 35, wherein the patient is refractory to prior treatment with an immune checkpoint inhibitor and / or has a mesenchymal subtype of a solid tumor. 37. The method of embodiment 35 or 36, wherein the cancer is selected from the group consisting of melanoma, lung cancer, squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma. 38. The method of any one of embodiments 35 to 37, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT. 39. The method of any one of embodiments 35 to 38, wherein the antibody or fragment alleviates an immunosuppressive tumor microenvironment. 40. The antibody or fragment according to any one of embodiments 1 to 6 for use in treating a patient in any of the above methods. 41. Use of an antibody or fragment according to any one of embodiments 1 to 6 for the manufacture of a medicament for treating a patient in any of the above methods. 42. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain, the light chain, or both, of the antibody or fragment of any one of embodiments 1 to 6. 43. An expression vector comprising the isolated nucleic acid molecule of embodiment 42. 44. A host cell comprising the expression vector of embodiment 43. 45. Providing a host cell comprising a first and a second nucleotide sequence encoding a heavy chain and a light chain, respectively, of an antibody or antigen-binding fragment; growing the host cells under conditions that allow for the production of the antibody or antigen-binding fragment; recovering the antibody or antigen-binding fragment; 7. A method for producing the antibody or antigen-binding fragment of any one of embodiments 1 to 6, comprising: 46. 1. A method for producing a pharmaceutical composition comprising: Providing an antibody or antigen-binding fragment according to any one of embodiments 1 to 6; mixing the antibody or antigen-binding fragment with a pharma- ceutically acceptable excipient; The method comprising: 47. A product or kit comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 6 and another therapeutic agent. 48. The product or kit of embodiment 47, wherein the other therapeutic agent is an immune checkpoint inhibitor as described herein.

[0084] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0085] [Example] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. EXAMPLES

[0086] TGF-β binding properties of Ab1 The affinity of Ab1 to all human and mouse TGF-β isoforms was determined by surface plasmon resonance on a Biacore T200 Biosensor instrument (GE Healthcare) using an S-series chip coated with carboxymethylated (CM5) dextran. A series of concentrations of Ab1 (1.11, 3.33, 10, and 30 nM) were injected onto immobilized recombinant TGF-β, and the binding interaction was measured in real time. TGF-β homodimers were immobilized at low density to reduce avidity effects. Injections were performed in triplicate, and binding assays were repeated three times. Data from kinetic experiments were processed using Biacore T200 Biaevaluation v2.0 software. The resulting sensorgrams were zeroed, aligned, double-referenced, and cropped for curve-fitting analysis using a 1:1 binding model to determine the association rate constant (k a ), dissociation rate constant (k d ), and the equilibrium dissociation constant (K D ) was decided.

[0087] Recombinant proteins were either produced in-house (human TGF-β1, 2 and 3) or obtained from R&D Systems (mouse TGF-β1 and 2). Table 1 below shows the amino acid sequence homology of the three active TGF-β isoforms between rhesus monkey, mouse or rat and human (homology is reported as the percentage of conserved amino acids out of the total amino acids).

[0088] [Table 1]

[0089] Human TGF-β3 and mouse TGF-β3 have identical amino acid sequences, so no different affinity measurements were calculated for these two proteins. Similarly, mouse and rat TGF-β1 and 2 have identical amino acid sequences, so no different affinity measurements were calculated.

[0090] The k of Ab1 determined by the above methoda , k d , and K D The values ​​are shown in Table 2 below. K of Ab1 for human TGF-β1, 2, and 3 D The K values ​​were determined to be 1.48, 3.00, and 1.65 nM, respectively. D The values ​​were determined to be 2.80 and 1.88 nM, respectively. These binding characteristics were similar to those of fresolimumab.

[0091] [Table 2]

[0092] The above data demonstrated that Ab1 is a potent and selective pan-TGF-β inhibitor. Measurements using surface plasmon resonance demonstrated that Ab1 has an affinity between 1 and 5 nM for all human and mouse TGF-β isoforms. The high level of specificity was confirmed by GLP immunohistochemistry (IHC) tissue cross-reactivity studies using normal rat, cynomolgus monkey and human tissues. EXAMPLES

[0093] TGF-β neutralizing ability of Ab1 The in vitro ability of Ab1 in neutralizing TGF-β activity was measured in a cell-based assay. This assay measured the ability of TGF-β to inhibit the proliferation of non-transformed mink lung epithelial cells (Mv 1Lu cells). See, e.g., WO 2006 / 086469 and Mazzieri, et al., Eds, “Methods in Molecular Biology,” Vol. 142, “Transforming Growth Factor-β Protocols”. The ability of Ab1, fresolimumab, and 1D11 (a mouse anti-TGF-β antibody, the heavy and light chain sequences of which are disclosed herein as SEQ ID NOs: 9 and 10) to neutralize human TGF-β1, 2, 3, and mouse TGF-β1 and 2 was evaluated. Recombinant TGF-β proteins were either produced in-house (human TGF-β1, 2, and 3) or obtained from R&D Systems (mouse TGF-β1 and 2).

[0094] All human and mouse TGF-β isoforms inhibited mink lung cell proliferation in a dose-dependent manner ranging from 0.02 pg / ml to 10 ng / ml. To quantify the potency of Ab1, fresolimumab, and 1D11, the indicated TGF-β and serially diluted antibodies at 1 ng / ml were incubated with mink lung cells. After 3 days of incubation, cell proliferation was quantified by CyQUANT dyes that fluoresce upon binding to DNA (Figure 1A-E). The data showed that Ab1, fresolimumab, and their mouse surrogate 1D11 inhibited all human and mouse TGF-β isoforms to a similar extent. EXAMPLES

[0095] Inhibition of inducible regulatory T cell differentiation by Ab1 Regulatory T cells (Tregs) are immunosuppressive and have been associated with negative outcomes in cancer patients. In the studies described below, the inventors demonstrated that Ab1 inhibits human CD4 + We investigated whether TGF-β-induced differentiation of T cells into induced regulatory T cells (iTregs) could be inhibited. +T cells were isolated from healthy donors. Human TGF-β1 was purchased from R&D Systems.

[0096] To examine the antagonistic activity of Ab1 against TGF-β produced endogenously by cultured cells, total CD4 + T cells were treated with 50 μg / ml of isotype control (human IgG4, kappa anti-hen egg lysozyme (HEL) antibody, Crown Biosciences), Ab1, or fresolimumab in the presence or absence of stimulation (anti-CD3, anti-CD28, and IL-2) for 6 days and then analyzed by flow cytometry. + FOXP3 + The mean percentage and standard deviation of the populations were calculated from the parent populations (lymphocytes / liver / single cells / CD4 + CD127 - ) were calculated in triplicate. Total human CD4+ with anti-CD3, anti-CD28, and IL-2 + Stimulation of T cells by FOXP3 in culture + CD25 + Treatment with Ab1 50 μg / ml or fresolimumab 50 μg / ml reduced the percentage of iTregs to a similar extent (8% and 7%, respectively; Figure 2). In contrast, treatment with human IgG4 (hIgG4) isotype control showed minimal effect on iTreg differentiation (20% iTreg) (Figure 2). CD4 + T cells produced similar results.

