KR20190105077A - Anti-TGF-beta Antibodies and Uses thereof - Google Patents

Abstract

The present invention provides improved pan-TGF-β antibodies for the treatment of diseases mediated by TGF-β, including autoimmune diseases, fibrotic diseases, and cancer. Also provided are methods and uses of antibodies for use with other immunomodulators, such as anti-PD-1 antibodies.

Description

Anti-TGF-beta Antibodies and Uses thereof

Cross Reference to Related Application

This application claims priority to US provisional application 62 / 448,800 and European application number 17305061.8, filed January 20, 2017. The disclosures of both priority applications are incorporated herein by reference in their entirety.

Sequence list

This application contains a list of sequences submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. Said ASCII copy, created on January 11, 2018, is named 022548_WO011_SL.txt and is 30,458 bytes in size.

Converting growth factor beta (TGF-β) is a cytokine that controls several key cellular functions, including proliferation, differentiation, survival, migration, and epithelial mesenchymal metastasis. It regulates a variety of biological processes such as extracellular matrix formation, wound healing, embryonic development, bone development, hematopoiesis, immune and inflammatory responses, and malignant cancerization. Dysregulation of TGF-β causes pathological diseases such as growth defects, cancer, chronic inflammation, and autoimmune and fibrotic diseases.

TGF-β has three known isotypes-TGF-β1, 2, and 3. All three isotypes are initially translated into pro-peptides. After cleavage, the mature C-terminal end remains associated with the N-terminus (referred to as the latent associated peptide or LAP) to form a small latent complex (SLC) secreted from the cells. Inability of SLC to bind TGF-β receptor II (TGFβRII) prevents receptor involvement. Activation by dissociation of the N- and C-terminus occurs by one of several mechanisms including proteolytic cleavage, acidic pH, or integrin structural alteration (Connolly et al., Int J Biol Sci (2012) 8 (7) (964-78).

TGF-β1, 2, and 3 are multi-expressed in their function and expressed in different patterns across cell and tissue types. They have similar in vitro activity, but individual knockouts in certain cell types suggest an unequal role in vivo despite their shared ability to bind to the same receptor (Akhurst et al., Nat Rev Drug Discov (2012). 11 (10): 790-811). Upon TGF-β binding to TGFβRII, the constitutive kinase activity of the receptor phosphorylates and activates TGFβRI, which phosphorylates SMAD2 / 3 to associate with SMAD4, localize to the nucleus, and transcription of the TGF-β-responsive gene Allow. Id. In addition to the normal signaling cascade, the non-normal pathway carries signals through other factors including p38 MAPK, PI3K, AKT, JUN, JNK, and NF-κB. TGF-β signaling is also mediated by other pathways including WNT, Hedgehog, Notch, INF, TNF, and RAS. The end result of TGF-β signaling is therefore crosstalk across these signaling pathways, integrating the state and environment of the cell. Id .

Because of the various functions of TGF-β, there is a need for pan-TGF-β-specific therapeutic antibodies that are safe for human patients (Bedinger et al., MAbs. (2016) 8 (2): 389-404). . However, TGF-β is highly conserved between species. As a result, the production of antibodies to human TGF-β in animals such as mice is a difficult task.

There is also a medical need for patients who currently lack effective treatment. For example, in a Phase III Checkmate-067 study treated with anti-PD1 antibody nivolumab monotherapy, more than 50% of patients with advanced melanoma had no complete or partial response to therapy (Larkin et al., N Engl J). Med (2015) 373: 23-34; Redman et al., BMC Med (2016) 14: 20-30).

Summary of the Invention

The present invention provides improved monoclonal antibodies that specifically bind (ie, pan-TGF-β-specific) to human TGF-β1, TGF-β2, and TGF-β3. These antibodies are less prone to forming half antibodies (ie, dimeric complexes with one heavy chain and one light chain) during preparation. They also have a better pharmacokinetic profile, such as increased half-life, thus conferring improved clinical benefit to the patient. We also found that TGF-β inhibition, e.g., effected by antibodies and antigen-binding fragments of the present invention alleviates immunosuppressive microenvironments in tumors, and immunotherapy, such as programmed cell death protein 1 (PD-1), It has been found to enhance the effectiveness of therapies targeting PD-1 ligand 1 (PD-L1) and 2 (PD-L2).

In one embodiment, the present invention provides human TGF-β1, TGF-β2, and comprising a heavy chain complementarity-determining region (CDR) 1-3 of SEQ ID NO: 1 and light chain CDR1-3 of SEQ ID NO: 2, and An isolated monoclonal antibody that specifically binds TGF-β3, wherein the antibody comprises a human IgG ₄ constant region with a mutation at position 228 (EU numbering). In some embodiments, the mutation is a mutation from serine to proline (S228P). In some embodiments, the antibody has a heavy chain variable domain (V _H ) amino acid sequence corresponding to residues 1-120 of SEQ ID NO: 1 and a light chain variable domain (V _L ) corresponding to residues 1-108 of SEQ ID NO: 2 Amino acid sequence. In further embodiments, the antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 1 (with or without C-terminal lysine) and a light chain amino acid sequence as shown in SEQ ID NO: 2. The invention also features an F (ab ') ₂ antigen-binding fragment of the antibody.

In a preferred embodiment, the antibodies or fragments of the invention have increased half-life, increased exposure, or both compared to presolimumab. For example, the increase is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Exposure of drugs, such as antibodies or fragments of the invention, is a function of drug concentration in the body over time. The concentration of drugs in the body is often expressed at drug levels in blood, plasma, or serum. Half-life and exposure (bio-exposure) of the drug can be measured by well known methods, as illustrated in Example 7 below.

The present invention further provides a composition comprising an antibody of the invention, wherein the composition comprises less than 1% half the antibody. Half antibody formation, for example, can be followed by SDS-capillary electrophoresis or non-reducing SDS-PAGE analysis under non-reducing conditions, followed by purity analysis of monoclonal antibody preparations using 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 pharmaceutically acceptable excipient.

In another aspect, the present invention provides a method of inhibiting TGF-β signal transduction in a patient (human) in need of inhibition of TGF-β signal transduction, the method comprising administering to the patient a therapeutic amount of an antibody or fragment of the present invention. It provides a method to include. In some embodiments, the patient has an immune-mediated disease (eg, scleroderma), a fibrotic disease (eg, renal fibrotic disease such as focal segmental glomerulosclerosis (FSGS), or pulmonary fibrotic disease such as idiopathic pulmonary fibrosis), Or birth or bone defects (eg, incomplete bone formation). In some embodiments, the patient has cancer. In some embodiments, the antibody or fragment used in the method inhibits the differentiation of CD4 ⁺ T cells into inducible regulatory T cells (iTreg). Antibodies or fragments can alleviate the immunosuppressive tumor microenvironment. This action of the antibody or fragment aids in the activation of the immune system and enhances the effectiveness of immunotherapy. The effectiveness of the treatment methods described herein can be suggested by one or more of, for example, in a patient (eg, in a patient's tumor tissue): (1) an increase in MIP2 and / or KC / GRO levels, ( 2) activation or infiltration of tumor tissue of CD8 ⁺ T cells, such as INF-γ-positive CD8 ⁺ T cells, and (3) increased clustering of natural killer (NK) cells.

The present invention provides a method of treating cancer in a patient (human), comprising administering to the patient a therapeutically effective amount of an antibody or fragment of the invention, and (2) an therapeutically effective amount of an inhibitor of an immune checkpoint protein. Additionally provided. These two agents may be administered simultaneously (eg, in a single composition or in separate compositions), or sequentially in any order. Two agents may be administered, for example, on the same day. In some embodiments, the therapeutic agent (1) is administered to the patient before the therapeutic agent (2) (eg, 1 day or more).

In some embodiments, the immune checkpoint protein is PD-1, PD-L1, or PD-L2. In further embodiments, the inhibitor of immune checkpoint protein is an anti-PD-1 antibody. In further embodiments, the anti-PD-1 antibody comprises (1) heavy chain CDR1-3 of SEQ ID NO: 5 and light chain CDR1-3 of SEQ ID NO: 6, (2) residues 1-117 of SEQ ID NO: 5 Either with or without the V _H amino acid sequence corresponding to the V _L amino acid sequence corresponding to residues 1 to 107 of SEQ ID NO: 6, or (3) the heavy chain amino acid sequence represented by SEQ ID NO: 5 (C-terminal lysine). And the light chain amino acid sequence shown in SEQ ID NO: 6. In one specific embodiment, the method comprises an anti-TGF-β comprising a heavy chain amino acid sequence as shown in SEQ ID NO: 1 (with or without a C-terminal lysine) and a light chain amino acid sequence as shown in SEQ ID NO: 2 Administering to the cancer patient an antibody and an anti-PD-1 antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO: 5 (with or without the C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO: 6 It includes a step. In some embodiments, the patient is refractory to anti-PD-1 antibody monotherapy. The patient may have advanced or metastatic melanoma, or cutaneous squamous cell carcinoma.

In some therapies, the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient every two or three weeks. In some therapies, the two agents are each administered at a dose of 0.01-40 (eg 0.02-20, 0.05-15, or 0.05-20) mg / kg body weight.

The invention also provides a method of increasing an immune response in a patient in need of an increase in the immune response, comprising administering an immune checkpoint inhibitor and an antibody or fragment of the invention to the patient. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, such as (1) HCDR1-3 of SEQ ID NO: 5 and LCDR1-3 of SEQ ID NO: 6; (2) 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 C-terminal lysine) and a light chain having the amino acid sequence of SEQ ID NO: 6.

The methods of the invention include, but are not limited to, melanoma (eg, metastatic or advanced), lung cancer (eg, non-small cell lung cancer), cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, fallopian tube cancer, uterine cancer, head and neck cancer ( For example, it can be used to treat a variety of cancers including head and neck squamous cell carcinoma), liver cancer (eg hepatocellular carcinoma), urinary tract epithelial cancer, and kidney cancer (eg renal cell carcinoma). In some embodiments, the patient has a solid tumor of mesenchymal tumor or mesenchymal subtype. Examples of such solid tumors include tumors in the colon (eg, colorectal cancer), ovary, head and neck (eg, head and neck squamous cell carcinoma), liver (eg, hepatocellular carcinoma), and urinary epithelium.

In some embodiments, the cancer comprising mesenchymal tumors includes ACTA2 (smooth muscle α2 actin), VIM (nonmentin), MGP (matrix Gla protein), ZWINT (ZW10 interacting analog protein), and ZEB2 (zinc finger E-box -Overexpression of one or more of the bound homeoboxes 2). The expression level of such biomarkers can be determined, for example, at the mRNA level or protein level in a biological sample from a patient, such as a tumor biopsy or circulating tumor cells.

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

Also included are nucleic acid expression vectors encoding the heavy or light chain or both of the antibodies of the invention; Host cells comprising heavy and light chain coding sequences for the antibody; And culturing the host cell in a suitable culture medium to allow expression of the antibody gene, and then harvesting the antibody, the method of producing the antibody using the host cell is included.

1a-e show 1 ng / ml of human TGF-β1 (a), human TGF-β2 (b), human TGF-β3 (c), murine TGF-β1 (d), or murine TGF-β2 (e) Is a graph showing the effect of Ab1, Presolimab, and 1D11 on proliferation of mink lung (Mv 1 Lu) cells treated with. Antibody concentrations are in μg / ml.
2 is a bar graph showing the effect of 50 μg / ml Ab1 on human inducible regulatory T cell (iTreg) differentiation. Stimulation provided to T cells was anti-CD3 and anti-CD28 antibody + IL-2.
3 is a bar graph showing the effect of Ab1 on human inducible regulatory T cell (iTreg) differentiation in human CD4 + T cell culture treated with 2 ng / ml human TGF-β1. Stimulation provided to T cells was anti-CD3 and anti-CD28 antibody + IL-2.
4 is a bar graph showing the effect of Ab1 (30 μg / ml) and human TGF-β1 (18 ng / ml) on NFATc-induced luciferase expression on Jurkat T cells after T cell stimulation and anti-PD-1 treatment. to be.
FIG. 5 is a graph showing median tumor volume with median absolute deviations (MAD) in the treatment groups shown using the C57BL / 6 MC38 colon mouse model. Vehicle: PBS. "Anti-PD-1": x-anti-mPD-1 Mab (see detailed description below). "Isotype control of Ab1": anti-HEL hIgG4.
FIG. 6 is a scatter point plot showing tumor volume changes from baseline at day 27 of treatment, shown using the C57BL / 6 MC38 colon mouse model. Control: PBS. "Anti-PD-1 RPM114 mIgG1": x-anti-mPD-1 Mab.
7A-F are graphs showing tumor volume over time for each indicated treatment group using the C57BL / 6 MC38 colon mouse model. Each line in the graph represents one animal. "mpk": mg / kg. "Ab1 isotype Ctrl": anti-HEL hIgG4. αPD1: x-anti-mPD-1 Mab.
8 is a graph showing the effect of Ab1 on active TGF-β1 concentration in LoVo tumor lysate.
9A is a graph showing serum concentrations of Ab1 and presolumimab over time in groups of five rats given a single dose of either antibody at 5 mg / kg. Three different batches (B1, B2, and B3) of Presolimumap were provided in Groups 1-3. Groups 4 and 5 were given Ab1 in two different batches (B1 and B2).
FIG. 9B is a graph showing serum concentrations of Ab1 and presolimab over time in monkeys given a single dose of either antibody at 1 mg / kg.
FIG. 9C shows Ab1 and presollimab over time in monkeys given a 5-weekly dose of Ab1 at 1 mg / kg per dose or a 2-weekly dose of presollimab at 1 mg / kg per dose for the study period shown. It is a graph showing the serum concentration of.
FIG. 9D is a graph showing serum concentrations of Ab1 and presolimab over time in monkeys given a single dose of either antibody at 10 mg / kg.
FIG. 9E shows Ab1 and presollimab over time in monkeys given a 5-weekly dose of Ab1 at 10 mg / kg / dose or a 2 weekly dose of presollimab at 10 mg / kg / dose during the study period shown It is a graph showing the serum concentration of.
10A is a graph showing changes in TGF-β1 levels in MC38 tumors after treatment with Ab1 (+/− anti-PD1) .
10B is a graph showing changes in MIP-2 levels in MC38 tumors after treatment with Ab1 (+/− anti-PD1) .
10C is a graph showing changes in KC / GRO levels in MC38 tumors after treatment with Ab1 (+/− anti-PD1) .
11A is a graph quantifying CellTrace Violet staining and IFN-γ staining of CD8 ^pos cells.
11B is a graph showing that Ab1 restored both proliferation and IFN-γ production in TGFβ-treated CD8 ⁺ T cells.
12A is a graph showing the relative abundance (log2-transformed) of CD8 ⁺ T cells over an outline of a syngeneic mouse tumor model for colon cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma and kidney cancer.
12B is a graph showing the TGFβ pathway activation over an outline of syngeneic mouse tumor models for colon cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma and kidney cancer.