[0097] To examine the antagonistic activity of Ab1 against exogenous TGF-β, total CD4 + T cells were treated for 6 days with various antibody concentrations of isotype control, Ab1, or fresolimumab in the presence or absence of stimulation (anti-CD3, anti-CD28, and IL-2) and then analyzed by flow cytometry. + FOXP3 +The mean percentage and standard deviation of the populations were calculated from the parent populations (lymphocytes / liver / single cells / CD4 + CD127 - ) were calculated in triplicate unless otherwise noted. Total human CD4 + Addition of Ab1 to T cells increased the percentage of iTregs in culture from 15% to 55%. Treatment with increasing concentrations of Ab1 decreased the percentage of iTregs in a follow-up manner from 55% to 15% at 200 μg / ml and 43% at 6.25 μg / ml. Treatment with fresolimumab decreased the percentage of iTregs to a similar extent as Ab1 (55% to 16% at 200 μg / ml and 32% at 6.25 μg / ml). Treatment with various concentrations of isotype control antibody had no effect on the percentage of iTregs, 60% at 200 μg / ml and 6.25 μg / ml. See Figure 3. CD4 isolated from a second healthy volunteer + T cells produced similar results.

[0098] This study demonstrated that Ab1 inhibits TGF-β-induced iTreg differentiation and thus may confer clinical benefit by alleviating the immunosuppressive tumor microenvironment. EXAMPLES

[0099] Effect of Ab1 and anti-PD-1 antibody combination in vitro In this study, we investigated whether TGF-β prevents maximal stimulation of T cells in vitro after anti-PD-1 treatment and, if so, whether Ab1 can counteract this prevention.Luciferase expression from an expression construct under the transcriptional control of the NFATc (nuclear factor of activated T cells, cytoplasmic 1) regulatory sequence was used to measure the level of T cell activation.

[0100] We used a cell assay system purchased from Promega for this study. The system contains two cell types: 1) Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element, and 2) CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate the cognate T cell receptor in an antigen-independent manner. When co-cultured, Jurkat T cells interacted with CHO-K1 cells, resulting in T cell receptor stimulation and translocation of NFATc to the nucleus, which drives luciferase expression. However, PD-1 / PD-L1 engagement recruited nonreceptor type 11 protein tyrosine phosphatase (SHP2) to the T cell receptor complex, inhibiting NFATc nuclear translocation and subsequent luciferase expression. Blockade of PD-1 signaling relieved SHP2-dependent inhibition, thus allowing maximal luciferase expression. Thus, the system provided a functional method to determine the effect of TGF-β on T cell signaling and the impact of Ab1 on anti-PD-1 treatment of T cells.

[0101] Due to the slow kinetics associated with TGF-β-dependent effects, Jurkat T cells were pretreated with TGF-β prior to T cell receptor stimulation. Human TGF-β1 was purchased from R&D Systems. Ab1 isotype control antibody (anti-HEL hlgG4) was purchased from Crown Bioscience (Cat#C0004-5). Mouse anti-hPD-1 IgG and its isotype control antibody were purchased from BioLegend (Cat#329912). Fourteen replicates were analyzed for each sample.

[0102] Results showed that addition of anti-hPD-1 antibody to Jurkat T cells cocultured with CHO-K1 cells for 24 h induced greater luciferase activity (865794 relative light units [RLU]) than addition of isotype control (234963 RLU, fold change = 3.685, p value < 0.0001) or simply no antibody (206043 RLU, fold change = 4.202, p value < 0.0001). Pretreatment of Jurkat T cells with TGF-β1 18 ng / ml for 12 days induced lower luciferase activity (638866 RLU) in CHO-K1 cell co-cultures in the presence of anti-hPD-1 antibody compared to Jurkat T cells not treated with TGF-β1 (865794 RLU, fold change = -1.355, p-value < 0.0001) (Figure 4).

[0103] To assess the antagonistic potential of Ab1, Jurkat T cells were pretreated with TGF-β1 18ng / ml in the presence of Ab1, isotype control Ab, or no Ab for 12 days and then co-cultured with CHO-K1 cells in the presence of anti-PD-1 for 24 hours. The presence of Ab1 (924186 RLU) attenuated the TGF-β-dependent inhibition of luciferase activity compared to the isotype control Ab (639440 RLU, fold change = 1.445, p value < 0.0001) and no Ab control (638866 RLU, fold change = 1.447, p value < 0.0001). Control groups in which Ab1 (975,654 RLU) or isotype control (955,717 RLU) was added to Jurkat T cells co-cultured with CHO-K1 cell co-cultures in the presence of anti-PD-1 Ab without pretreatment with TGF-β1 showed a statistically increased luciferase activity compared to the no Ab control, but with a minimal fold change (865,794 RLU, fold change = 1.127 and 1.104, respectively, p-value = 0.0023 and 0.001284) (Figure 4). RLU values ​​are also shown in Table 3 below.

[0104] [Table 3]

[0105] To exclude the possibility that TGF-β1 pretreatment of Jurkat T cells resulted in reduced proliferation or viability and therefore reduced luciferase activity during the 24-h coculture with CHO-K1 cells, we incubated Jurkat T cells with TGF-β1 18 ng / ml or PBS for 7 days in the presence of Ab1, anti-HEL hlgG4, or no antibody (vehicle). Every 2-3 days, an equal amount of each group was used to seed new flasks, at which time both TGF-β1 and antibody were renewed (a total of two reseeding events occurred). Assessment of the final cultures demonstrated that the viability of all treatment groups was altered (ranging from 94% to 96%). Furthermore, the total number of Jurkat T cells in the final cultures of each treatment group was very similar (ranging from 25 to 29 million).

[0106] The above studies demonstrated that the increase in downstream signaling of the T cell receptor after anti-PD-1 treatment was suppressed by TGF-β, resulting in suboptimal T cell stimulation. Our data suggested that inhibition of TGF-β could alleviate the immunosuppressive tumor microenvironment, allowing checkpoint modulators such as anti-PD-1 agents to induce better immune responses, thus increasing the proportion of patients who benefit from immuno-oncological treatment. EXAMPLES

[0107] Effect of Ab1 and anti-PD1 antibody combination in vivo We next studied the effect of combined anti-TGF-β and anti-PD-1 treatment in a C57BL / 6 mouse cancer model.

[0108] Tolerability / Preliminary Safety material and method The tolerability of Ab1 and anti-mouse PD-1 (mPD-1) monoclonal antibodies (mAbs) as single agents and in combination was evaluated in female C57BL / 6 mice. Ab1 (10, 20, and 50 mg / kg) or an isotype control Ab (anti-HEL hlgG4 was purchased from Crown Bioscience; used at 10 and 20 mg / kg) was administered IV every 3 days (Q3D) as a single agent or in combination with anti-PD1 Mab 5 mg / kg IV twice weekly. The anti-PD-1 Ab used in this study is denoted as "anti-mPD1_hyb_RMP114_mIgG1LCfullrat" (or x-anti-mPD-1 Mab). It is a rat IgG 2a It was a chimeric rat anti-mPD-1 antibody generated by replacing the rat Fc region of clone RMP1-14 (BioXcell, Cat.#BE0146) with the mouse IgG1 Fc region. The heavy and light chain amino acid sequences of this chimeric antibody are shown in SEQ ID NOs: 7 and 8. Tolerability was assessed by measuring the body weight of the animals and clinical observations. At the end of the 3-week treatment, 4 hours after the last treatment, a final sampling was performed and tissues (heart, kidney, liver, lung, and spleen) were fixed in formaldehyde and sent for histopathological analysis.