The present invention also provides an improved pan-TGF-β-specific monoclonal antibody with a better pharmacokinetic profile, such as a lower likelihood of forming half antibody with higher exposure in the body compared to antibodies known in the prior art. It features. Antibodies of the invention are collectively referred to as "Ab1 and related antibodies" and they have heavy chain CDRs (HCDR) 1-3 of SEQ ID NO: 1 and light chain CDRs (LCDR) 1-3 of SEQ ID NO: 2, and in the hinge region Residue 228 (EU numbering) shares a common structural feature that has a human IgG ₄ constant region mutated from serine to proline. P228 is shown in bold in the box in the sequence of SEQ ID NO: 1 shown below.

Antibody Ab1, when aglycosylated, has an estimated molecular weight of 144 KD. Its heavy and light chain amino acid sequences are SEQ ID NOs: 1 and 2, respectively. These two sequences are shown below. Variable domains are shown in italics. CDRs are shown in boxes. Glycosylation sites in the constant domain of the heavy chain are shown in bold (N297).

In some embodiments, antibodies of the invention, such as anti-TGF-β antibodies, do not have a C-terminal lysine in the heavy chain. C-terminal lysine may be removed during preparation or by recombinant techniques (ie, the coding sequence of the heavy chain does not include codons for the C-terminal terminal lysine). Thus, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO: 1 without C-terminal lysine are also contemplated within the present invention.

Ab1 and related antibodies specifically bind to human TGF-β1, -β2, and -β3. “Specifically” means that the binding is less than 10 ⁻⁷ M, such as less than 10 ⁻⁸ M, such as determined by surface plasmon resonance (eg, see Example 1 below), or by Bio-Layer interferometry (eg, , it means having a K _D of 1 ~ 5 nM). Ab1 and related antibodies may also be assayed in a mink lung epithelial cell assay (see, eg, Example 2 below), either a strong TGF-β neutralizing titer, or an A549 cell IL-11 induction assay (eg, as described in its entirety herein). It can have an EC50 of about 0.05 to 1 μg / ml, as determined in Example 6 of PCT publication WO 2006/086469, which is incorporated herein by reference.

These antigen-binding and neutralizing properties of Ab1 and related antibodies are comparable with the anti-TGF-β antibody prelimimab (the germline IgG ₄ PET1073G12 antibody described in WO 2006/086469). The heavy and light chain sequences of Presolimumab comprising the leader sequence are shown in SEQ ID NOs: 3 and 4, respectively. As shown in SEQ ID NO: 3, Presolimumap has no proline at position 228 (EU numbering, which corresponds to actual position 247 in SEQ ID NO: 3). Ab1 and related antibodies have some improved features compared to presolimumab.

During preparation, presolimumab is a dimer with one heavy chain and one light chain, not a tetramer with two heavy chains complexed with two light chains, as much as 6-18% half antibody under non-reducing denaturing conditions. ) Can be formed. In contrast, Ab1 produces substantially fewer half antibodies (<1%). Thus, Ab1 and related antibodies provide a pureer finished product during manufacture.

In addition, Ab1 and related antibodies may have improved pharmacokinetic (PK) profiles compared to presolimumab. They have linear PK behavior with much longer half-life and lower elimination rates than presolimumab, resulting in about 1.7 times higher in vivo exposure than presolimumab. For example, in rats, Ab1 has an average half-life of 7.1 days compared to 4.3 days of presolimumab, and a clearance (CL) of 0.30 ml / hr / kg compared to 0.51 ml / hr / kg of presolimumab. (Example 7, below). In cynomolgus monkeys, Ab1 has been shown to have an average half-life of 13 days compared to 4.5 days of presolimumab, and a removal rate (CL) of 0.40 ml / hr / kg compared to 0.66 ml / hr / kg of presolimumab. . Id. These improved PK properties are at lower doses and / or less frequently than presolimumab to achieve the same or better clinical efficacy while Ab1 and related antibodies induce fewer adverse side effects and fewer anti-drug antibody responses. It is presented to the patient, suggesting that a longer treatment period may be allowed if necessary.

In addition, during toxicological studies of presolimab in nonhuman primates, a correlation between drug exposure and adverse events such as anemia was observed. However, this case was not observed in similar studies at equivalent or even higher exposures with Ab1.

Without being bound by theory, we assume that residue 228 mutations in the heavy chains of Ab1 and related antibodies result in improved stability as well as improved PK and toxicological profiles.

The constant domains of Ab1 and related antibodies can be further modified as needed, for example at Kabat residue L248 (eg, by introducing a mutation L248E) to reduce any undesirable effector function of the molecule.

As used herein, the terms “antibody” (Ab) or “immunoglobulin” (Ig) refer to two heavy chains (H) (about 50-70 kDa) and two light chains (L) (about 25) interconnected by disulfide bonds. tetrameric proteins, including kDa). Each heavy chain consists of a heavy chain variable domain (V _H ) and a heavy chain constant region (C _H ). Each light chain consists of a light chain variable domain (V _L ) and a light chain constant region (C _L ). The V _H and V _L domains can be further subdivided into hypervariable regions, called “complementarity determining regions” (CDRs), interspersed with more conserved regions, called “framework regions” (FR). Each V _H or V _L consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each region is defined in the IMGT® definition (Lefranc et al., Dev Comp Immunol 27 (1): 55-77 (2003)); Or Kabat, 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).

The term “human antibody” refers to an antibody in which the variable domain and constant region sequences are derived from human sequences. The term encompasses antibodies with sequences derived from human genes, but these sequences have been modified, for example, to reduce immunogenicity, increase affinity, and increase stability. The term encompasses antibodies produced recombinantly in non-human cells, which can impart glycosylation that is not typical of human cells.

The term “chimeric antibody” refers to an antibody comprising sequences from two different animal species. For example, chimeric antibodies are immunized using a murine antibody (ie, an antibody encoded by a murine antibody gene, such as a hybridoma technique) linked to a constant region of the antibody from another species (eg, human, rabbit, or rat). V _H and V _L of antibodies obtained from mice).

The term “antigen-binding fragment” of an antibody refers to a fragment of the antibody that retains the ability to specifically bind an antigen. In some embodiments, the antigen-binding fragment of the invention is an F (ab ') ₂ fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region (Fab is V _L , V _Monovalent antibody fragment consisting of _H , C _L and C _H1 domains. In some embodiments, antigen-binding fragments of the invention may also comprise a C _H2 or C _H3 domain.

The antibodies and antigen-binding fragments described herein can be isolated. The term “isolated protein”, “isolated polypeptide” or “isolated antibody”, due to its origin or origin, is either (1) not associated with the naturally associated component involved in its original state, or (2) from the same species. Refers to a protein, polypeptide or antibody that is substantially free of (3) is expressed by cells from different species or (4) does not occur in nature. Thus, a polypeptide synthesized in a cell system different from a chemically synthesized or naturally derived cell will be "isolated" from its naturally associated component. Proteins may also be substantially free of naturally related components by isolation using protein purification techniques well known in the art.

I. Use of Ab1 and Related Antibodies

TGF-β receptors are widely expressed in immune cells, resulting in various effects of TGF-β in both the innate and adaptive immune systems. TGF-β has been linked to several morbid diseases, such as birth defects, cancer, chronic inflammation, autoimmune, and fibrotic diseases. A therapeutic amount of Ab1 or related antibodies can be used to treat these diseases. A “therapeutically effective” amount refers to the amount of Ab1, related antibody, or another therapeutic agent referred to herein that rescues one or more symptoms of the disease being treated. The amount may vary depending on the disease or patient being treated and may be determined by a healthcare professional using well established principles.

In some embodiments, the Ab1 or related antibody is 40, 20, or 15 mg / kg or less (eg, 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, once daily, once every two days, once every three days, once every four days, or once every five days, once a week, once every two weeks, or once every three weeks, It can be once a month or once every two months. The antibody can be administered intravenously (eg, intravenous infusion over 0.5-8 hours), subcutaneous, topical, or by any other route of administration appropriate for the disease and drug formulation.

Ab1 and related antibodies are derived from human antibody genes and thus have low immunogenicity in humans. Toxicological studies of Ab1 are described in detail in Example 8 below. Certain heart and lung side effects were observed in rats. Thus, when treating a patient with Ab1 or related antibodies, the patient can be monitored for adverse events.

In some embodiments, the effectiveness of an antibody of the invention may be suggested by one or more of the following in a patient (eg, in a diseased tissue, such as a tumor tissue in a patient): (1) the level or activity of TGF-β Decrease, (2) increase MIP2 and / or KC / GRO levels, (3) activate or infiltrate CD8 ⁺ T cells, such as INF-γ-positive CD8 ⁺ T cells, or invade tumor tissue, and (4) natural killing ( NK) increased clustering of cells.

A. Non-Tumor Disease

Diseases that can be treated by Ab1 and related antibodies include, but are not limited to, bone defects (eg, incomplete bone formation), glomerulonephritis, nerve or skin scarring, lung or lung fibrosis (eg idiopathic pulmonary fibrosis), radiation-induced Fibrosis, liver fibrosis, myeloid fibrosis, scleroderma, immune-mediated diseases (including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Burger's disease, and transplant rejection) and Duputran contractile diseases.

They also include but are not limited to focal segmental glomerulosclerosis (FSGS), diabetic (type I and II) nephropathy, radiation nephropathy, obstructive nephropathy, widespread systemic sclerosis, hereditary kidney disease (e.g., polycystic kidney disease, Treats, prevents, and renal dysfunction, including spongy nephropathy), glomerulonephritis, nephropathy, nephropathy, systemic or glomerular hypertension, tubulointerstitial nephropathy, renal tubuloacidosis, renal tuberculosis and renal infarction This may be useful in reducing the risk of their occurrence. In particular, they are combined with antagonists of the renin-angiotensin-aldosterone system, including but not limited to renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, Ang II receptor antagonists (also known as "Ang II receptor blockers") and aldosterone antagonists. This is useful if See, for example, WO 2004/098637, which is incorporated herein by reference in its entirety.

Ab1 and related antibodies are associated with diseases and disorders associated with the deposition of ECM, such as systemic sclerosis, postoperative adhesions, keloids and thickening scarring, proliferative vitreoretinopathy, glaucoma drainage surgery, corneal injury, cataracts, Feroni disease, adult respiratory distress syndrome, liver Sclerosis, scar formation after myocardial infarction, restenosis after angioplasty, scar formation after subarachnoid hemorrhage, fibrosis after annuloplasty, fibrosis after tendon and other recovery, biliary sclerosis (including sclerocholangitis), pericarditis, pleurisy, tracheostomy It is useful for treating invasive CNS injury, eosinophilic myalgia syndrome, vascular restenosis, venous obstruction, pancreatitis and psoriatic arthrosis.

Ab1 and related antibodies are further useful in diseases where the promotion of re-epithelialization is beneficial. Such diseases include, but are not limited to, skin diseases such as venous ulcers, ischemic ulcers (bedsores), diabetic ulcers, transplant sites, transplant donor sites, abrasions and burns, diseases of bronchial epithelium such as asthma, ARDS, intestinal epithelium, Such as mucositis, esophageal ulcers (reflux disease), gastric-esophageal reflux disease, gastric ulcers, small and large intestine lesions (inflammatory bowel disease) associated with cytotoxic therapy.

Further uses of Ab1 and related antibodies may be used in diseases in which epithelial cell proliferation is desired, for example in stabilizing atherosclerotic plaques, promoting healing of vascular anastomosis, or in diseases in which smooth muscle cell proliferation is desired, such as arterial disease, re Are in stenosis and asthma.

Ab1 and related antibodies are also known as macrophage-mediated infections, such as Leishmania spp. Protozoan Toxoplasma gondii , fungal histoplasmas , as well as Trypanosorna cruzi , Mycobacterium tuberculosis and Mycobacterium leprae Useful for augmenting the immune response to those induced by Histoplasma capsulatum , Candida albicans , Candida parapsilosis , and Cryptococcus neoformans Do. They are also useful for reducing immunosuppression induced by, for example, tumor, AIDS or granulomatous diseases.

Ab1 and related antibodies are also useful for the prevention and / or treatment of ophthalmic diseases such as scarring after glaucoma and trabeculectomy.

B. Tumor-Related Diseases

TGF-β regulates several biological processes including cell proliferation, epithelial-mesenchymal metastasis (EMT), matrix remodeling, angiogenesis, and immune function. Each of these processes contributes to tumor progression. The widespread and detrimental role of TGF-β in cancer patients across several indications is also shown by elevations not only in the tumor microenvironment but also systemically. See, eg, Kadam et al., Mol. Biomark. Diagn. (2013), 4 (3)]. Studies have shown that in a malignant state, TGF-β can induce EMT and the resulting mesenchymal phenotype causes increased cell migration and invasion.

Ab1 and related antibodies include hyperproliferative diseases such as skin cancer (eg, melanoma, including unresectable or metastatic melanoma, cutaneous squamous cell carcinoma, and keratinocytes), lung cancer (eg, non-small cell lung cancer), esophageal cancer, Gastric cancer, colorectal cancer, pancreatic cancer, liver cancer (eg hepatocellular carcinoma), primary celiac cancer, bladder cancer, kidney cancer or kidney cancer (eg kidney cell carcinoma), urinary tract carcinoma, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer , Prostate cancer, testicular cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), brain cancer, glioblastoma, glioma, mesothelioma, leukemia, and lymphomas.

In some embodiments, Ab1 and related antibodies are patients that have failed or are expected to fail previous therapy based on anti-PD-1, anti-PD-L1 or anti-PD-L2 therapeutics, ie anti-PD-1, anti Useful for treating cancer in patients expected to be non-responders or anti-responders to -PD-L1, or anti-PD-L2 therapy. In some embodiments, Ab1 and related antibodies are useful in the treatment of cancer in patients who relapse from previous anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy. As used herein, the term “expected” refers to one's general medical knowledge and to the patient's specific disease, whether the patient is a responder or non-responder and whether the treatment fails or will not be effective, without administration of the therapy. That means you can expect based on that.