[0109] A dose was considered highly toxic if there was 15% weight loss on three consecutive days, 20% weight loss on one day, or 10% or more drug-related deaths in individual mice, unless tumor-induced cachexia resulting in weight loss was observed in the control vehicle-treated group. Animal weights included tumor weight.

[0110] Toxicity / safety results Tolerability studies in C57BL / 6 mice showed that all dose levels tested of Ab1 and x-anti-mPD-1 Mab single agents and combinations were well tolerated. No significant changes in body weight were observed at all doses tested in any treatment group. No serious or gross clinical observations were observed during the study. Histopathological analysis identified increased numbers of lymphocytes in the spleen (white pulp) of all treatment groups, including the isotype control antibody treatment group, without any dose-related association in any combination. No other significant microscopic findings were observed. Two mice in the isotype control Ab (10 mg / kg) and anti-PD-1 Mab (5 mg / kg) combination group were found dead after the last dose on the last day of the study. Histopathological analysis did not find any drug associated cause of death.

[0111] Efficacy Testing The effect of combined anti-TGF-β and anti-PD-1 treatment in C57BL / 6 mice bearing subcutaneous MC38 syngeneic colon tumors was evaluated. Mice received Ab1 25mg / kg, x-anti-mPD-1 Mab 5mg / kg, or both Q3D for 3 weeks. This study demonstrated that the combination of Ab1 and anti-mPD-1 Mab had significantly greater antitumor activity than either single agent alone. The materials and methods and the date of this study are detailed below.

[0112] material and method animal Female C57BL / 6 mice were obtained from Charles River Labs (Wilmington, MA, USA). Animals were acclimated for at least 3 days before inclusion in the study. Mice were 11 weeks old at the start of the study and weighed between 17.0 and 20.9 g. They were allowed free access to chow (Harlan 2916 rodent chow, Massachusetts, USA) and sterile water and were housed on a 12-h light / dark cycle.

[0113] Tumor cells MC38 is a colon adenocarcinoma cell line. Cells were obtained from the National Cancer Institute (Bethesda, MD, USA) and cultured at 37°C and 5% CO2 in complete medium (CM) containing Roswell Park Memorial Institute medium (RPMI)-1640 with L-glutamine (Gibco, Cat#11875) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) (Gibco, Cat#10438026). Cells were harvested, resuspended in Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Cat#14190), and cultured at 1 × 10 per mouse. 6 Cells / 200 μl were implanted subcutaneously (SC) into the right flank of female C57BL / 6 mice.

[0114] compound Ab1 was administered to animals in an aqueous solution that was passed through a 0.22 μm filter through PES and stored in sterile water at 2–10° C. Antibody was given to animals intraperitoneally (IP) at 10 ml / kg, 25 mg / kg.

[0115] Anti-HEL hlgG4 (Crown Bioscience) was used as an isotype control for Ab1 and was given by IP at 10 ml / kg to control animals and by IP at 25 mg / kg.

[0116] x-anti-mPD-1 Mab (above) was provided in DPBS (Gibco, Cat#14190-094) and given to animals IP at 10 mg / kg, 5 mg / kg.

[0117] Study design On day 0, 60 animals were implanted with MC38 tumor cells. On day 8 after implantation, the average tumor size was 50–75 mm. 3Mice bearing the IL-1 IgG1 gene were pooled and randomly divided into control and treatment groups (10 mice per group). Treatment with vehicle (PBS, pH 7.2), anti-HEL hlgG4, Ab1, and anti-mPD-1 Mab at the doses mentioned above was initiated on day 9 and repeated on days 12, 15, 18, 21, and 27. Vehicle and anti-HEL hlgG4 treated animals were used as controls. Mice were checked daily and clinically adverse reactions were recorded. Individual mice were weighed 3 to 4 times per week until the end of the experiment.

[0118] Mice were euthanized if ≥20% morbidity or weight loss was observed. Tumors were measured by caliper twice weekly until terminal sacrifice. 3 When the tumor volume reached 100 mg / kg / day or when the animal had health problems (20% of the tumor area was ulcerated), the animal was euthanized and the date of death was recorded. Solid tumor volume was estimated from bilateral tumor measurements and calculated using the following formula: Tumor volume (mm 3 ) = [Length (mm) x Width 2 (mm 2 )] / 2 The calculation was made according to

[0119] The median percent regression of a group on a given day was then obtained by taking the median of the individual percent regressions calculated for each animal in the group on that day. The day of calculation was determined as the day on which ΔT / ΔC (i.e., the ratio of the median tumor volume change from baseline between the treatment and control groups) was calculated, unless the median percent regression was not representative of the group activity. In this case, the day was determined by the first day when the median percent regression was greatest. Tumor volume was defined as partial (PR) if it was reduced to 50% of the tumor volume at the start of treatment. Complete regression (CR) was defined as a tumor volume of 14 mm or more. 3 was considered to have been achieved if it was less than or not recorded.

[0120] efficacy The primary efficacy endpoint was tumor volume change from baseline as indicated by ΔT / ΔC, median percent regression, partial regression, and complete regression. Changes in tumor volume for each treatment (T) and control (C) group were calculated for each animal each day by subtracting the tumor volume on the day of first treatment (staging day) from the tumor volume on the particular observation day. Median ΔT was calculated for the treatment groups and median ΔC was calculated for the control group. ΔT / ΔC ratios were calculated and expressed as percentages. ΔT / ΔC = (median delta T / median delta C) x 100

[0121] A ΔT / ΔC ratio ≦40% was considered therapeutically active. A ΔT / ΔC ratio of 0% was considered as tumor stasis. A ΔT / ΔC ratio <0% was considered as tumor regression.

[0122] Percent tumor regression was defined as the percentage of tumor volume reduced in a treatment group on a particular observation day compared to the tumor volume at the start of the study (t0). At a particular time point (t), for each animal, the percent regression was calculated using the following formula: Retraction%(t)=[(Volume t0 -volume t ) / volume t0 ] x 100 was used to calculate.

[0123] The median percent regression for a group on a given day was then calculated by taking the median of the individual % regression values ​​calculated for each animal in the group. The day of calculation was determined by the day on which ΔT / ΔC was calculated, unless the median percent regression was not representative of the activity of the group. In this case, the day was determined by the first day when the median percent regression was greatest.

[0124] statistical analysis A two-way ANOVA with factors treatment and day (replicate) was performed on the tumor volume change from baseline. Treatment with significant interaction or treatment effect *For days, all treatment groups were compared to the control group on each of days 8 to 27, following comparative analysis by Bonferroni-Holm correlation for multiplexing. Tumor volume change from baseline was calculated for each animal and day by subtracting the tumor volume on the first treatment day (day 8) from the tumor volume on the specified observation day.

[0125] When heterogeneity of variances was observed between groups, the symmetric compound by group (CS) covariance structure=option was selected for ANOVA-type models (SAS Institute Inc. (2008) SAS / STAT 9.2 User's Guide by Cary NC). In Figures 5 and 6, the median and median absolute deviation (MAD) for each group are shown for each day of treatment. In Tables 4-6 below, the median and normalized MAD (nMAD=1.4826) for each group are shown. * The mean average average deviation (MAD) was reported for each measurement day. All statistical analyses were performed using SAS version v9.2 software. Probabilities less than 5% (p<0.05) were considered significant.

[0126] Efficacy Results Treatment of tumor-bearing C57BL / 6 mice with Ab1, anti-PD-1 Mab, or a combination of the two was also well tolerated and non-toxic as indicated by the normal health and activity of the animals and the lack of significant changes in body weight. As single agents, Ab1 25 mg / kg Q3D and anti-PD-1 Mab 5 mg / kg Q3D caused body weight loss values ​​of only 3.4% (day 9) and 2.1% (day 9), respectively. The combination of Ab1 (25 mg / kg Q3D) and anti-PD-1 Mab (5 mg / kg Q3D) was also well tolerated, showing body weight loss values ​​of only 1.3% (day 9) (Table 4).

[0127] As single agents, Ab1 (25 mg / kg Q3D) and anti-PD-1 Mab (5 mg / kg Q3D) showed no perturbation in tumor growth compared to animals treated with Ab1 isotype control (anti-HEL hlgG4). The ΔT / ΔC ratios at day 27 of treatment were 93% and 109%, respectively (Table 4). The combination of anti-PD-1 Mab and anti-HEL hlgG4 demonstrated minimal anti-tumor activity with a ΔT / ΔC of 31% at day 27 of treatment (not statistically different from the control group), with complete regressions observed in only 2 of 10 mice. However, the combination of anti-PD-1 Mab and Ab1 demonstrated superior anti-tumor activity from days 15 to 27 with a -1 ΔT / ΔC at day 27 of treatment (statistically different from the control group), with complete regressions observed in 6 of 10 mice (Table 4).

[0128] [Table 4]

[0129] Tables 5 and 6 and Figures 5-7 provide further data showing the activity of antibodies alone or in combination on tumor volume in mouse models.

[0130] [Table 5]

[0131] [Table 6]

[0132] Data in the tables and figures are for Ab1 25mg / kg Q3D and x-anti-mPD-1 Mab The results show that the combination with 5 mg / kg Q3D had a greater antitumor effect than either antibody at those doses. When compared to the combination with Ab1 as a single agent, this difference was statistically significant, with p values ​​of 0.0007, <0.0001, and <0.0001 on days 19, 23, and 27, respectively. When compared to the combination with x-anti-mPD-1 Mab as a single agent, this difference was also statistically significant, with p values ​​of 0.0276, 0.0004, and 0.0024 on days 19, 23, and 27 (Table 6). For the combination groups of anti-HEL hlgG4 2 5 mg / kg Q3D and x-anti-mPD-1 Mab 5 mg / kg Q3D, the treatment effect on tumor volume change from baseline was not significantly different from the effect of either agent alone on any day of measurement.

[0133] In summary, the combination of Ab1 25 mg / kg Q3D and x-anti-mPD-1 Mab 5 mg / kg Q3D had a significantly greater antitumor effect than either agent used alone from days 15 to 27.

[0134] In another study, we evaluated the antitumor activity of Ab1 at doses of 1, 10, or 25 mg / kg in combination with a murine PD-1 antibody at a dose of 5 mg / kg against the subcutaneous MC38 murine colon cancer model in C57BL / 6J mice. Exponentially growing MC38 colon adenocarcinoma cells (NCl, Frederick, MD) were cultured in RPMI-1640 supplemented with 10% FBS in a humidified incubator with 5% CO2 and then implanted subcutaneously (1 × 10 6 The average size of the tumor is 50-75 mm. 3Once tumor volume reached 100 mg / kg, mice were pooled and randomly divided into control and treatment groups (10 mice per group). Tumor-bearing mice were then treated intraperitoneally three times a week with PBS, IgG4 isotype control antibody (25 mg / kg), or Ab1 (1, 10, and 25 mg / kg) until each animal had received a total of 6-7 doses. Tumors were measured twice a week by digital calipers and tumor volumes were calculated (mm 3 = L × W × H) and graphed using GraphPad Prism. 3 Mice were euthanized by CO2 at the end of the study if tumors grew to any size or if tumors showed ulceration of >20% of the tumor surface.

[0135] As single agents, a dose of Ab1 25 mg / kg Q3D and a dose of murine α-PD-1 antibody 5 mg / kg demonstrated partial activity in MC38 tumor-bearing mice with 2 / 8 and 4 / 8 complete regressions, respectively. Combinations of Ab1 1, 10, or 25 mg / kg Q3D with murine α-PD-1 antibody 5 mg / kg Q3D were therapeutically active. When comparing tumor volume changes from baseline at day 24 post-implantation, the effect of combinations of all tested doses of Ab1 with murine α-PD-1 antibody 5 mg / kg Q3D was greater than the effect of each single agent, with 5 / 8, 6 / 8, and 7 / 8 complete regressions for Ab1 1, 10, and 25 mg / kg, respectively. Table 6A provides a summary of the results.

[0136] [Table 7]

[0137] In summary, these preclinical data demonstrated that combining PD-1 inhibition with TGF-β inhibition can inhibit tumor growth to a greater extent than checkpoint inhibitor blockade alone. EXAMPLES

[0138] Intratumoral TGF-β1 levels Intratumoral TGF-β1 levels were studied in a subcutaneous xenograft BALB / c mouse model of LoVo colorectal cancer. Mice were treated with either Ab1 or an isotype control Mab at 10, 25, or 50 mg / kg until tumor volume reached 100 mm 3 Starting when he was younger, he was given intravenous injections every three days for a total of eight IV doses.

[0139] Tumor samples stored at -80°C in 2 ml plastic tubes with 2.8 mm ceramic balls (MoBio 13114-50) were thawed at room temperature. One milliliter (ml) of cold Meso Scale Diagnostic (MSD) Tris lysis buffer (R60TX-2) supplemented with 1× Halt™ protease and phosphatase inhibitor cocktail (Thermo 78440) was added to the tissue, which was then homogenized using a Precellys® 24 Dual homogenizer (Bertin Instruments) for two cycles at 6500 rpm for 20 seconds each at 4°C. Lysates were clarified by centrifugation at 20,000 x g for 10 min in an Eppendorf 5417C centrifuge at 4 °C. The supernatants were transferred to clean, chilled Eppendorf tubes and further purified by centrifugation for an additional 20 min as above. The supernatants were then transferred to plastic 96-well storage blocks, flash frozen in liquid nitrogen, and stored at -80 °C.

[0140] The following day, samples were thawed at room temperature and placed on ice. Protein concentrations of lysates were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo 23225) according to the manufacturer's instructions. Lysates were normalized to a protein concentration of approximately 8 mg / ml using MSD Tris lysis buffer containing protease and phosphatase inhibitors (see above) and distributed into plastic microtubes.

[0141] TGF-β1 concentrations in normalized tumor lysates were measured using a human TGF-β1 kit with electrochemiluminescence assay (MSD, K151IUC-2) according to the manufacturer's instructions. Recombinant mouse TGF-β1 (R&D Systems, Cat.#7666-MB-005) serially diluted in MSD lysis buffer was used as a calibrator. Samples were loaded on the plate in duplicate. Electrochemiluminescence signals were measured using MESO Measurements were performed using a SECTOR S 600 plate reader (MSD), and TGF-β1 concentrations in samples were quantified based on a standard curve using MSD Discovery Workbench software v4.0.

[0142] The average concentration of duplicate samples was calculated by the software. Concentration values ​​determined by the software as "below the fitted curve range" or "below the detection range" were replaced with a zero value. To calculate the TGF-β1 concentration per mg of total protein, the concentration measured in the assay (pg / ml) was divided by the protein concentration of the sample (mg / ml).