In some embodiments, the cancer is a solid tumor of the mesenchymal subtype, including but not limited to mesenitis colorectal cancer, mesenchymal ovarian cancer, mesenchymal lung cancer, mesenchymal head cancer and mesenchymal cervical cancer. Epithelial mesenchymal metastasis (EMT) promotes cell migration and invasive properties by downregulating epithelial cell genes and enhancing mesenchymal gene expression. EMT is a hall mark of tumor progression and invasion. Up to one quarter of colorectal and ovarian cancers are mesenchyme. Thus by inhibiting the induction of TGF-β and EMT, Ab1 or related antibodies can be used to treat mesenchymal solid tumors. Solid tumors of the mesenchymal subtype can be identified by several genetic markers and pathological evaluation. Markers include ACTA2, VIM, MGP, ZEB2, and ZWINT, which can be detected by qRT-PCR or immunohistochemistry. Such markers can be used to select patients for anti-TGF-β monotherapy or combination therapy of the present invention.

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

Ab1 and related antibodies can also be used in the treatment of hematopoietic disorders or tumors 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.

Ab1 and related antibodies may also be useful for inhibiting cyclosporin-mediated tumor or cancer progression (eg, metastasis).

Naturally, in the context of cancer therapy, “treatment” includes any medical intervention that causes partial remission of the cancer as well as a decrease in tumor growth, a delay in tumor progression or recurrence, or a reduction in tumor metastasis to prolong the patient's life expectancy. This includes.

C. Combination Therapy in Oncology

It has been observed that the level of cytotoxic T cell infiltration in cancer is associated with desirable clinical outcomes (Fridman et al., Nat Rev Cancer (2012) 12 (4): 298-306; and Galon et al., Immunity (2013) 39 ( 1): 11-26). In addition, T helper cells that assist cytotoxic T cells (CD4 ⁺ T _H1 ) and cytokines they produce (eg IFN-γ) are 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).

TGF-β inhibits almost all aspects of the anti-tumor immune response. Cytokines promote iTreg differentiation and reduce cytotoxicity (CD8 ⁺ ) cell proliferation and invasion. Inhibition of TGF-β by Ab1 or related antibodies will mitigate the immunosuppressive tumor microenvironment as described above and will have a positive result for cancer patients.

In addition, the inventors have found that by alleviating the immunosuppressive tumor microenvironment, Ab1 and related antibodies allow a checkpoint modulator, such as an anti-PD-1 antibody, to better induce an immune response. As a result, more patients may benefit from immunotherapy, such as anti-PD-1, anti-PD-L1, or anti-PD-L2 treatment.

With or without a therapeutic agent that targets an immune checkpoint molecule, Ab1 and related antibodies can also be used for other cancer therapies, such as chemotherapy (eg, platinum- or taxoid-based therapies), radiation therapy, and cancer antigens or tumors. It can be used in conjunction with therapies that target formation inducers.

Cancers that can be treated by a combination involving Ab1 or related antibodies and immune checkpoint inhibitors, such as anti-PD-1 antibodies, include the cancers described in the subsections above.

In some embodiments, the cancer, such as advanced or metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and Hodgkin's lymphoma are previously anti-PD-1, anti-PD-L1, or anti -Refractory to PD-L2 therapy. A refractory patient is a patient whose disease progression is confirmed radiologically, eg, within 12 weeks of treatment start without any evidence of response.

In some embodiments, the Ab1 or related antibodies 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 skin squamous cell carcinoma Can be used in conjunction with another cancer therapy, such as anti-PD-1 therapy. See also discussion above.

Examples of anti-PD-1 antibodies include nivolumab, pembrolizumab, pidilizumab, MEDI0608 (formerly AMP-514; see eg WO 2012/145493 and US Pat. No. 9,205,148), PDR001 (see, eg, WO 2015/112900). PF-06801591 (see, eg, WO 2016/092419) and BGB-A317 (see, eg, WO 2015/035606). In some embodiments, the anti-PD-1 antibodies include those disclosed in WO 2015/112800 (e.g., H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7795N, H2M7796, H2M7796, and H2M7796 in Table 1 of PCT Publications). , as H4xH9034P2, H4xH9035P2, H4xH9037P2, H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P and in Table 3, the ones that are referred to as H4xH9008P, and publications PCT H4H7798N, H4H7795N2, H4H9008P and H4H9048P2 Mentioned). The entirety of the disclosure of WO 2015/112800 is incorporated herein by reference.

For example, in the PCT publications as well as related antibodies, including antibodies and antigen-binding fragments having the antibodies disclosed in WO 2015/112800 and the CDR, V _H and V _L sequences disclosed in the PCT publication or heavy and light chain sequences. Antibodies and antigen-binding fragments that bind to the same PD-1 epitope as the disclosed antibodies can be used with the Ab1 or related antibodies of the invention to treat cancer. In related embodiments, useful anti-PD-1 antibodies include the heavy and light chain amino acid sequences shown below as SEQ ID NOs: 5 and 6, respectively; The V _H and V _L sequences in SEQ ID NOs: 5 and 6 (in italics); Or one or more (eg, all six) CDRs (indicated by boxes) of SEQ ID NOs: 5 and 6.

In some embodiments, the antibodies of the invention, such as anti-PD-1 antibodies, do not have a C-terminal lysine in the heavy chain. C-terminal lysine may be removed during preparation or by recombinant techniques (ie, the coding sequence of the heavy chain does not include codons for the C-terminal terminal lysine). Thus, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO: 5 without C-terminal lysine are also contemplated within the present invention.

In some embodiments, anti-TGF-β antibodies or fragments of the invention may also be used with antibodies against immunomodulatory antigens such as PD-L1 and CTLA-4. Exemplary anti-PD-L1 antibodies are atezolizumap, avelumab, duvalumab, LY3300054 and BMS-936559. Exemplary anti-CTLA-4 antibodies are Ipilimumab or Tremelimumab.

D. Biomarkers of Therapeutic Effectiveness

The effectiveness of Ab1 and related antibodies can be determined by biomarker or target occupancy. For example, in tumor tissues, target occupancy can be assayed by evaluating active TGF-β levels in biopsies using meso scale search (MSD) assays. In blood, target involvement can be assayed by assessing the effect of reduced circulating TGF-β on peripheral blood monocytes such as lymphocytes (T cells, B cells, NK cells) and monocytes. For example, increased proliferation of circulating CD8 ⁺ T cells can be assessed using CD45 ⁺ RO ⁺ CCR7 ⁺ CD28 ⁺ Ki67 ⁺ as a marker in flow cytometry. Activation of circulating NK cells can be assessed using CD3 ^- CD56 ^{high / dim} CD16 ⁺ or CD137 ⁺ as markers in flow cytometry. Ki-67, PD-1, and ICOS can also be used as PD markers associated with T cell activation.

Immune modulation upon treatment with Ab1 or related antibodies can be assayed by assessing changes in infiltrating immune cells and immune markers, for example, by multiplex immunohistochemistry (IHC) assay using the NeoGenomics platform. Specifically, NeoGenomic's MultiOmyx TIL panel stains immune marker panels, allowing quantitative determination of the density and localization of various immune cells. Immune markers include the differentiation of iTreg; Infiltration and proliferation of CD8 ⁺ T cells; And production of IFNγ by CD8 ⁺ T cells. Ab1 has been shown to inhibit the differentiation of CD4 ⁺ T cells into iTreg (eg, see Example 3 below) and to increase CD8 ⁺ T cell proliferation and its IFN γ production (as shown in the mixed lymphocyte response assay; data Not shown). Thus, the therapeutic efficacy by Ab1 or related antibodies may be associated with inhibition of iTreg, induction of CD8 ⁺ T cell proliferation and invasion into tumors or other affected tissues, increased IFNγ production and / or increased ratio of CD8 ⁺ T cells to Treg cells. May be suggested. Immune modulation upon treatment with Ab1 or related antibodies can also be assayed in peripheral blood by methylation-PCR based quantitative immune cell counts of CD8 ⁺ T cells, Treg cells, NK cells, and other immune cells. The therapeutic efficacy can be clinically expressed with disease progression, such as delaying or reversing tumor progression.

II. Method of producing antibody

Antibodies that target Ab1 and related antibodies as well as other co-targets such as PD-1, PD-L1 or PD-L2 can be prepared by methods well established in the art. DNA sequences encoding the heavy and light chains of an antibody can be inserted into an expression vector such that the gene is operably linked to the required expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmid, YAV, EBV derived episomes and the like. The antibody light chain coding sequence and the antibody heavy chain coding sequence can be inserted into separate vectors and operably linked with the same or different expression control sequences (eg, promoters). In one embodiment, both coding sequences are inserted into the same expression vector and in the same expression control sequence (e.g., common promoter), in separate identical expression control sequences (e.g., promoter), or in different expression control sequences (e.g., promoter). Can be operatively connected. Antibody coding sequences can be inserted into expression vectors by standard methods (eg, ligation of complementary restriction enzyme sites on antibody gene fragments and vectors, or blunt end ligation if no restriction enzyme sites are present).

In addition to the antibody chain genes, the recombinant expression vector can carry regulatory sequences that control the expression of the antibody chain genes in the host cell. Examples of regulatory sequences for mammalian host cell expression include promoters and / or enhancers (such as CMV promoters / enhancers) derived from viral elements that direct high levels of protein expression in mammalian cells such as retrovirus LTR, cytomegalovirus (CMV). , Promoters and / or enhancers derived from Simian virus 40 (SV40) (such as the SV40 promoter / enhancer), promoters and / or enhancers derived from adenovirus (eg, adenovirus major late promoters (AdMLP)), polyomas, and Potent mammalian promoters such as intact immunoglobulins and actin promoters are included.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that control the replication of the vector in a host cell (eg, origin of replication) and selectable marker genes. For example, the selectable marker gene confers resistance to drugs such as G418, hygromycin or methotrexate on host cells into which the vector has been introduced. Selection marker genes can include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells for methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthase gene.

Expression vectors encoding the antibodies of the invention are introduced into host cells for expression. Host cells are 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 include several immortalized cell lines that are well known in the art and are 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), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney Cells (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells, and several other cell lines. Cell lines can be selected based on their expression level. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells.

In addition, expression of antibodies can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a general approach to enhance expression under certain conditions.

Tissue culture medium for host cells may or may not include animal-derived components (ADCs) such as bovine serum albumin. In some embodiments, ADC-free culture medium is preferred for human safety. Tissue culture can be performed using a feed-batch method, a continuous perfusion method, or any other method appropriate for the host cell and desired yield.

III. Pharmaceutical composition

Antibodies of the invention may be formulated for suitable storage stability. For example, the antibody can be lyophilized, stored or reconstituted for use using pharmaceutically acceptable excipients. In combination therapy, two or more therapeutic agents, such as antibodies, may be co-formulated, such as mixed and provided in a single composition.

As used herein, the term “excipient” or “carrier” describes any ingredient that is not a compound (s) of the invention. The choice of excipient (s) will depend in large part on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. "Pharmaceutically acceptable excipients" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In some cases, the composition will include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Further examples of pharmaceutically acceptable ingredients are small amounts of accessory ingredients, such as a hydrate or emulsifier, preservative or buffer, which enhance the shelf life or effectiveness of the hydrate or antibody.

The pharmaceutical compositions of the invention may be prepared, packaged, or sold in bulk, in single unit doses, or in multiple single unit doses. As used herein, “unit dose” is a discrete amount of a pharmaceutical composition comprising a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient to be administered to the subject or a convenient fraction of such dosage, such as 1/2 or 1/3 of such dosage.

Pharmaceutical compositions of the invention are typically suitable for parenteral administration. As used herein, “parenteral administration” of any of the pharmaceutical compositions includes administration of the pharmaceutical composition via any route of administration characterized by physical leakage of the subject tissue and leakage into the tissue, and thus generally into the bloodstream, into the muscle, Or direct administration into internal organs. Accordingly, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, by application of the composition via surgical resection, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, but not limited to, subcutaneous, intraperitoneal, intramuscular, sternum, intravenous, intraarterial, intradural, intraventricular, intrauterine, intracranial, intratumoral, and intravitreal injections or infusions; And parenteral administration including renal dialysis infusion techniques. Area perfusion is also considered. Preferred embodiments may include intravenous and subcutaneous routes.

Formulations of pharmaceutical compositions suitable for parenteral administration typically comprise the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in multidose containers containing ampoules or preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In one embodiment of the formulation for parenteral administration, the active ingredient is in dry (ie powder or granule) form for reconstitution of a reconstituted composition to a suitable vehicle (eg, sterile pyrogen-free water) prior to parenteral administration. Is provided. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (such as having a pH of 3 to 9), but for some applications, they are more suitably in sterile non-aqueous solutions or It may be formulated in a dry form for use with a suitable vehicle, such as sterile, pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions such as aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered if desired. Other parenteral-administrable formulations useful include those comprising the active ingredient in microcrystalline form or in a liposome preparation. Formulations for parenteral administration may be formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-releases.

IV. Example implementation

Further specific embodiments of the present invention are described as follows.

1. Specific to human TGF-β1, TGF-β2, and TGF-β3, comprising heavy chain complementarity-determining regions (CDRs) 1-3 of SEQ ID NO: 1 and light chain CDR1-3 of SEQ ID NO: 2 An isolated monoclonal antibody that binds to an antibody, wherein the antibody comprises a human IgG ₄ constant region having proline at position 228 (EU numbering).

2. The antibody of embodiment 1, wherein the antibody comprises a heavy chain variable domain (V _H ) amino acid sequence corresponding to residues 1-120 of SEQ ID NO: 1 and a light chain variable domain corresponding to residues 1-108 of SEQ ID NO: 2 ( V _L ) an antibody comprising an amino acid sequence.

3. The antibody of embodiment 2, wherein the antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 1 (with or without C-terminal lysine) and a light chain amino acid sequence as shown in SEQ ID NO: 2.

4. The antigen-binding fragment of the antibody of embodiment 3, wherein the fragment is F (ab ') ₂ .

5. The antibody or fragment according to any one of embodiments 1 to 4, wherein the antibody or fragment has increased half-life or increased exposure compared to presolimumab.

6. The antibody or fragment of any one of embodiments 1-5 wherein the antibody or fragment has one or more of the following properties:

a) inhibits the differentiation of CD4 ⁺ T cells into inducible regulatory T cells (iTreg);

b) increases CD8 ⁺ T cell proliferation;

c) increased clustering of natural killer (NK) cells;

d) increasing the level of MIP-2; And

e) increase the level of KC / GRO.