[0143] The results showed that in mice injected with isotype control, intratumoral TGF-β1 levels were a median of 21.4 pg / mg of total protein, with corresponding levels undetectable in mice injected with Ab1 (Fig. 8 ).

[0144] To demonstrate the relevance of the above findings in humans, we used the above methods to test 10 human colorectal tumor samples and 10 human melanoma tumor samples for their intratumoral TGF-β1 levels as described above. In human CRC samples, TGF-β1 levels ranged from about 7 to 25 pg / mg. In human melanoma samples, TGF-β1 levels ranged from about 1 pg / ml to as high as 43 pg / ml. These data further support the use of anti-TGF-β1 therapeutics such as Ab1 in treating tumors, either alone or in conjunction with other immune checkpoint inhibitors such as anti-PD-1 antibodies. EXAMPLES

[0145] Pharmacokinetic study of Ab1 This example describes a study that characterized the pharmacokinetic (PK) profile of Ab1 and compared it with that of fresolimumab. In one study, five groups of cannulated Sprague-Dawley rats were given a single dose of Ab1 or fresolimumab 5 mg / kg intravenously. Each group had five females and five males. Blood from the rats was collected at 0.25, 6, 24, 48, 72, 144, 192, and 240 hours after administration. Ab1 and fresolimumab serum concentrations were determined by ELISA. Comparability was determined when the 90% confidence intervals of the AUC ratios (of test substance to reference) were within the range of 80% to 125%.

[0146] Antibody serum concentrations over time from the five groups of rats are shown in Figure 9A. PK parameters from groups 2, 4, and 5 (see legend to Figure 9A) are shown below in Table 7. This study demonstrated that PK parameters from 100 mg / kg / day of 100% CI 0.01 to 0.25% were significantly lower than those from 100 mg / kg / day of 100% CI 0.01 to 0.25%. 1 / 2 ) and a slower elimination rate (CL of 0.30 ml / hr / kg vs. 0.51 ml / hr / kg), indicating that Ab1 exhibited linear PK behavior. Data showed that Ab1 had a 1.7-fold higher exposure than fresolimumab in rats.

[0147] [Table 8]

[0148] Further PK studies with Ab1 (Study 2) were performed in groups of cynomolgus monkeys. Each group was 5 females and 5 males and received a single dose of Ab1 at 1 mg / kg (Figure 9B) or 10 mg / kg (Figure 9D) or 5 weekly doses of Ab1 at 1 mg / kg (Figure 9C) or 10 mg / kg (Figure 9E) per dose by intravenous infusion. Serum concentrations of Ab1 over time in monkeys are shown in Figures 9B-E. Serum concentrations over time of fresolimumab given to monkeys in a single or repeated Q2W (biweekly) dose in a previous study are also shown in the figures for comparison. These data showed that Ab1 also showed linear PK behavior in monkeys, with higher exposure after single or repeated dosing than fresolimumab at both 1 mg / kg and 10 mg / kg per dose. At a single dose of 10 mg / kg, Ab1 had a half-life of 13 days while fresolimumab had a half-life of 4.5 days; Ab1 had a CL of approximately 0.40 ml / hr / kg while fresolimumab had a CL of 0.66 ml / hr / kg. Similar to the rat studies, monkey studies also showed that Ab1 had approximately 1.7-fold higher exposure than fresolimumab.

[0149] The above studies demonstrated that Ab1 exhibits a statistically significantly longer half-life, longer clearance time, and higher biological exposure in vivo than fresolimumab.

[0150] Furthermore, studies in Ab12 tumor-bearing Balb / C mice showed that Ab1 had a similar PK profile whether administered intravenously or intraperitoneally.

[0151] Using allometric scaling in a 2-compartment model, we predicted the following PK parameters in a 70 kg man based on the monkey data (Table 8):

[0152] [Table 9]

[0153] The predicted PK parameters of Ab1 were also more favorable than those of fresolimumab in humans. For example, fresolimumab, with a CL of 12.3 ml / hr / kg in humans, showed a faster clearance rate than Ab1. EXAMPLES

[0154] Toxicity study of Ab1 Toxicity studies of Ab1 were performed in rats and cynomolgus monkeys. Drug safety assurance endpoints were evaluated under GLP (Good Laboratory Practice) at repeated weekly doses for 5 weeks. No Ab1-related histopathological findings were noted at the injection site at doses up to 10 mg / kg / dose (concentration 2 mg / ml) in monkeys and up to 30 mg / kg / dose (concentration 6 mg / ml) in rats. No Ab1-related effects on body temperature, respiratory rate, blood pressure, and ECG parameters were recorded in this study at any dose level tested in the neurological investigations.

[0155] The NOAEL (no observed adverse effects level) in rats was found to be 3 mg / kg / dose with repeated weekly dosing for 5 weeks, and the STD10 (severely toxic dose causing death or irreversible severe toxicity in 10% of animals) was found to be between 3 and 10 mg / kg / dose in rats. Toxicities included cardiac valve enlargement characterized by multiple hypertrophic nodules; as well as abnormal pulmonary conditions such as mixed cell alveolar exudates, mixed cell perivascular infiltrates, muscular arterial hypertrophy, hemorrhage, and / or increased lung weight.

[0156] The NOAEL and HNSTD (i.e., highest non-severe toxic dose above which death, lethal toxicity, or irreversible toxicity occurs) dose was found to be 10 mg / kg / dose in monkeys for repeated weekly dosing for 5 weeks (by comparison, the NOAEL in monkeys for fresolimumab was shown to be 1 mg / kg when administered every other week for 7 or 13 doses, or Q3D for 4 weeks). See also the data presented in Table 9 below.

[0157] [Table 10]

[0158] Based on the above toxicity data, Ab1 is expected to be safely administered to human patients at dosage levels of about 0.05 mg / kg to 0.5 mg / kg weekly, or less frequently, such as every other week. EXAMPLES

[0159] In vivo efficacy of anti-TGF-β monotherapy In this study, we investigated the effect of 1D11, a murine IgG1 anti-bovine TGF-β antibody that cross-reacts with human and murine TGF-β1, 2, and 3, in a metastatic syngeneic tumor model. In this model, B16-F10 murine melanoma cells were introduced IV into the footpads of C57BL / 6 mice and allowed to form metastases in the draining lymph nodes of the mice. Treatment with a control antibody, 13C4, had no effect, but treatment with 1D11 50 mg / kg three times a week starting one day after tumor inoculation completely suppressed metastases.

[0160] To investigate the role of the immune response, we used mice lacking the β2-microglobulin gene and therefore lacking CD8 + Mice lacking cytotoxic T cell responses were implanted with B16-F10 in the footpad and treated as above. In contrast to the results seen in immune-competent mice, 1D11 had no effect on the number of metastases in the draining lymph nodes of these mice. These results suggest that the mechanism of action of TGF-β inhibition is through adaptive cell-mediated immunity. EXAMPLES

[0161] TGF-β characteristics in cancer Previous studies have shown that melanoma patients who do not respond to anti-PD-1 treatment have the transcriptional signature IPRES (Hugo et al., Cell (2016) 165:35-44). To investigate the mechanism of natural resistance to anti-PD-1 monotherapy, we studied the transcriptional signatures of non-responders versus responders. We found that comparison of these profiles using Gene Set Enrichment Analyses on a database of over 1M profiles revealed a strong correlation between anti-PD-1 response and activation of TGF-β signaling in tumors. These data suggested that TGF-β is associated with natural resistance to anti-PD-1 monotherapy at baseline in melanoma.

[0162] Furthermore, we found that not only is there a correlation between anti-PD-1 responses and activation of TGF-β signaling, but that the correlation is strong (R=0.59, p-value by t-test<9E-4). Therefore, we propose the following gateway indicators: We arrived at application 1: TGF-β-mediated immune suppression in melanoma (e.g., metastatic melanoma) can contribute to natural resistance. Furthermore, we found that TGF-β-induced gene expression changes could be quenched by 1D11 treatment, confirming the specificity of the TGF-β activation signature. These results supported the benefit of using anti-TGF-β and anti-PD-1 therapeutic agents in combination to treat cancer patients who do not respond to anti-PD-1 monotherapy.

[0163] Beyond melanoma, analysis of this correlation across other tumor types revealed that mesenchymomas (e.g., CRC, HCC, head and neck squamous cell carcinoma, and ovarian cancer) were also enriched for both TGF-β activation and predicted anti-PD-1 resistance. This finding was consistent with a role for TGF-β signaling in EMT. Thus, we arrived at our gateway indication 2: mesenchymomas, especially those with immune infiltration, will benefit from anti-TGF-β and anti-PD-1 combination therapy. Using machine learning techniques, we identified a small number of genes used to select mesenchymomas from over 30 EMT marker genes; e.g., ACTA2, VIM, MGP, ZEB2, and ZWINT. ACTA2 and VIM were found to be transportable across tumor types, for example. Thus, genes within the transcriptional signature and signature of TGF-β activation may contribute as useful biomarkers for cancer patient selection at baseline for anti-TGF-β and anti-PD-1 antibody combination therapy.

[0164] To study biomarkers in the tumor microenvironment, the immune architecture of patient tumors was evaluated using MultiOmyx, a multiplex IHC assay, in CRC and melanoma. Multiplexing was performed with 12 biomarkers in one FFPE section from each tumor sample (together accounting for 22 immune cell types). The study included the extent of inflammation and assessed to what extent the analysis assessed each tumor type and correlated to possible treatment effects. Statistical methods were developed to evaluate differences at the cell population level, including replicate matching, volcano-type ordination for analysis of variance, and correlation matrices. The MultiOmyx assay demonstrated excellent technical reproducibility and precision, favorable dynamic range, select immune cells and differences in inflammatory status in the region of interest, including both positive and negative correlations between cell populations. EXAMPLES

[0165] Changes in TGF-β1, MIP-2, and KC / GRO in MC38 tumors after treatment with Ab1 with or without anti-PD-1 To demonstrate neutralization of TGF-β, the ability of Ab1 (with or without anti-PD-1) to affect cytokine expression in tumors was assessed.

[0166] MC38 tumor-bearing mice had tumors ranging in volume from 61 to 110 mm 3 If tumors were positive, they were treated with a single dose of either PBS or anti-PD-1 (5 mg / kg) alone, or with increasing doses of Ab1 (10, 25, or 50 mg / kg, ip) in combination with anti-PD-1 (5 mg / kg). Tumors were harvested at 1, 6, 10, 24, 72, and 168 hours after treatment, flash frozen in 2 ml plastic tubes containing 2.8 mm ceramic balls (Precyllys KT3961-1007.2), and stored at -80°C. To prepare lysates, tumors were thawed at room temperature. One mL of cold Meso Scale Diagnostics (MSD) Tris lysis buffer (R60TX-2) supplemented with 1× Halt™ protease and phosphatase inhibitor cocktail (Thermo78440) was added to the tissue, which was then homogenized using a Precelly® 24Dual homogenizer (Bertin Instruments) for two cycles of 6500 rpm, 20 s each at 4° C. Lysates were clarified by centrifugation at 20,000×g for 10 min in an Eppendorf 5417C centrifuge at 4° C. The supernatant was transferred to a clean, chilled Eppendorf tube. , and further purified by centrifugation for an additional 30 min as above. The supernatant was transferred to a plastic 96-well storage block and placed on ice. The protein concentration of the lysates was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo 23225) according to the manufacturer's instructions. Lysates were normalized to a protein concentration of approximately 5 mg / ml using MSD Tris lysis buffer containing protease and phosphatase inhibitors (see above), distributed into plastic microtubes, flash frozen in liquid nitrogen, and stored at -80°C.

[0167] The concentration of activated TGF-β1 in tumor lysates was measured using a human TGF-β1 kit (MSD, K151IUC-2) with an electrochemiluminescence assay. Recombinant mouse TGF-β1 (R&D Systems, 7666-MB-005) serially diluted in MSD lysis buffer was used as a calibrator. Normalized tumor lysates prepared as described above were lysed and the assay was performed according to the manufacturer's instructions. To quantify only the active form of TGFβ-1 present in the tumor rather than total TGFβ-1, including TGFβ-1 complexed with the latency-associated peptide, samples were not acid-treated. Samples were loaded on the plate in duplicate. Electrochemiluminescence signals were measured using a MESO SECTOR S 600 plate reader (MSD) and TGFβ-1 concentrations in samples were quantified based on a standard curve using MSD Discovery Workbench software v.4.0.

[0168] Compared with animals treated with either PBS or anti-PD-1 alone, animals treated with Ab1 at all dose levels (10, 25, or 50 mg / kg) together with anti-PD-1 (5 mg / kg) showed reduced levels of activated TGF-β in tumors, demonstrating engagement of Ab1 with its target in vivo (Figure 10A). Reduced levels of active TGF-β1 were observed within 1 hour and persisted for at least 168 hours.

[0169] MIP-2 (CXCL2) and KC / GRO (CXCL1) are chemotactic chemokines for granulocytes, including neutrophils. The levels of MIP-2 and KC / GRO were also assessed in these same samples. After treatment with Ab1 together with anti-PD-1, the intratumoral levels of MIP-2 showed at least a four-fold increase in animals treated with Ab-1 together with anti-PD-1 compared to those of animals treated with either PBS or anti-PD-1 alone; the increase in MIP-2 levels was shown to last for at least 168 hours (Figure 10B). Similarly, the levels of KC / GRO were also shown to increase, but at later time points of 72 and 168 hours compared to those of MIP-2 (Figure 10C). Thus, the combination of Ab1 and anti-PD-1 mAb induced a decrease in the levels of active TGF-β1 earlier than the increase in the levels of MIP-2 and KC / GRO. These results demonstrated that Ab1 can reduce and inhibit the levels of TGF-β within the tumor microenvironment. Furthermore, the observed increase in MIP-2 and KC / GRO levels indicated that they are cytokines affected by neutralization of TGF-β and therefore could serve as potential biomarkers in patients treated with Ab1. EXAMPLES

[0170] Restoration of NK cell clustering by Ab1 treatment TGF-β is known to affect the immune system by inhibiting the activity of different immune cell types. TGF-β has been reported to inhibit natural killer (NK) cell activity and NK cell-mediated ADCC (Trotta et al., Journal of Immunology (2008) 181:3784-3792). NK cells have a dense cluster of receptors as a mechanism to enhance their activity. It has recently been reported that these tightly packed clusters form IL-2 receptors and are activated via the localization of IL-2 within these clusters (Kim et al., Scientific Reports (2017) 7:40623). Purified human NK cells clustered in vitro in the presence of IL-2 showed that these It has been shown to form tightly packed clusters.