7. The composition comprising the antibody or fragment of any one of embodiments 1 to 6, wherein the composition comprises less than 1% half antibody.

8. The antibody or fragment according to any one of embodiments 1 to 6 as a medicament.

9. A method of inhibiting TGF-β signaling in a patient in need thereof, comprising administering to the patient a therapeutic amount of an antibody or fragment of any one of embodiments 1-6. .

10. The method of embodiment 9, wherein the patient has cancer.

11. The cancer cell of embodiment 10, wherein the cancer is selected from the group consisting of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma, Way.

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 10-13, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

15. A method of treating cancer in a patient, the method comprising administering to the patient an antibody or fragment of any one of embodiments 1-6, and (2) an inhibitor of an immune checkpoint protein.

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 immune checkpoint protein is an anti-PD-1 antibody.

18. The method of embodiment 17, wherein the anti-PD-1 antibody comprises heavy chain CDR1-3 of SEQ ID NO: 5 and light chain CDR1-3 of SEQ ID NO: 6.

19. The antibody of embodiment 17, wherein the anti-PD-1 antibody comprises a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO: 5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO: 6 Including, method.

20. The antibody of embodiment 17, wherein the anti-PD-1 antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 5 (with or without C-terminal lysine) and a light chain amino acid sequence as shown in SEQ ID NO: 6 How to.

21. The method according to any of embodiments 15-20, wherein the anti-TGF-β antibody is shown in the heavy chain amino acid sequence shown in SEQ ID NO: 1 (with or without C-terminal lysine) and in SEQ ID NO: 2 Comprising a light chain amino acid sequence.

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 15-22, wherein the cancer is advanced or metastatic melanoma, or cutaneous squamous cell carcinoma.

24. The method of any one of embodiments 15-23, wherein the cancer is a solid tumor of the mesenchymal subtype.

25. The method of any one of embodiments 15-24, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT.

26. The cancer according to any one of embodiments 15-25, wherein the cancer consists of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma Selected from the group.

27. The method of any one of embodiments 15-26, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

28. The method of any one of embodiments 15-27, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient the same day.

29. The method of any one of embodiments 15-28, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient once every two weeks.

30. The method of any one of embodiments 15-29, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered at a dose of 0.05-20 mg / kg body weight, respectively.

31. A method of increasing an immune response in a patient in need of an increase in the immune response, comprising administering an immune checkpoint inhibitor and the antibody or fragment of any one of embodiments 1-6 to the patient.

32. The method of embodiment 31, wherein the checkpoint inhibitor is an anti-PD-1 antibody.

33. The method of embodiment 32, wherein the anti-PD-1 antibody comprises:

a) HCDR1-3 of SEQ ID NO: 5 and LCDR1-3 of SEQ ID NO: 6;

b) V _H and V _L corresponding to residues 1-117 of SEQ ID NO: 5 and residues 1-107 of SEQ ID NO: 6, respectively; or

c) a heavy chain having the amino acid sequence shown in SEQ ID NO: 5 (with or without the C-terminal lysine) and a light chain having the amino acid sequence shown in SEQ ID NO: 6.

34. The antibody of any one of embodiments 31-33, wherein the anti-TGF-β antibody is shown in the heavy chain amino acid sequence shown in SEQ ID NO: 1 (with or without C-terminal lysine) and in SEQ ID NO: 2 Comprising a light chain amino acid sequence.

35. The method of embodiments 31-34, wherein the patient has cancer.

36. The method of embodiment 35, wherein the patient is refractory to previous treatment with an immune checkpoint inhibitor and / or has a solid tumor of the mesenchymal subtype.

37. The cancer cell of embodiment 35 or 36, wherein the cancer is selected from the group consisting of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma How.

38. The method of any one of embodiments 35-37, 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 35-38, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

40. The antibody or fragment of any one of embodiments 1-6 for use in treating a patient in any of the above methods.

41. Use of the antibody or fragment of any one of embodiments 1-6 for the manufacture of a medicament for the treatment of a patient in any of the above methods.

42. An isolated nucleic acid molecule, the molecule comprising a nucleotide sequence encoding the heavy chain, light chain, or both of the antibody or fragment of any one of embodiments 1-6.

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. A method of making an antibody or antigen-binding fragment of any one of embodiments 1-6,

Providing a host cell comprising first and second nucleotide sequences encoding the heavy and light chains of the antibody or antigen-binding fragment, respectively,

Growing a host cell under conditions that allow the production of an antibody or antigen-binding fragment, and

Recovering the antibody or antigen-binding fragment.

46. A method of preparing a pharmaceutical composition,

Providing the antibody or antigen-binding fragment of any one of embodiments 1-6, and

Mixing the antibody or antigen-binding fragment with a pharmaceutically acceptable excipient.

47. An article of manufacture or kit comprising the antibody or antigen-binding fragment of any one of embodiments 1-6 and another therapeutic agent.

48. The article of manufacture or kit of embodiment 47, wherein the other therapeutic agent is an immune checkpoint inhibitor described herein.

The invention will be further illustrated in the following examples, which do not limit the scope of the invention described in the claims.

Example

In order that the present invention may be better understood, the following examples are presented. These examples are for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 TGF-β-Binding Properties of Ab1

The affinity of Ab1 for all human and murine TGF-β isoforms was determined by surface plasmon resonance on a Biacore T200 Biosensor instrument (GE Healthcare) using dextran-coated carboxy-methylated (CM5) series S chips. A series of concentrations (1.11, 3.33, 10, and 30 nM) were injected on immobilized recombinant TGF-β to measure binding interactions in real time. TGF-β homodimer was fixed at low density to reduce the binding effect. Injections were performed three times and the binding assay repeated three times. Data from kinetic experiments were processed using Biacore T200 Biaevaluation v2.0 software. The generated sensograms are zeroed, aligned, double-referenced, and shortened for curve fitting analysis using a 1: 1 binding model so that the association rate constant (k _a ), dissociation rate constant (k _d ), and equilibrium dissociation constant (K _D ) was determined.

Recombinant proteins were produced internally (human TGF-β1, 2 and 3) or obtained from R & D Systems (murine TGF-β1 and 2). Table 1 below shows the amino acid sequence homology of the three active TGF-β isoforms between Rhesus monkeys, mice, or rats and humans (homology is reported as the percentage of amino acids conserved over the entire amino acid).

Homology of TGF-β Active Isotypes with Humans Homology percentage TGF-β1 TGF-β2 TGF-β3 Rhesus monkey 100% (112/112) 100% (112/112) 100% (112/112) mouse 99.1% (111/112) * 97% (109/112) * 100% (112/112) Rat 99.1% (111/112) * 97% (109/112) * 99.1% (111/112) Rat and mouse TGFβ1 are 100% homologous to each other, and rat and mouse TGFβ2 are 100% homologous to each other

Since human TGF-β3 and murine TGF-β3 have the same amino acid sequence, no separate affinity measurements were calculated for these two proteins. Likewise, murine and rat TGF-β1 and 2 have identical amino acid sequences and did not calculate separate affinity measurements.

The k _a , k _d , and K _D values of Ab1 determined by this method are shown in Table 2 below. K _D values of Ab1 for human TGF-β1, 2, and 3 were determined to be 1.48, 3.00, and 1.65 nM, respectively. K _D values of Ab1 for murine / rat TGF-β1 and 2 were determined to be 2.80 and 1.88 nM, respectively. These binding properties are similar to those in Presolimumab.

Equilibrium Constants and Affinity of Ab1 for TGF-β human Rats and Rats Human and rat TGF-β1 TGF-β2 TGF-β1 TGF-β2 TGF-β3 k _a (× 10 ⁵ M ^-1 s ^-1 ) 3.11 ± 0.3 2.86 ± 0.3 2.98 ± 0.07 3.48 ± 0.24 2.23 ± 0.6 k _d (× 10 ^-4 s ^-1 ) 3.43 ± 2.9 8.23 ± 5.0 8.35 ± 0.38 6.45 ± 1.44 3.72 ± 2.3 K _D (nM) 1.48 ± 1.1 3.00 ± 2.0 2.80 ± 0.08 1.88 ± 0.56 1.65 ± 1.1

The data demonstrate that Ab1 is a potent and selective pan-TGF-β inhibitor. Measurements using surface plasmon resonance demonstrate that Ab1 has an affinity of 1 to 5 nM for all human and murine TGF-β isotypes. High levels of specificity were confirmed by GLP immunohistochemistry (IHC) tissue cross-reactivity studies using normal rats, cynomolgus monkeys, and human tissues.

Example 2: TGF-β-Neutralizing Titer of Ab1

In vitro titers of Ab1 in neutralizing TGF-β activity were measured in cell-based assays. This assay measured the ability of TGF-β to inhibit proliferation of untransformed mink lung epithelial cells (Mv 1 Lu cells). See, eg, WO 2006/086469 and Mazzieri, et al., Eds, "Methods in Molecular Biology," Vol. 142, "Transforming Growth Factor-β Protocols". Ab1, Presolimumab, and 1D11 (murine anti-TGF-β antibody, whose heavy and light chain sequences are disclosed herein as SEQ ID NOs: 9 and 10) are human TGF-β1, 2, 3 and murine TGF-β1 and The ability to neutralize 2 was evaluated. Recombinant TGF-β protein was produced internally (human TGF-β1, 2 and 3) or obtained from R & D Systems (murine TGF-β1 and 2).

All human and murine TGF-β isotypes inhibited the growth of mink lung cells in a dose-dependent manner in the range of 0.02 pg / ml to 10 ng / ml. To quantify the titer of Ab1, presolimab, and 1D11, 1 ng / ml of the designated TGF-β and serially-diluted antibodies were incubated with mink lung cells. After 3 days incubation, proliferation of cells was quantified by CyQUANT dye which fluoresces upon binding to DNA (FIGS. 1A-E). The data show that Ab1, presolimab, and murine surrogate 1D11 inhibited all human and murine TGF-β isotypes to a similar degree.

Example 3: Inhibition of Inducible T Regulatory 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, we investigated whether Ab1 can inhibit TGF-β-induced differentiation of human CD4 ⁺ T cells into inducible regulatory T cells (iTreg). Primary human CD4 ⁺ T cells were isolated from healthy general donors. Human TGF-β1 was purchased from R & D Systems.

To investigate Ab1 antagonist activity on TGF-β produced endogenously by cultured cells, whole CD4 ⁺ T cells were stimulated for 6 days without addition of exogenous TGF-β (anti-CD3, anti-CD28, And flow cytometry after treatment with 50 μg / ml isotype control (human IgG4, kappa anti-egg lysozyme (HEL) antibody, Crown Biosience), Ab1 or presolumimab in the presence or absence of IL-2). The mean percentage and standard deviation of the CD25 ⁺ FOXP3 ⁺ populations were calculated from three populations (lymphocytes / live / single cells / CD4 ⁺ CD127 ⁻ ). Stimulation of total human CD4 ⁺ T cells with anti-CD3, anti-CD28, and IL-2 increased the percentage of FOXP3 ⁺ CD25 ⁺ (iTreg) in culture from 0% to 15%. Treatment with 50 μg / ml Ab1 or 50 μg / ml presollimab reduced the percentage of iTreg to a similar extent (8% and 7%, respectively; FIG. 2). In contrast, treatment with isotype human IgG ₄ (hIgG ₄ ) control had a minimal effect on iTreg differentiation (20% iTreg) (FIG. 2). CD4 ⁺ T cells isolated from a second healthy general volunteer produced similar results.

To investigate the antagonist activity of Ab1 against exogenous, TGF-β, whole CD4 ⁺ T cells incubated with 2 ng / ml human TGF-β1 was stimulated (anti-CD3, anti-CD28, and IL-2 for 6 days). ) Was analyzed by flow cytometry after treatment with isotype control, Ab1, or presolumimab at various antibody concentrations in the presence or absence of a). The mean percentage and standard deviation of the CD25 ⁺ FOXP3 ⁺ population were calculated from three populations (lymphocytes / live / single cells / CD4 ⁺ CD127 ⁻ ) except where noted. Addition of exogenous TGF-β1 (2 ng / ml) to stimulated total human CD4 ⁺ T cells increased the percentage of iTreg in culture from 15% to 55%. Treatment with increasing concentrations of Ab1 reduced the percentage of iTreg in a simultaneous manner from 55% to 15% at 200 μg / ml and 43% at 6.25 μg / ml. Treatment with presolimumab reduced the percentage of iTreg to a similar level as Ab1 (55% to 16% at 200 μg / ml and 32% at 6.25 μg / ml). Treatment with varying concentrations of isotype control antibodies had no effect on the percentage of iTreg with 60% at 200 μg / ml and 6.25 μg / ml. See FIG. 3. CD4 ⁺ T cells isolated from a second healthy general volunteer produced similar results.

This study demonstrates that Ab1 inhibits TGF-β-induced iTreg differentiation and thus may have clinical benefit by reducing the immunosuppressive tumor microenvironment.

Example 4 Effect of Combination of Ab1 and Anti-PD-1 Antibodies in Vitro

In this study, we investigated whether TGF-β would prevent maximal stimulation of T cells in vitro after anti-PD-1 treatment, and if so Ab1 could react to the prevention. T cell activation levels were measured using luciferase expression from expression constructs under transcriptional control of NFATc (activated T cell nuclear factor, cytoplasm 1) regulatory sequences.

We used a cell assay system purchased from Promega for this study. The system comprises two cell types: 1) luciferase reporter induced by the NFAT response element and Jurkat T cells expressing human PD-1, and 2) activating the cognitive T cell receptor in an antigen-independent manner. CHO-K1 cells expressing engineered cell surface proteins and human PD-L1 designed to. In co-culture, Jurkat T cells interact with CHO-K1 cells to induce T cell receptor stimulation and translocation of NFATc into the nucleus, which induces luciferase expression. However, the involvement of PD-1 / PD-L1 recruits tyrosine-protein phosphatase non-receptor 11 (SHP2) into the T cell receptor complex, inhibiting NFATc nuclear translocation and subsequent luciferase expression. Blocking PD-1 signaling mitigates SHP2-dependent inhibition and thus allows maximal luciferase expression. The system thus provides a functional method for determining the effect of TGF-β on T cell signaling and the effect of Ab1 on anti-PD-1 treatment of T cells.

Due to the slow kinetics associated with TGF-β-dependent effects, Jurkat T cells were pre-treated with TGF-β prior to T cell receptor stimulation. Human TGF-β1 was purchased from R & D Systems. Isotype control antibodies against Ab1 (anti-HEL hIgG ₄ ) were purchased from Crown Bioscience (Cat # C0004-5). Mouse anti-hPD-1 IgG and its isotype control antibodies were purchased from BioLegend (Cat # 329912). Fourteen replicates were analyzed for each sample.

The results indicate that the addition of anti-hPD-1 antibody to Jurkat T cells co-cultured with CHO-K1 cells for 24 hours resulted in luciferase activity (865794 relative luminescence unit [RLU]) and addition of isotype control (234963). RLU, magnification change = 3.685, p-value <0.0001) or only absence of antibody (206043 RLU, magnification change = 4.202, p-value <0.0001). Pre-treatment of Jurkat T cells with 18 ng / ml TGF-β1 for 12 days compared to Jurkat T cells (865794 RLU, magnification change = -1.355, p-value <0.0001) not treated with TGF-β1 Less luciferase activity (638866 RLU) was induced in CHO-K1 cell co-culture in the presence of hPD-1 antibody (FIG. 4).

To assess antagonistic titers of Ab1, Jurkat T cells were pre-treated with 18 ng / ml TGF-β1 for 12 days in the presence of Ab1, isotype control Ab, or without Ab, followed by anti-PD-1 for 24 hours. Co-culture with CHO-K1 cells in the presence of The presence of Ab1 (924186 RLU) was compared to the isotype control Ab (639440 RLU, magnification change = 1.445, p-value <0.0001) and the absence of Ab control (638866 RLU, magnification change = 1.447, p-value <0.0001) Mitigated TGF-β-dependent inhibition of activity. Control group comprising isotype control (955717 RLU) or Ab1 added to Jurkat T cells co-cultured with CHO-K1 cell co-culture in the presence of anti-PD-1 Ab and not pre-treated with TGF-β1 (975654 RLU) had statistically elevated luciferase activity compared to the absence of the Ab control, but the magnification change was minimal (865794 RLU, magnification-change = 1.127 and 1.104, p-value = 0.0023 and 0.001284, respectively) 4). RLU values are also presented in Table 3 below.

Relative Luminescence Units in T Cell Activation Assays Anti-PD-1 Ab Anti-PD-1 Ab isotype control Vehicle
(PBS) No TGF-β1 pre-treatment TGF-β1 pre-processing Ab1 absence 865794 638866 234963 206043 Ab1 975654 924186 - - Anti-HEL hIgG ₄ 955717 639440 - -

In order to rule out the possibility that TGF-β1 pre-treatment of Jurkat T cells resulted in reduced proliferation or bioactivity and thus reduced luciferase activity during 24 hour co-culture with CHO-K1 cells, Jurkat T cells were incubated with 18 ng / ml TGF-β1 or PBS in the presence of Ab1, anti-HEL hIgG ₄ , or in the absence of antibody (vehicle). Each group was inoculated into fresh flasks at the time when both TGF-β1 and antibody were refreshed using the same volume every two to three days (there were two total re-inoculation cases). Evaluation of the final culture demonstrated that the bioactivity of all treatment groups did not change (range of 94% to 96%). In addition, the total number of Jurkat T cells in the final culture of each treatment group was very similar (25 million to 29 million).

The study demonstrates that the increase in signaling downstream of the T cell receptor after anti-PD-1 treatment is inhibited by TGF-β, resulting in sub-optimal T cell stimulation. Our data indicate that inhibition of TGF-β ameliorates immunosuppressive tumor microenvironments and that checkpoint modulators, such as anti-PD-1 agents, induce an immune response better, and thus benefit from immune oncology treatment. It is suggested that the ratio can be increased.

Example 5: Effect of Ab1 and Anti-PD1 Antibody Combination In Vivo

Next we studied the effects of combined anti-TGF-β and anti-PD-1 treatments in a C57BL / 6 mouse cancer model.

Tolerance / Preliminary Safety

Substances and Methods

Tolerance of Ab1 and anti-mouse PD-1 (mPD-1) monoclonal antibody (mAb) as a single agent and combination was evaluated in C57BL / 6 female mice. Ab1 (10, 20, and 50 mg / kg) or isotype control Ab (anti-HEL hIgG ₄ purchased from Crown Bioscience; used at 10 and 20 mg / kg) as a single formulation every three days (Q3D) IV Or IV twice a week in combination with 5 mg / kg of anti-PD1 Mab. The anti-PD1 Ab used in this study was named "antimPD1_hyb_RMP114_mIgG1LCfullrat" (or x-anti-mPD-1 Mab). It was a chimeric rat anti-mPD-1 antibody generated by replacing the rat Fc region of the rat IgG _2a clone RMP1-14 (BioXcell, Cat. # BE0146) with mouse IgG ₁ Fc. The heavy and light chain amino sequences of the chimeric antibodies are shown in SEQ ID NOs: 7 and 8. Tolerability was assessed by measuring animal weight and clinical observations. At the end of the 3 week treatment, 4 hours after the last treatment, terminal sampling was performed and tissues (heart, kidney, liver, lung, and spleen) were fixed in formaldehyde and sent for histopathological analysis.

If individual mice cause 15% weight loss for 3 consecutive days, 20% weight loss for 1 day, or more than 10% drug-related death, no tumor-induced cachexia causing weight loss is observed in the control vehicle-treated group. However, the dose was considered excessively toxic. Animal weights included tumor weight.

Toxicity / Safety Results

Tolerability studies in C57BL / 6 mice showed that all evaluated dose levels of single agents and combinations of Ab1 and x-anti-mPD-1 Mab were well tolerated. No significant body weight change was observed in any treatment group at all doses evaluated. No serious or major clinical observations were observed during the study. Histopathological analysis identified an increased number of lymphocytes in the spleen (white stromal) of all treatment groups, including the isotype control antibody treatment group, regardless of any combination, whatever the combination. No other significant microscopic findings were observed. Two animals from the combination group, the isotype control Ab (10 mg / kg) and the anti-PD-1 Mab (5 mg / kg) were found dead after the last dose on the last day of the study. Histopathological analysis did not identify any drug-related causes of death.

Effectiveness study

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

Substances and Methods

animal

Female C57BL / 6 mice were obtained from Charles River Labs (Wilmington, Mass., USA). Animals were allowed to acclimate for at least 3 days prior to study enrollment. Mice were 11 weeks old and weighed 17.0-20.9 g at the start of the study. They had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water and were housed in a 12 hour light / dark cycle.

Tumor cells

MC38 is a colon adenocarcinoma cell line. Cells were obtained from the National Cancer Institute (Bethesda, MD, USA) and Roswell Park Memorial containing L-glutamine (Gibco, Cat # 11875) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) (Gibco, Cat # 10438026). Incubated at 37 ° C. with 5% CO ₂ in complete medium (CM) containing Institute medium (RPMI) -1640. Cells were harvested and resuspended in Dulbecco phosphate-buffered saline (DPBS) (Gibco, Cat # 14190) and 1 × 10 ⁶ cells / 200 μl per mouse subcutaneously (SC) implanted into the right thigh of female C57BL / 6 mice It was.

compound

Ab1 was administered to the animals in aqueous solution. It was 0.22 μm-filtered through PES and stored as a sterile aliquot at 2-10 ° C. Antibodies were given to animals at 10 ml / kg intraperitoneally (IP), 25 mg / kg.

Anti-HEL hIgG ₄ (Crown Bioscience) was used as the isotype control for Ab1. The antibody was given to control animals at 10 mg / kg by IP and 25 mg / kg by IP.

x-anti-mPD-1 Mab (supra) was given in DPBS (Gibco, Cat # 14190-094) and given to animals by IP at 10 ml / kg, 5 mg / kg.

Study design

On day 0, 60 animals were implanted with MC38 tumor cells. On day 8 post-transplant, mice with mean tumor size of 50-75 mm 3 were pooled and randomized to control and treatment groups (10 mice per group). Treatment with vehicle (PBS, pH 7.2), anti-HEL hIgG ₄ , Ab1, and anti-mPD-1 Mab at the above-described doses was initiated on day 9 and repeated on days 12, 15, 18, 21, and 27. Vehicle- and anti-HEL hIgG ₄ -treated animals were used as controls. Mice were checked daily and adverse clinical responses were recorded. Individual mice were weighed 3-4 times a week until the end of the experiment.

Mice were euthanized if pathological conditions or at least 20% weight loss were observed. Tumors were measured twice a week with calipers until the final sacrifice. When tumor size reached approximately 2000 mm 3 or animal health problems (20% area of tumor ulcerated) were present, the animals were euthanized and the date of death was recorded. Solid tumor volume was estimated from two-dimensional tumor measurements and calculated according to the following formula:

Tumor volume = [length (mm) × width ² (mm2)] / 2

The median of the individual regression percentages calculated for each animal in the group on a given day was then taken to obtain the median regression percentage for the group on that day. Except when the median regression percentage did not indicate activity of the group, the date of calculation was determined on the day when ΔT / ΔC (ie, the ratio of median tumor volume changes from baseline between treatment and control) was calculated. In that case, this day was determined on the first day with the highest median regression percentage. Regression was defined as partial (PR) when tumor volume decreased by 50% of tumor volume at the start of treatment. Complete regression (CR) was considered achieved when the tumor volume was less than 14 mm 3 or could not be recorded.

effectiveness

Primary efficacy endpoints were tumor volume change from baseline, expressed as ΔT / ΔC, median regression percentage, partial regression, and complete regression. Tumor volume changes for each treatment group (T) and control group (C) were calculated daily for each animal by subtracting the tumor volume of the first treatment day (start date) from the tumor volume of the specified observation day. Median ΔT was calculated for the treatment group and median ΔC was calculated for the control group. Calculate the ratio ΔT / ΔC and express it as a percentage:

ΔT / ΔC = (median delta T / median delta C) × 100

A ΔT / ΔC ratio of up to 40% was considered therapeutic activity. The ΔT / ΔC ratio 0% was considered tumor retention. A ΔT / ΔC ratio of less than 0% was considered tumor regression.

Tumor regression percentage was defined as the percentage of tumor volume reduction in the treatment group at the specified observation day relative to the tumor volume at the start of the study (t ₀ ). For each animal at the specified time point t, the percent regression was calculated using the following formula:

(in t) degeneration% = _[(t0 volume - volume _t) / volume _t0] × 100

The median of individual regression values calculated for each animal in the group was then taken to calculate the median regression percentage for the group on a given day. The date of calculation was determined to be the day at which ΔT / ΔC was calculated, except when the median regression percentage did not indicate activity of the group. In that case, this day was determined as the first day with the highest median regression percentage.

Statistical analysis

Two-way ANOVA types, including treatment and date (repeat) factors, were performed for tumor volume changes from baseline. Control, including Bonferroni-Holm correction for multiplicity, to compare all treatment groups with controls on each day from day 8 to day 27, if there were significant treatment * date interactions or treatment effects An analysis followed. Tumor volume change from baseline for each animal and each day was calculated by subtracting the tumor volume of the first treatment day (day 8) from the tumor volume of the specified observation day.

Since heterogeneity of variance was observed between groups, a group = optional compound symmetry (CS) covariance structure was chosen for the ANOVA type model (SAS Institute Inc. (2008) SAS / STAT 9.2 User's Guide by Cary NC). 5 and 6, median and median absolute deviations (MAD) of each group are shown for each measurement day. In Tables 4-6 below, the median and normalized MAD (nMAD = 1.4826 * MAD) of each group is reported for each measurement day. All statistical analyzes were performed using SAS version v9.2 software. A probability (p <0.05) of less than 5% was considered significant.

Validity result

Treatment of tumor-bearing C57BL / 6 mice with Ab1, anti-PD-1 Mab, or a combination of both was well tolerated and nontoxic as suggested by the absence of significant changes in the general health and activity and body weight of the animals. As a single formulation, Ab1 25 mg / kg Q3D and anti-PD-1 Mab 5 mg / kg Q3D induced only 3.4% (day 9) and 2.1% (day 9) nadir weight loss values, respectively. The combination of Ab1 (25 mg / kg Q3D) and anti-PD-1 Mab (5 mg / kg Q3D) was also well tolerated, indicating a weight loss value of day 9 (day 9) of nadir (Table 4).

As a single agent, Ab1 (25 mg / kg Q3D) and anti-PD-1 Mab (5 mg / kg Q3D) had a disturbance on tumor growth compared to animals treated with Ab1 isotype control (anti-HEL hIgG ₄ ). Not shown. 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 hIgG ₄ demonstrated 31% ΔT / ΔC with minimal anti-tumor activity on day 27 of treatment (not statistically different from control) and 2 of 10 mice Only complete degeneration was observed. However, the combination of anti-PD-1 Mab and Ab1 demonstrated -1 ΔT / ΔC at day 27 and demonstrated better anti-tumor activity from day 15 to day 27 (statistically different from control) out of 10 mice. Complete regression was observed in 6 rats (Table 4).

Activity of Ab1 and X-anti-mPD-1 Mab in C57BL / 6 MC38 Cancer Model Formulation ΔW *
(nadir date) ΔT / ΔC%
(Day 27) Median Degeneration%
(Day 27) p-value **
(Day 27) Regression
(Day 27) PR CR Vehicle -1.8 (D9) 100 - - 0/10 0/10 Anti-HEL hIgG ₄
(25 mg / kg) - 104 - 0.7914 1/10 1/10 Ab1 (25 mg / kg) -3.4 (D9) 93 - 0.9953 0/10 0/10 x-anti-mPD-1 (5 mg / kg) -2.1 (D9) 109 - 0.5435 2/10 1/10 Ab1 (25 mg / kg) +
x-anti-mPD-1 (5 mg / kg) -1.3 (D9) -One 24.5 <0.0001 6/10 6/10 Anti-HEL hIgG ₄ (25 mg / kg) + x-anti-mPD-1 (5 mg / kg) -3.0 (D9) 31 - 0.4020 2/10 2/10

* ΔW represents the average weight change per group in nadir.

** p-values in a control assay using Bonferroni-Holm adjustment for multi-way after 2-way ANOVA-type with repeated measurements of tumor volume change from baseline to compare each treatment group with the control group Obtained. A probability (p <0.05) of less than 5% was considered significant.

Tables 5 and 6 and FIGS. 5-7 provide additional data showing the activity of antibodies alone or in combination on tumor volume in mouse models.