[0171] In this study, we evaluated the effect of TGF-β on NK cell "clustering" in the absence or presence of Ab1. NK cells were freshly isolated from the blood of healthy donors by negative selection with NK cell RosetteSep reagent (Stem Cell Technologies) according to the manufacturer's protocol. NK cells were cultured at 1.2 × 10 in IL-2 (100 IU / mL) supplemented with Myelocult (Stem Cell Technologies) in round-bottom assay plates (Costar). 5 Cells were cultured at 100 cells / well. TGF-β1 was added at a final concentration of 0.1, 1 or 10 ng / mL in the presence of either irrelevant IgG4 or Ab1 at 100 μg / mL as indicated. Cells were cultured for 72 hours and NK cell clustering was visualized by capturing images with a Nikon microscope.

[0172] The addition of increasing doses of TGF-β1 was shown to inhibit NK cell clustering. When Ab1, but not the IgG4 control antibody, was added to NK cell cultures, NK cell clusters were shown to occur. The results demonstrated that TGF-β neutralization affects NK cell activation, leading to increased activation and proliferation of NK cells, supporting the antitumor response of the immune system. EXAMPLES

[0173] Expanded CD8 by Ab1 treatment + Reversion of TGF-β-mediated suppression of IFN-γ production in T cells In addition to the innate immune system, TGF-β also mediates the activation of CD8 + It has been reported that CD4+ inhibits T cell activity (Flavell et al., Nature Reviews Immunology (2010) 10:554-567). 8+ To explore the role of TGF-β and Ab1 in T cell activation, purified human CD3 + An MLR (mixed lymphocyte reaction) assay system was established in which CD8 cells were mixed with BLCL cells. + Cell proliferation and IFN-γ production were first assessed in the presence of TGF-β. + CD3 cells were isolated from PBMCs fractionated from healthy donors after Ficoll gradient separation using the EasySep T Cell enrichment kit (StemCell Technologies). + Cells were then labeled with CellTrace Violet (ThermoFisher) according to the manufacturer's protocol. MLR assays were performed using labeled CD3 + cells (2×10 5 BLCL cells (Astarte Bio)(2×10 4 100 cells; 2 min). TGF-β1, IgG4 control antibody and / or Ab1 were added to the cultures as indicated, and the cultures were incubated at 37°C with 5% CO2 for 4 days. Cells were then stimulated for 4 hours in the presence of PMA cell stimulation cocktail (eBioscience) and protein transporter inhibitor cocktail (eBioscience). Live cells were identified by staining with Zombie NIR viability dye (BioLegend) on ice and washed with FACs buffer. Cells were fixed with True-Nuclear buffer (BioLegend), washed, pelleted, and resuspended in FACs buffer. Cells were prepared for flow cytometry by staining with BV650 anti-huCD4, PERCP / Cy5.5 anti-huCD8, FITC anti-huCD3, and PE anti-huIFNγ (BioLegend). Flow cytometry was performed on a BD Canto and results were analyzed with FlowJo software to identify live, single, and CD3 + Cells were gated. IFNγ + CD8 +The percentage of T cells was decreased CD8 cells undergoing proliferation based on Violet staining and positive for INF-γ staining + Cells were quantified by gating. FMO was performed as a control for all antibody staining.

[0174] Inclusion of TGF-β in the MLR assay increased the number of IFN-γ-positive CD8 + Inclusion of the Ab or control Ab in the absence of TGFβ reduced the percentage of these IFNγ T cells (Figure 11A). + The inclusion of Ab1, but not a control antibody, had no effect on the generation of proliferating CD8+ cells (Figure 11B). However, the inclusion of Ab1, but not a control antibody, increased IFNγ production in a dose-dependent manner. + CD8 + These results suggest that TGF-β neutralization can restore cell proliferation by inhibiting the expression of effector CD8 + They demonstrated that they could affect the adaptive immune system by blocking the immunosuppressive effects of TGF-β on cell proliferation. + CD8 + It has been suggested that T cells play an important role in antitumor immunity (Ikeda et al., Cytokine Growth Factor Rev (2002) 13:95-109). EXAMPLES

[0175] Response of syngeneic mouse models to anti-TGF-β treatment In this study, we investigated which syngeneic mouse models could be used to predict response to treatment with anti-TGF-β antibody Ab1 and anti-PD-1. To stratify the mouse models, we used CD8 + T cell infiltration and TGF-β pathway activation were assessed. CD8 + T cell infiltration was determined by RNASeq data. +T cell signatures were assayed. Seventeen different mouse syngeneic models with tumor cells originating from several indications (indicated under the x-axis in Figures 12A and 12B) were transcriptionally profiled using whole-transcriptome RNAseq. This "list" of syngeneic models was constructed in a common background strain C57 / BL6, with 5 to 7 biological replicates used per model. After Illumina2000 sequencing, gene expression profiles expressed in transcripts per million reads (TPM) were generated by standard processing of the raw sequence reads using the STAR aligner and Cufflinks transcript estimator. The resulting multi-sample data matrix was finally quantile normalized.

[0176] Figure 12A shows the CD8 + Relative abundance of T cells is shown (log2 transformed). Relative CD8 + T cell abundance was estimated using the unique marker gene CD8B, which has been shown to be a highly specific indicator of the presence of CD8 T cells (Becht et al. al., Curr Opin Immunol (2016) 39:7-13; and Becht et al., Genome Biol (2016) 17:218). Each box plot summarizes the range of values ​​across biological replicates. The MC38 model , approximately twice as many CD8 + T cell infiltration (left and right boxes, respectively). The A20 and EL4 lymphoma models had the highest and lowest overall levels of CD8 + T cell infiltration was observed in EL4 and CD8 + T cells are negligible.

[0177] The MC38, MC38.ova, CT26, and L1210 mouse cell lines showed the highest levels of Furthermore, the EMT-6 breast cancer cell line was shown to display near-baseline T cell infiltration, consistent with recent reports that EMT6 tumors have an immune-exclusion phenotype (S. Mariathasan et al. 2017, ESMO Immuno-Oncology Congress, Geneva, Geneva Switzerland).

[0178] Figure 12B shows TGF-β pathway activation across the enumeration. A transcriptional signature of 170 genes of TGF-β pathway activation derived from in vitro stimulation of MCF7 cells with TGFβ and confirmed by comparison with several other TGF-β signatures was used to assign a pathway activation score to each profile in the enumeration. Scores were calculated using "Regulated Gene Set Enrichment Analysis" (rGSEA, Theilhaber et al. 2014) and were based on gene background. Enrichment of signature genes relative to the mean was expressed as log2. The MC38 model showed average activation, whereas the EMT6 model showed very high TGF-β pathway activation (left and right boxes, respectively). EXAMPLES

[0179] Effect of Ab1 and anti-PD1 antibody combination on mouse breast cancer models In this study, we investigated the therapeutic effects of Ab1 with or without anti-PD-1. Exponentially growing EMT-6 breast cells (CRL-2755, ATCC) were cultured in RPMI-1640 supplemented with 10% FBS in a humidified incubator with 5% CO2, and then implanted subcutaneously (0.5 × 10 6 cells / mouse). Tumors were 68-116 mm in average size. 3Upon reaching a tumor mass of 100 mg / kg, mice were pooled and randomly divided into control and treatment groups (10 mice per group). Tumor-bearing mice were then treated intraperitoneally with PBS, Ab1 (10 and 25 mg / kg) three times a week for a total of six doses per animal. Tumors were measured twice a week by digital calipers and tumor volumes were calculated (mm 3 = L × W × H) and graphed using Graph Pad Prism. 3 Mice were euthanized by CO2 at the end of the study if tumors grew to any size or if tumors showed ulceration of >20% of the tumor surface.