Treatment vs. Control Tumor volume change from baseline ㎣: median (nMAD), n, and p-value * Treatment group all Day 12 Day 15 Day 19 Day 23 Day 27 Vehicle - 26.0
(20.02)
n = 10 71.5
(53.37)
n = 10 220.5
(117.87)
n = 10 528.0
(157.16)
n = 10 1610.0
(413.65)
n = 9 Anti-HEL hIgG ₄
(25 mg / kg) -
0.9629 17.0
(17.79)
n = 10
0.5197 65.5
(51.89)
n = 10
1.0000 182.5
(113.42)
n = 10
0.8858 491.5
(358.05)
n = 10
1.0000 1675.0
(621.21)
n = 9
0.7914 Ab1
(25 mg / kg) -
0.9629 17.0
(15.57)
n = 10
0.4754 79.0
(43.74)
n = 10
1.0000 218.0
(83.03)
n = 10
0.8858 567.0
(344.70)
n = 10
1.0000 1496.5
(472.21)
n = 8
0.9953 X-anti-mPD1 Mab
(5 mg / kg) -
0.2439 -2.0
(21.50)
n = 10
0.0224 38.5
(68.20) n = 10
0.1235 147.5
(149.74)
n = 10
0.2259 458.0
(280.21)
n = 10
0.2228 1748.0
(851.01)
n = 9
0.5435 Ab1
(25 mg / kg) +
x-anti-mPD-1 Mab
(5 mg / kg) -
0.0007 -3.5
(10.38)
n = 10
0.0743 20.0
(20.02)
n = 10
0.0121 -0.5
(69.68)
n = 10
<.0001 -17.0
(51.15)
n = 10
<.0001 -9.0
(86.73)
n = 10
<.0001 Anti-HEL hIgG ₄
(25 mg / kg) +
x-anti-m-PD-1
(5 mg / kg) -
0.2531 19.0
(37.81)
n = 10
0.5197 61.5
(65.98)
n = 10
0.2848 145.0 (160.12) n = 10
0.2259 266.0
(398.82)
n = 10
0.1979 503.0
(841.38)
n = 8
0.4020

P-values were obtained by a control analysis of each day using Bonferroni-Holm adjustment for multiplicity after two-way Anova-type for tumor volume change from baseline.

Ab1 and X-anti-mPD-1 Mab as Single Agent vs. Combination Tumor volume change from baseline ㎣: median (nMAD), n and p-value * Treatment group all Day 12 Day 15 Day 19 Day 23 Day 27 Ab1
(25 mg / kg) +
x-anti-mPD-1 Mab
(5 mg / kg) - -3.5
(10.38)
n = 10 20.0
(20.02)
n = 10 -0.5
(69.68)
n = 10 -17.0
(51.15)
n = 10 -9.0
(86.73)
n = 10 X-anti-mPD-1
(5 mg / kg) -
0.0405 -2.0
(21.50)
n = 10
1.0000 38.5
(68.20)
n = 10
0.7631 147.5
(149.74)
n = 1
0.0276 458.0
(280.21)
n = 10
0.0004 1748.0 (851.01)
n = 9
0.0024 Ab1
(25 mg / kg) -
0.0017 17.0
(15.57)
n = 10
1.0000 79.0
(43.74)
n = 10
0.0694 218.0
(83.03)
n = 10
0.0007 567.0
(344.70)
n = 10
<.0001 1496.5 (472.21)
n = 8
<.0001 Anti-HEL hIgG ₄ (25 mg / kg) +
x-anti-mPD-1 Mab
(5 mg / kg) - 19.0
(37.81)
n = 10 61.5
(65.98)
n = 10 145.0
(160.12)
n = 10 266.0
(398.82)
n = 10 503.0
(841.38)
n = 8 X-anti-mPD-1 Mab
(5 mg / kg) -
0.8680 -2.0
(21.50)
n = 10 0.2024 38.5
(68.20)
n = 10
0.7631 147.5
(149.74)
n = 10
0.9476 458.0
(280.21)
n = 10
0.8576 1748.0 (851.01)
n = 9
0.8491 Anti-HEL hIgG ₄
(25 mg / kg) -
0.5691 17.0
(17.79) n = 10
1.0000 65.5
(51.89)
n = 10
0.5743 182.5
(113.42)
n = 10
0.4761 491.5
(358.05)
n = 10
0.2357 1675.0 (621.21)
n = 9
0.8491

* Bonperoni-Holm adjustments for two-way Anova-type post-multiplicity for tumor volume change from baseline using Ab1, anti-HEL hIgG ₄ and x-anti-mPD-1 combination versus combination on each day P-values were obtained in a control assay to compare each single agent of the dose involved.

The data shown in the tables and figures show that the combination of Ab1 25 mg / kg Q3D and x-anti-mPD-1 Mab 5 mg / kg Q3D had greater antitumor effect than either antibody at these doses. When comparing the combination with Ab1 as a single agent, the p values at days 19, 23, and 27 were 0.0007, <0.0001, and <0.0001, respectively, with the difference being statistically significant. When comparing the combination to the x-anti-mPD-1 Mab as a single agent, the p values at days 19, 23 and 27 were 0.0276, 0.0004, and 0.0024, respectively, with the difference being statistically significant (Table 6). . In the combination group of anti-HEL hIgG ₄ 25 mg / kg Q3D and x-anti-mPD-1 Mab 5 mg / kg Q3D, the effect of treatment on tumor volume change from baseline was only with either agent on any measurement day. It did not differ significantly from the effect of.

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

In another study, we found antitumor activity of a combination of 1, 10, or 25 mg / kg Ab1 and 5 mg / kg mouse PD-1 antibody in a subcutaneous MC38 mouse colon cancer model in C57BL / 6J mice. Evaluated. Exponentially growing MC38 colon adenocarcinoma cells (NCI, Frederick, MD) were cultured in RPMI-1640 supplemented with 10% FBS in a humidified 5% CO ₂ incubator and then in female C57 / Bl6J mice (Jackson Laboratory, Bar Harbor, ME). Subcutaneously transplanted into the thighs (1 × 10 ⁶ cells). When tumors reached an average size of 50-75 mm 3, mice were pooled and randomized to control and treatment groups (10 mice per group). Tumor-bearing mice were then treated with PBS, IgG4 isotype control antibody (25 mg / kg), or Ab1 (1, 10, and 25 mg / t) three times a week until each mouse received a total of 6-7 doses. Kg) was treated intraperitoneally. Tumors were measured twice a week with digital calipers and tumor volumes were calculated (㎣ = L × W × H) and graphed using GraphPad Prism. At the end of the study, mice were euthanized with CO ₂ if the tumor grew above 2000 mm 3, or if the tumor exhibited ulcers greater than 20% of the tumor surface.

Ab1 25 mg / kg Q3D and 5 mg / kg mouse α-PD-1 antibody as a single agent demonstrated that they were partially active with 2/8 and 4/8 complete regression, respectively, in MC38 tumor bearing mice. The combination of Ab1 1, 10, or 25 mg / kg Q3D and mouse α-PD-1 antibody 5 mg / kg Q3D was therapeutically active. At day 24 post-transplantation, when comparing tumor volume from baseline, the effect of all assessment doses of Ab1 and the mouse α-PD-1 antibody 5 mg / kg Q3 combination was 1, 10, and 25 mg / kg of Ab1, respectively. Were greater than the effect of each single agent with 5/8, 6/8 and 7/8 complete regression in. Table 6a provides a summary of the results.

TABLE 6a

In summary, these preclinical data demonstrate that the combination of PD-1 inhibition and TGF-β inhibition can significantly inhibit tumor growth compared to the checkpoint inhibitor block alone.

Example 6: Intratumoral TGF-β1 Levels

Intratumoral TGF-β1 levels were studied in LoVo colorectal cancer subcutaneous xenograft-grafted BALB / c mouse model. Starting with tumor volume <100 mm 3, mice were injected intravenously with Ab1 or isotype control Mab 10, 25, or 50 mg / kg every 3 days for a total of 8 IV administrations.

Tumor samples stored at −80 ° C. in 2 ml plastic tubes containing 2.8 mm ceramic balls (MoBio 13114-50) were thawed at room temperature. One milliliter (ml) of low temperature 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 and 20 seconds each at 4 ° C. Homogenized using Precellys® 24 Dual homogenizer (Bertin Instruments) twice at 6500 rpm. The lysates were clarified by centrifugation at 20,000 × g for 10 minutes at 4 ° C. in an Eppendorf 5417C centrifuge. The supernatant was transferred into a clean cold Eppendorf tube and further clarified by centrifugation for an additional 20 minutes as described above. The supernatant was then transferred into a plastic 96-well storage block, flash frozen in liquid nitrogen and stored at -80 ° C.

The next day the samples were thawed at room temperature and placed on ice. Protein concentration in the lysate was measured using the Non-Succinic 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 (see above) containing protease and phosphatase inhibitors and aliquoted into plastic microtubes.

TGF-β1 concentrations in normalized tumor lysates were measured using the human TGF-β1 kit (MSD, K151IUC-2) using an electrochemiluminescent assay 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 correction factor. Samples were loaded onto plates two by two. Electrochemiluminescence signals were measured using a MESO SECTOR S 600 plate reader (MSD) and the TGF-β1 concentration in the sample was quantified using MSD Discovery Workbench software v4.0 based on a standard curve.

Average concentrations for the same samples were calculated by software. Concentration values determined by the software to be "below the fitting curve range" or "below the detection range" were replaced with zero values. To calculate the TGF-β1 concentration per mg of total protein, the concentration (pg / ml) measured in the assay was divided by the protein concentration (mg / ml) in the sample.

The results showed that intratumoral TGF-β1 levels in mice injected with the isotype control had a median value of 21.4 pg / total protein mg, and corresponding levels in mice injected with Ab1 were undetectable (FIG. 8).

To demonstrate the relevance of this finding in humans, we evaluated 10 human colorectal tumor samples and 10 human melanoma tumor samples for intratumoral TGF-β1 levels as described above using the methods described above. . For human CRC samples, TGF-β1 levels ranged from about 7 to 25 pg / mg. For human melanoma samples, TGF-β1 levels ranged as high as about 1 pg / mg to 43 pg / mg. These data further support the use of anti-TGF-β1 therapeutics, such as Ab1, alone or in combination with other immune checkpoint inhibitors, such as anti-PD-1 antibodies.

Example 7: Pharmacokinetic Studies of Ab1

This example describes a study that characterizes the pharmacokinetic (PK) profile of Ab1 and compares it to the profile of presolimab. In one study, five groups of cannula intubated Sprague-Dawley rats were given intravenously with a single dose of Ab1 or presolumimab 5 mg / kg. Each group consisted of 5 females and 5 males. Blood from rats was collected 0.25, 6, 24, 48, 72, 144, 192, and 240 hours after administration. Ab1 and presolimab serum concentrations were determined by ELISA. Comparability was determined when the 90% confidence interval for the AUC ratio (of evaluation material to reference) was in the range of 80% to 125%.

Over time antibody serum concentrations from five groups of rats are shown in FIG. 9A. PK parameters from groups 2, 4, and 5 (see note in FIG. 9A) are shown in Table 7 below. This study showed that Ab1 linearly behaves with a much longer half-life (half-life T _1/2 7.1 days versus 4.3 days) and lower elimination rates (CL 0.30 ml / hr / kg vs. 0.51 ml / hr / kg) than presolimumab. It was shown to have. This data indicated that Ab1 had 1.7 times higher exposure in rats than presolumimab.

Comparison of PK between Presolimumap and Ab1 PK parameters Presolimumap
(Batch 2) Ab1
(Batch 1) Ab1
(Batch 2) t _1/2 (hr) 103.21 ± 12.98 181.83 ± 80.92 * 158.38 ± 66.73 Cmax (μg / ml) 114.56 ± 16.42 116.60 ± 16.78 109.72 ± 18.49 Vz (ml / kg) 75.22 ± 15.21 78.35 ± 26.33 63.47 ± 10.65 CL (ml / hr / kg) 0.51 ± 0.10 0.31 ± 0.04 * 0.30 ± 0.05 * AUC _last (hr * µg / ml) 8046 ± 1210 10007 ± 1180 * 11223 ± 1035 * AUC _0-INF (hr * μg / ml) 10107 ± 1613 16322 ± 2523 * 17553 ± 4130 *

* This group means that the mean ± SD is statistically different from the group of “presolimumap (B2)” (group 2 in FIG. 9A).

Additional PK studies on Ab1 (Study 2) were performed in cynomolgus monkey groups. Each group consisted of 5 females and 5 males, with a single dose of Ab1 1 mg / kg (FIG. 9B) or 10 mg / kg (FIG. 9D), or Ab1 1 mg / kg per dose (FIG. 9C). Or 5 weekly doses of 10 mg / kg (FIG. 9E) were given by intravenous infusion. The serum concentrations of Ab1 in monkeys are shown in Figs. 9B-E. Over time serum concentrations of presolimab given to monkeys in single or repeated Q2W (once weekly) doses in previous studies are also shown for comparison. These data showed that Ab1 also had linear PK behavior in monkeys and showed higher exposures after both single and repeated administrations compared to presolimab at both 1 mg / kg and 10 mg / kg per dose. After a single dose of 10 mg / kg, Ab1 had a half-life of 13 days, while presolimumab had a half-life of 4.5 days; Ab1 had a CL of about 0.40 ml / hr / kg, while Presolimumab had a CL of 0.66 ml / hr / kg. Similar to the rat study, the monkey study showed that Ab1 had about 1.7 times higher exposure than presolimumab.

The studies described above demonstrate that Ab1 has a statistically significant longer half-life, longer elimination time, and higher biological exposure compared to presolimumab.

In addition, studies in Ab12 tumor bearing Balb / C mice showed that Ab1 had a similar PK profile, whether administered intravenously or intraperitoneally.

Using relative growth scaling for the two-compartment model, we predicted the following PK parameters in 70 kg of human based on monkey data (Table 8):

Relative Growth Modeling for PK Parameters PK parameters Ab1 in monkeys Ab1 in humans T _1/2 (day) 13.1 20.9 CL (ml / hr / kg) 0.392 5.7 V1 (center compartment) (L) 0.104 2.43 Q (ml / hr) 3.18 46 V2 (peripheral compartment) (L) 0.0693 1.62

Predicted PK parameters of Ab1 are also preferred over presolimumab in humans. For example, presolimumab with a CL of 12.3 ml / hr / kg in humans has a faster clearance than Ab1.