[0180] As single agents, Ab1 at a dose of 10 or 25 mg / kg Q3D and murine α-PD-1 antibody at a dose of 5 mg / kg demonstrated partial activity in EMT-6 tumor-bearing mice with 1 / 10, 2 / 10, and 2 / 10 complete regressions, respectively. The combination of Ab1 at a dose of 10 or 25 mg / kg Q3D and murine α-PD-1 antibody at 5 mg / kg Q3D was therapeutically active. When comparing tumor volume changes from baseline at day 31 post-implantation, the effect of the combination of Ab1 at all doses tested and murine α-PD-1 antibody at 5 mg / kg Q3D was greater than the effect of each single agent with 7 / 10 and 4 / 10 complete regressions for Ab1 at 10 and 25 mg / kg, respectively. Table 10 summarizes the results.

[0181] [Table 11]

[0182] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below. Methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All literature and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, controls. Although a number of texts are cited herein, this citation does not acknowledge that any of these texts form part of the common general knowledge in the art. Furthermore, unless otherwise required by context, the singular includes the plural and plural terms also include the singular. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques were performed according to manufacturer's instructions as commonly accomplished in the art or as described herein. Throughout this specification and the embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises" or "comprising", are understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0183] The sequences described herein are listed below.

[0184] [Sequence Listing] SEQ ID NO:1 (Ab1 heavy chain) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SNVISWVRQA PGQGLEWMGG VIPIVDIANY AQRFKGRVTI TADESTSTTY MELSSLRSED TAVYYCASTL GLVLDAMDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK SEQ ID NO:2 (Ab1 light chain) ETVLTQSPGT LSLSPGERAT LSCRASQSLG SSYLAWYQQK PGQAPRLLIY GASSRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYADSPITFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC SEQ ID NO:3 (Fresolimumab heavy chain, including leader sequence residues 1 to 19) MGWSCIILFL VATATGVHSQ VQLVQSGAEV KKPGSSVKVS CKASGYTFSS NVISWVRQAP GQGLEWMGGV IPIVDIANYA QRFKGRVTIT ADESTSTTYM ELSSLRSEDT AVYYCASTLG LVLDAMDYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK YGPPCPSCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK SEQ ID NO: 4 (Fresolimumab light chain, including leader sequence residues 1 to 19) MGWSCIILFL VATATGVHSE TVLTQSPGTL SLSPGERATL SCRASQSLGS SYLAWYQQKP GQAPRLLIYG ASSRAPGIPD RFSGSGSGTD FTLTISRLEP EDFAVYYCQQ YADSPITFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO:5 (anti-PD-1 Mab heavy chain) EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA PGKGLEWVSG ISGGGRDTYF ADSVKGRFTI SRDNSKNTLY LQMNSLKGED TAVYYCVKWG NIYFDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK SEQ ID NO:6 (anti-PD-1 Mab light chain) DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP GKAPNLLIYA ASSLHGGVPS RFSGSGSGTD FTLTIRTLQP EDFATYYCQQ SSNTPFTFGP GTVVDFRRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO:7 (x-anti-mPD-1 Mab heavy chain) EVQLQESGPG LVKPSQSLSL TCSVTGYSIT SSYRWNWIRK FPGNRLEWMG YINSAGISNY NPSLKRRISI TRDTSKNQFF LQVNSVTTED AATYYCARSD NMGTTPFTYW GQGTLVTVSS AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV PSSTWPSETV TCNVAHPASS TKVDKKIVPR DCGCKPCICT VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEV QFSWFVDDVE VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW NGQPAENYKN TQPIMDTDGS YFVYSKLNVQ KSNWEAGNTF TCSVLHEGLH NHHTEKSLSH SPG SEQ ID NO:8 (x-anti-mPD-1 Mab light chain) DIVMTQGTLP NPPVPSGESVS ITCRSSKSLL YSDGKTYLNW YLQRPGQSPQ LLIYWMSTRA SGVSDRFSGS GSGTDFTLKI SGVEAEDVGI YYCQQGLEFP TFGGGTKLEL KRADAAPTVS IFPPSTEQLA TGGASVVCLM NNFYPRDISV KWKIDGTERR DGVLDSVTDQ DSKDSTYSMS STLSLTKADY ESHNLYTCEV VHKTSSSPVV KSFNRNEC SEQ ID NO:9 (1D11 heavy chain) HVQLQQSGPE LVRPGASVKL SCKASGYIFI TYWMNWVKQR PGQGLEWIGQ IFPASGSTNY NEMFEGKATL TVDTSSSTAY MQLSSLTSED SAVYYCARGD GNYALDAMDY WGQGTSVTVS SAKTTPPSVY PLAPGSAAQT NSMVTLGCLV KGYFPEPVTV TWNSGSLSSG VHTFPAVLQS DLYTLSSSVT VPSSTWPSQT VTCNVAHPAS STKVDKKIVP RDCGCKPCIC TVPEVSSVFI FPPKPKDVLT ITLTPKVTCV VVDISKDDPE VQFSWFVDDV EVHTAQTKPR EEQFNSTFRS VSELPIMHQD WLNGKEFKCR VNSAAFPAPI EKTISKTKGR PKAPQVYTIP PPKEQMAKDK VSLTCMITDF FPEDITVEWQ WNGQPAENYK NTQPIMDTDG SYFVYSKLNV QKSNWEAGNT FTCSVLHEGL HNHHTEKSLS HSPGK SEQ ID NO: 10 (1D11 light chain) NIVLTQSPAS LAVSLGQRAT ISCRASESVD SYGNSFMHWY QQKSGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YCQQNNEDPL TFGAGTKLEL KRADAAPTVS IFPPSEQLT SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC

Claims

1. 1. Use of an anti-TGF-β antibody that specifically binds to human TGF-β1, TGF-β2, and TGF-β3 in the manufacture of a medicament for treating a disease selected from focal segmental glomerulosclerosis (FSGS), idiopathic pulmonary fibrosis, radiation-induced fibrosis, hepatic fibrosis, myelofibrosis, and osteogenesis imperfecta in a patient, wherein the antibody comprises heavy chain complementarity-determining regions (CDRs) 1-3 of SEQ ID NO: 1 and light chain CDRs 1-3 of SEQ ID NO: 2, and wherein the antibody is a human IgG1A antibody having a proline at position 228 (EU numbering). 4 The above use, wherein the antibody comprises a constant region.

2. The antibody comprises a heavy chain variable domain (V) corresponding to residues 1 to 120 of SEQ ID NO:

1. H ) amino acid sequence and a light chain variable domain (V) corresponding to residues 1 to 108 of SEQ ID NO:2 L ) the amino acid sequence of claim 1.

3. The use according to claim 1 or 2, wherein the antibody comprises a heavy chain amino acid sequence set forth in SEQ ID NO: 1 and a light chain amino acid sequence set forth in SEQ ID NO:

2.

4. IgG 4 The use according to claim 1 or 2, wherein the constant region comprises residues 121 to 447 of SEQ ID NO:1.

Images