Example 8: Toxicological Study of Ab1

Toxicological studies of Ab1 were performed in rat and cynomolgus monkeys. Safety pharmacology endpoints were assessed with weekly repeat-dose GLP (Drug Clinical Trial Control). At up to 10 mg / kg / dose (2 mg / ml concentration) in monkeys and up to 30 mg / kg / dose (6 mg / ml concentration) in rats, there was no Ab1-related histopathological findings at the injection site. Ab1-related effects on body temperature, respiratory rate, blood pressure, and ECG parameters were not noted at all dose levels evaluated in neurological tests.

NOAEL (no harmful effect level observed) on rats was found to be 3 mg / kg / dose at weekly repeated doses for 5 weeks, and STD10 (severe to induce death or irreversible severe toxicity in 10% of animals) Toxic doses) were identified at 3 to 10 mg / kg / dose for rats. Toxicities include cardiac valve proliferation characterized by multiple thickening nodules; And abnormal lung diseases such as mixed cell alveolar exudate, mixed cell perivascular infiltrates, enlarged muscle arteries, bleeding, and / or increased lung weight.

The dose of NOAEL and HNSTD (ie, the highest non-emetic toxicity dose that goes beyond lethal, life-threatening, or irreversible toxicity) doses to monkeys at weekly repeated doses of 5 weeks were identified as 10 mg / kg / dose. It was (cf. Solid pre mumaep 7 or when administered in two or once a week for four weeks for the 13 Q3D capacity, appears the NOAEL in monkeys with 1 ㎎ / ㎏). See also the data in Table 9 below.

Summary of Toxicological Studies in Rats and Monkeys Toxicological parameters Rat monkey LD (lethal dose) 50 mg / kg / dose ¹ Over 10 mg / kg / dose Highest Non-lethal Capacity (HNLD) 30 mg / kg / dose 10 mg / kg / dose STD10 3-10 mg / kg / dose NA HNSTD NA 10 mg / kg / dose NOAEL Less than 3 mg / kg / dose 10 mg / kg / dose Major target organ (histopathology) Heart, lungs, bones, teeth Not verified ¹ : Based on exploratory research
NA: Not applicable

Based on the toxicological data, it is expected that Ab1 may be safely administered to human patients at a dosage level of about 0.05 mg / kg to 0.5 mg / kg once a week or less frequently, such as once a week. .

Example 9: In vivo efficacy of anti-TGF-β monotherapy

In this study, we investigated the effects of 1D11, mouse IgG ₁ anti-bovine TGF-β antibodies that cross-react with human and mouse TGF-β1, 2, and 3 in metastatic syngeneic tumor models. In this model, B16-F10 mouse melanoma cells were introduced by IV into the plantar of C57BL / 6 mice to form metastases in drained lymph nodes of mice. Treatment with the control antibody, 13C4, was ineffective, but treatment with 50 mg / kg 1D11 three times a week beginning one day after tumor inoculation completely eliminated metastasis.

To investigate the role of the immune response, B16-F10 was implanted into the soles of mice lacking the β2-microglobulin gene and thus lacking a CD8 ⁺ cytotoxic T cell response and treated as before. In contrast to the results seen in immunocompetent mice, 1D11 had no effect on the number of metastases in draining lymph nodes of these mice. These results suggest that the mechanism of action of TGF-β inhibition depends on adaptive cellular immunity.

Example 10 TGF-β Features in Cancer

Previous studies have shown that patients with melanoma who do not respond to anti-PD-1 therapy have transcriptional feature IPRES (Hugo et al., Cell (2016) 165: 35-44). To investigate the innate resistance mechanism for anti-PD-1 monotherapy, we studied the transcriptional features of non-responders versus responders. We found that a comparison of these profiles with a database with greater than 1 M profile using the Gene Set Enrichment assay revealed a strong correlation between anti-PD-1 response and activation of TGF-β signaling in tumors. These data suggest that, at baseline of melanoma, TGF-β is associated with innate resistance to anti-PD-1 monotherapy.

In addition, the present inventors confirmed that not only the correlation between the anti-PD-1 response and the activation of TGF-β signaling exists but also that the correlation is strong (R = 0.59, p-value <9E by t-test). -4). Thus, we have reached that Gate Preview 1: TGF-β mediated immunosuppression in melanoma (eg, metastatic melanoma) may contribute to innate resistance. In addition, the inventors have confirmed that TGF-β induced gene expression changes can be quenched by 1D11 treatment, confirming the specificity of the TGF-β activation feature. These results support the benefit of using anti-TGF-β and anti-PD-1 therapeutic combinations to treat cancer patients who do not respond to anti-PD-1 monotherapy.

Analysis of this correlation across tumor types other than melanoma shows that anti-PD-1, in which mesenchymal tumors (eg CRC, HCC, head and neck squamous cell carcinoma, and ovarian cancer) are also activated and predicted by TGF-β It has been shown to be strengthened against both resistance. The findings were consistent with the role of TGF-β signaling in EMT. Thus, the inventors have arrived at Gateway Preview 2: mesenchymal tumors, particularly tumors with immune infiltration, can benefit from anti-TGF-β and anti-PD-1 combination therapy. Machine learning methods were used to identify fewer genes that could be used to select mesenchymal tumors from more than 30 EMT marker genes, such as ACTA2, VIM, MGP, ZEB2, and ZWINT. ACTA2 and VIM have been found to be transportable, for example, across several tumor types. Thus, TGF-β activating transcriptional features and genes within these features can serve as useful biomarkers for cancer patient selection at baseline for anti-TGF-β and anti-PD-1 antibody combination therapy.

To study biomarkers in the tumor microenvironment, the immune structures of patient tumors were evaluated using MultiOmyx, multiplex IHC assays in CRC and melanoma. Multiplexing was performed with 12 biomarkers (in conjunction with describing 22 immune cell types) on a single FFPE section from each tumor sample. The study included a range of inflammation to assess how well the assay assesses each tumor type and correlates it with possible therapeutic effects. Statistical methods were developed to assess differences at the cell population level, including replicate samples, volcano graphs for variance analysis, and correlation matrices. The MultiOmyx assay demonstrated excellent technical reproducibility and precision, preferred dynamic range, selected immune cells and inflammatory status differences in the region of interest and included both positive and negative correlations between cell populations.

Example 11 Changes in TGF-β1, MIP-2 and KC / GRO in MC38 Tumors After Treatment with Ab1 With or Without Anti-PD1

To demonstrate neutralization of TGF-β, the ability of Ab1 (with or without anti-PD-1) to assess cytokine expression in tumors was evaluated.

When the tumor volume is 61-110 mm 3, MC38 tumor-bearing mice are combined with a single dose of PBS or anti-PD-1 alone (5 mg / kg), or anti-PD-1 (5 mg / kg). Treatment with increasing doses of Ab1 (10, 25 or 50 mg / kg, ip). Tumors were collected after 1, 6, 10, 24, 72, and 168 hours of treatment, rapidly frozen in 2 ml plastic tubes containing 2.8 mm ceramic balls (Precyllys KT3961-1007.2) and at -80 ° C. Stored. To prepare lysates, tumors were thawed at room temperature. 1 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 tissues and then 20 s and 6,500 rpm, respectively, at 4 ° C. Homogenized using Precellys® 24 Dual homogenizer (Bertin Instruments) twice. The lysates were clarified by centrifugation at 20,000 × g for 10 minutes in an Eppendorf 5417C centrifuge at 4 ° C. The supernatant was transferred into a clean cold Eppendorf tube and further clarified by centrifugation for an additional 30 minutes as described above. Supernatants were transferred into 96-well storage blocks and placed on ice. Protein concentration in the lysate was measured using the Non-Succinic Acid (BCA) Protein Assay Kit (Thermo 23225) according to the manufacturer's instructions. Lysates were normalized to approximately 5 mg / ml protein concentration using MSD Tris Lysis Buffer (see above) containing protease and phosphatase inhibitors, aliquoted into plastic microtubes, flash frozen in liquid nitrogen and at -80 ° C. Stored.

The concentration of active TGFβ-1 in tumor lysate was measured using the human TGFβ-1 kit (MSD, K151IUC-2) using an electrochemiluminescent assay. Recombinant mouse TGFβ-1 (R & D Systems 7666-MB-005) serially diluted in MSD lysis buffer was used as a correction factor. Normalized tumor lysates prepared as described above were thawed and assayed according to the manufacturer's instructions. In order to quantify only the active form of TGFβ-1 present in the tumor but not the total TGFβ-1 including TGFβ-1 in complex with a latent associated peptide, no acid treatment of the sample was performed. Samples were loaded onto plates two by two. Electrochemiluminescent signals were measured using a MESO SECTOR S 600 plate reader (MSD), and TGFβ-1 concentrations in the samples were quantified using MSD Discovery Workbench software v4.0 based on a standard curve.

Compared to animals treated with PBS or anti-PD-1 alone, animals treated with Ab1 at all dose levels (10, 25, or 50 mg / kg) with anti-PD-1 (5 mg / kg) were tumors. It appears to have a reduced level of active TGF-β1 at, demonstrating the involvement of Ab1 with its target in vivo (FIG. 10A). Reduced levels of active TGF-β1 were observed within 1 hour and lasted for at least 168 hours.

MIP-2 (CXCL2) and KC / GRO (CXCL1) are chemotactic chemokines for granulocytes, including neutrophils. Levels of MIP-2 and KC / GRO were also evaluated in these same samples. After treatment of Ab1 with anti-PD-1, the intratumoral level of MIP-2 was at least in animals treated with Ab1 with anti-PD-1, as compared to animals treated with PBS or anti-PD-1 alone. 4 times increase; And an increase in MIP-2 levels persisted for at least 168 hours (FIG. 10B). Similarly, the level of KC / GRO was shown to increase, albeit at 72 and 168 hours later, compared to MIP-2 (FIG. 10C). Thus, the Ab1 and anti-PD-1 mAb combination induced a decrease in active TGF-β1 levels faster than the increase in MIP-2 and KC / GRO levels. These results demonstrate that Ab1 can reduce and inhibit TGF-β levels in the tumor microenvironment. In addition, the observed increases in MIP-2 and KC / GRO levels suggest that they may be affected by neutralization of TGF-β and thus function as potential biomarkers in patients treated with Ab1.

Example 12 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 recently been reported to form dense clusters as a mechanism to enhance their activity and activation through localization of IL-2 in tightly packed clusters (Kim et al. , Scientific Reports (2017) 7: 40623) . ). Purified human NK cells cultured in vitro in the presence of IL2 appear to form such densely packed clusters.

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

Addition of increasing doses of TGF-β1 has been shown to inhibit NK cell clustering. When Ab1 was added to NK cell culture but not the IgG4 control antibody, NK cell clusters appeared to occur. The results demonstrate that TGF-β neutralization affects NK cell activation, leading to increased activity and proliferation of NK cells, thereby supporting the anti-tumor response of the immune system.

Example 13: Proliferation CD8 by Ab1 Treatment ⁺ Reversal of TGF-β-mediated Inhibition of IFN-γ Production in T Cells

In addition to the innate immune system, TGF-β has been reported to inhibit the activity of CD8 ⁺ T cells (Flavell et al. , Nature Reviews Immunology (2010) 10: 554-567). To explore the role of TGF-β and Ab1 on CD8 ⁺ T cell activity, we established an MLR (Mixed Lymphocyte Response) assay system in which purified human CD3 ⁺ cells were mixed with BLCL cells. CD8 ⁺ cell proliferation and IFN-γ production were first evaluated in the presence of TGF-β. Specifically, CD3 ⁺ cells were isolated using EasySep T cell enrichment kit (StemCell Technologies) from PBMCs fractionated from healthy general donors after Ficoll gradient isolation. CD3 ⁺ cells were then labeled with CellTrace Violet according to the manufacturer's protocol (ThermoFisher). MLR assays were performed by mixing labeled CD3 ⁺ cells (2 × 10 ⁵ cells) in RPMI supplemented with 10% FBS with irradiated BLCL cells (Astarte Bio) (2 × 10 ⁴ cells; 2 minutes). TGF-β1, IgG4 control antibody and / or Ab1 were added to the culture as shown and the culture was incubated at 37 ° C. with 5% CO ₂ 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 distinguished by staining with Zombie NIR bioactive dye (BioLegend) on ice and washing 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 BD Canto and the results were analyzed by cross-culture on live cells, singlets, and CD3 ⁺ cells in FlowJo software. Percentages of IFNγ ⁺ CD8 ⁺ T cells were proliferated based on reduced CellTrace Violet staining and quantified by CD8 ⁺ cell correlation culture positive for IFN-γ staining. FMO was used as a control for all antibody staining.

The introduction of TGF-β in the MLR assay was shown to reduce the percentage of CD8 ⁺ T cells positive for IFN-γ by approximately 4 fold (FIG. 11A). Introduction of Ab1 or control Ab had no effect on the development of these IFNγ ⁺ proliferating CD8 + cells in the absence of TGF-β (FIG. 11B). However, introduction of Ab1 but not the control antibody could restore proliferation of IFNγ ⁺ CD8 ⁺ cells in a dose-dependent manner. These results demonstrate that TGF-β neutralization can affect the adaptive immune system by blocking the immunosuppressive effects of TGF-β on proliferation of effector CD8 ⁺ cells expressing IFN-γ. These IFNγ ⁺ CD8 ⁺ T cells have been shown to play an important role in anti-tumor immunity (Ikeda et al. , Cytokine Growth Factor Rev (2002) 13: 95-109).

Example 14 Response of Syngeneic Mouse Model to Anti-TGF-β Therapy

In this study, we investigated which syngeneic mouse models can be used to predict the response to treatment with anti-TGF-β antibodies Ab1 and anti-PD-1. To stratify the mouse model, we assessed CD8 ⁺ T cell infiltration and TGF-β pathway activation into tumors in mice. CD8 ⁺ T cell infiltration was assayed based on CD8 ⁺ T cell features from data obtained on RNASeq. Seventeen individual mouse syngeneic models, including tumor cells resulting from several indications (shown below the x-axis in FIGS. 12A and 12B), were transcribed profiled using whole-transcriptome RNAseq. The "outline" of the syngeneic model was built on a common background line C57 / BL6 and contained 5 to 7 biological replicates per model. After Illumina 2000 sequencing, a STAR aligner and Cufflinks transcript abundance estimator were used to generate gene expression profiles expressed in per million transcripts (TPM) by standard processing of raw sequence readings. The resulting multi-sample data matrix was final quartile normalized.

12A shows the relative abundance of CD8 ⁺ T cells (log2-transformed) over an outline. Relative CD8 ⁺ T cell abundance was estimated using the unique marker gene CD8B, which appears to be a highly specific indicator of the presence of CD8 T cells (Becht et al. , Curr Opin Immunol (2016) 39: 7-13; and Becht et al. , Genome Biol (2016) 17: 218). Each box graph summarizes the range of values across biological replicates. The MC38 model showed about 2 times more CD8 ⁺ T cell infiltration compared to the EMT6 model (left and right boxes respectively). The A20 and EL4 lymphoma models had negligible CD8 ⁺ T cells in EL4 and displayed overall highest and lowest levels of CD8 ⁺ T cell infiltration, respectively.

The MC38, MC38.ova, CT26, and L1210 murine cell lines showed the highest levels of CD8 gene features. In addition, EMT-6 breast cancer cell lines have been shown to have near-baseline T cell infiltration, consistent with recent reports that EMT6 tumors have an immuno-exclusion phenotype (S. Mariathasan et al. 2017, ESMO Immuno-Oncology Congress, Geneva, Geneva Switzerland).

12B shows TGF-β pathway activation over schematic. Pathway activation for each profile in the outline using 170-gene transcriptional features of TGFβ pathway activation, derived from in vitro stimulation of MCF7 cells by TGF-β and verified by comparison with several other TGF-β features Scores were assigned. Scores were calculated using “Regulatory Gene Set Enrichment Analysis” (rGSEA, Theilhaber et al. 2014) and expressed as log2 enrichment of feature genes against the genetic background. The MC38 model displayed mean activation, but the EMT6 model displayed very high TGF-β pathway activation (left and right boxes respectively).

Example 15 Effect of Ab1 and Anti-PD1 Antibody Combination on a Mouse Breast Cancer Model

In this study, we investigated the therapeutic effect of Ab1 with or without anti-PD-1. Exponentially growing EMT-6 breast cancer cells (CRL-2755, ATCC) were cultured in RPMI-1640 supplemented with 10% FBS FBS in a humidified 5% CO ₂ incubator and then female BALB / c mice (Shanghai Lingchang Bio-Technology Co. Ltd, Shanghai, China) subcutaneously implanted (0.5 × 10 ⁶ cells / mouse). When tumors reached an average size of 68-116 mm 3, mice were pooled and randomized to control and treatment groups (10 mice per group). Tumor-bearing mice were then intraperitoneally treated with PBS, Ab1 (10 and 25 mg / kg) three times a week for each animal for a total of six doses. Tumors were measured twice a week with digital calipers and tumor volumes were calculated (㎣ = L × W × H) and graphed using GraphPad Prism. At the end of the study, mice were euthanized with CO ₂ if the tumor grew above 3000 mm 3, or if the tumor exhibited ulcers greater than 20% of the tumor surface.

As a single formulation, 10 or 25 mg / kg doses of Q3D Ab1 and 5 mg / kg doses of mouse α-PD-1 antibody were 1/10, 2/10 and 2/10 complete in EMT-6 tumor-bearing mice Each regression and partial activity were demonstrated. The combination of 10 or 25 mg / kg dose of Q3D Ab1 and 5 mg / kg of Q3D mouse α-PD-1 antibody was therapeutically active. When comparing tumor volume changes from baseline on day 31 post-transplantation, the effects of all assessment doses of Ab1 and mouse α-PD-1 antibody 5 mg / kg Q3D combination were on 10 and 25 mg / kg of Ab1, respectively. Were greater than the effect of each single formulation with 7/10 and 4/10 complete regression for. Table 10 is a summary of the results.

Effect of Ab1 / anti-mPD-1 Combination on EMT-6 Mouse Model group process Total number of mice Number of complete responses (complete response rate) One. PBS 10 0 (0%) 2. 5 mg / kg x-anti-mPD-1 Mab 10 2 (20%) 3. 10 mg / kg Ab1 10 1 (10%) 4. 25 mg / kg Ab1 10 2 (20%) 5. 10 mg / kg Ab1 +
5 mg / kg x-anti-mPD-1 Mab 10 7 (70%) 6. 25 mg / kg Ab1 +
5 mg / kg x-anti-mPD-1 Mab 10 4 (40%)

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those of ordinary skill in the art. Although exemplary methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or evaluation of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Although several documents are cited herein, such citations do not constitute an admission that any of these documents form a common general knowledge in the art. Also, singular terms shall include the plural and plural terms shall include the singular unless the context otherwise requires. Generally, the nomenclature and techniques used for cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are known in the art. Are widely known and commonly used ones. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as is generally accomplished in the art or as described herein. Throughout this specification and embodiments, the words “have” and “comprise” or variations (“has”, “having”, “comprises” or “comprising”) refer to the mentioned integers or groups of integers. It is to be understood that the term includes, but does not exclude any other integer or group of integers.

The sequences described herein are described below.

Sequence list

SEQ ID NO: 1 (Ab1 heavy chain)

SEQ ID NO: 2 (Ab1 light chain)

SEQ ID NO: 3 (Presolimumab heavy chain, leader sequence including residues 1-19)

SEQ ID NO: 4 (Presolimumab light chain, leader sequence-containing residues 1-19)

SEQ ID NO: 5 (anti-PD-1 Mab heavy chain)

SEQ ID NO: 6 (anti-PD-1 Mab light chain)

SEQ ID NO: 7 (x-anti-mPD-1 Mab heavy chain)

SEQ ID NO: 8 (x-anti-mPD-1 Mab Light Chain)

SEQ ID NO: 9 (1D11 heavy chain)

SEQ ID NO: 10 (1D11 light chain)

                               SEQUENCE LISTING <110> SANOFI   <120> ANTI-TGF-BETA ANTIBODIES AND THEIR USE <130> 022548.WO011 <140> <141> <150> EP 17305061.8 <151> 2017-01-20 <150> 62 / 448,800 <151> 2017-01-20 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 447 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Asn             20 25 30 Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe     50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Thr Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ser Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val         115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro     210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser     290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 2 <211> 215 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 2 Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser             20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Asp Ser Pro                 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala             100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser         115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu     130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu                 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val             180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys         195 200 205 Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 3 <211> 466 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 3 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe         35 40 45 Ser Ser Asn Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu     50 55 60 Glu Trp Met Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala 65 70 75 80 Gln Arg Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser                 85 90 95 Thr Thr Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Ser Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val     210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 225 230 235 240 Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu         275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu     450 455 460 Gly lys 465 <210> 4 <211> 234 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 4 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu             20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu         35 40 45 Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro     50 55 60 Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                 85 90 95 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala             100 105 110 Asp Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 5 <211> 444 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 5 Glu Val Gln Leu Leu Glu Ser Gly Gly Val Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe             20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Gly Ile Ser Gly Gly Gly Arg Asp Thr Tyr Phe Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Gly Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Lys Trp Gly Asn Ile Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu         115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys     130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser                 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser             180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro     210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val                 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe             260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro         275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr     290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala                 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln             340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly         355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro     370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu                 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His             420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 <210> 6 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Ile Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Asn Thr Phe             20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu His Gly Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Thr Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Asn Thr Pro Phe                 85 90 95 Thr Phe Gly Pro Gly Thr Val Val Asp Phe Arg Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 7 <211> 443 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 7 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ser             20 25 30 Tyr Arg Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Trp         35 40 45 Met Gly Tyr Ile Asn Ser Ala Gly Ile Ser Asn Tyr Asn Pro Ser Leu     50 55 60 Lys Arg Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Val Asn Ser Val Thr Thr Glu Asp Ala Ala Thr Tyr Tyr Cys                 85 90 95 Ala Arg Ser Asp Asn Met Gly Thr Thr Pro Phe Thr Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val         115 120 125 Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr     130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser             180 185 190 Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala         195 200 205 Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys     210 215 220 Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val                 245 250 255 Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe             260 265 270 Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro         275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro     290 295 300 Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val 305 310 315 320 Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr                 325 330 335 Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys             340 345 350 Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp         355 360 365 Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro     370 375 380 Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser 385 390 395 400 Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala                 405 410 415 Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His             420 425 430 His Thr Glu Lys Ser Leu Ser His Ser Pro Gly         435 440 <210> 8 <211> 218 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 8 Asp Ile Val Met Thr Gln Gly Thr Leu Pro Asn Pro Val Pro Ser Gly 1 5 10 15 Glu Ser Val Ser Ile Thr Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser             20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Gly Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Gln Gln Gly                 85 90 95 Leu Glu Phe Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg             100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Thr Glu Gln         115 120 125 Leu Ala Thr Gly Gly Ala Ser Val Val Cys Leu Met Asn Asn Phe Tyr     130 135 140 Pro Arg Asp Ile Ser Val Lys Trp Lys Ile Asp Gly Thr Glu Arg Arg 145 150 155 160 Asp Gly Val Leu Asp Ser Val Thr Asp Gln Asp Ser Lys Asp Ser Thr                 165 170 175 Tyr Ser Met Ser Ser Thr Leu Ser Leu Thr Lys Ala Asp Tyr Glu Ser             180 185 190 His Asn Leu Tyr Thr Cys Glu Val Val His Lys Thr Ser Ser Ser Pro         195 200 205 Val Val Lys Ser Phe Asn Arg Asn Glu Cys     210 215 <210> 9 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 9 His Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ile Thr Tyr             20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe     50 55 60 Glu Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser         115 120 125 Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val     130 135 140 Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro             180 185 190 Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro         195 200 205 Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly     210 215 220 Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys                 245 250 255 Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln             260 265 270 Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys         275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu     290 295 300 Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg 305 310 315 320 Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys                 325 330 335 Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro             340 345 350 Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr         355 360 365 Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln     370 375 380 Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly 385 390 395 400 Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu                 405 410 415 Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn             420 425 430 His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys         435 440 445 <210> 10 <211> 218 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note = "Description of Artificial Sequence: Synthetic       polypeptide " <400> 10 Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr             20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro         35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala     50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn                 85 90 95 Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg             100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln         115 120 125 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr     130 135 140 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145 150 155 160 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr                 165 170 175 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg             180 185 190 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro         195 200 205 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys     210 215

Claims (86)

Specifically binds human TGF-β1, TGF-β2, and TGF-β3, comprising heavy chain complementarity-determining regions (CDRs) 1-3 of SEQ ID NO: 1 and light chain CDR1-3 of SEQ ID NO: 2 An isolated monoclonal antibody comprising a human IgG ₄ constant region having proline at position 228 (EU numbering). The heavy chain variable domain (V _H ) amino acid sequence of claim 1 corresponding to residues 1 to 120 of SEQ ID NO: 1 and the light chain variable domain (V _L ) amino acid corresponding to residues 1 to 108 of SEQ ID NO: 2. An antibody comprising a sequence. 3. The antibody of claim 2 comprising the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2. An antigen-binding fragment of the antibody of any one of claims 1 to 3, wherein the fragment is F (ab ') ₂ . The antibody or fragment according to any one of claims 1 to 4, having an increased half-life or increased exposure compared to presolimumab. The antibody or fragment according to any one of claims 1 to 5, having one or more of the following properties:
a) inhibits the differentiation of CD4 ⁺ T cells into inducible regulatory T cells (iTreg);
b) increases CD8 ⁺ T cell proliferation;
c) increased clustering of natural killer (NK) cells;
d) increasing the level of MIP-2; And
e) increase the level of KC / GRO. The antibody or fragment according to any one of claims 1 to 6 as a medicament. A composition comprising the antibody or fragment of any one of claims 1 to 6, wherein the composition comprises less than 1% half an antibody or fragment. A method of inhibiting TGF-β signal transduction in a patient in need thereof, comprising administering to the patient a therapeutic amount of an antibody or fragment of any one of claims 1-6. . The method of claim 9, wherein the patient has cancer. The method of claim 10, wherein the cancer is selected from the group consisting of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma. The method of claim 10 or 11, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. The method of claim 10, wherein the cancer is a mesenchymal tumor. The method of any one of claims 10-13, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. A method of treating cancer in a patient, the method comprising administering to the patient an antibody or fragment of any one of claims 1 to 6, and (2) an inhibitor of an immune checkpoint protein. The method of claim 15, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2. The method of claim 16, wherein the inhibitor of immune checkpoint protein is an anti-PD-1 antibody. The method of claim 17, wherein the anti-PD-1 antibody comprises heavy chain CDR1-3 of SEQ ID NO: 5 and light chain CDR1-3 of SEQ ID NO: 6. 18. The antibody of claim 17, wherein the anti-PD-1 antibody comprises a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO: 5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO: 6 How to. The antibody of claim 17, wherein the anti-PD-1 antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 5 and a light chain amino acid sequence as shown in SEQ ID NO: 6. 18. The method of claim 15, wherein the anti-TGF-β antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2. 21. The method of any one of claims 15-21, wherein the cancer is refractory to anti-PD-1 antibody treatment. 23. The method of any one of claims 15-22, wherein the cancer is advanced or metastatic melanoma or cutaneous squamous cell carcinoma. 24. The method of any one of claims 15-23, wherein the cancer is a solid tumor of the mesenchymal subtype. The method of claim 15, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. The cancer according to any one of claims 15 to 25, wherein the cancer is composed of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma. Selected from the group consisting of: The method of any one of claims 15-26, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. The method of any one of claims 17-27, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient the same day. The method of any one of claims 17-28, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient once every two weeks. The method of any one of claims 17-29, wherein the anti-TGF-β antibody and anti-PD-1 antibody are each administered at a dose of 0.05-20 mg / kg body weight. A method of increasing an immune response in a patient in need of an increase in the immune response, the patient comprising (1) an antibody or fragment of any one of claims 1 to 6, and (2) an inhibitor of an immune checkpoint protein Comprising the steps of: The method of claim 31, wherein the inhibitor of immune checkpoint protein is an anti-PD-1 antibody. The method of claim 32, wherein the anti-PD-1 antibody comprises:
a) HCDR1-3 of SEQ ID NO: 5 and LCDR1-3 of SEQ ID NO: 6;
b) a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO: 5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO: 6; or
c) the heavy chain amino acid sequence shown in SEQ ID NO: 5 and the light chain amino acid sequence shown in SEQ ID NO: 6. 34. The method of any one of claims 31-33, wherein the anti-TGF-β antibody comprises a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2. The antibody or fragment according to any one of claims 1 to 6 for use in inhibiting TGF-β signal transduction in a patient in need of inhibition of TGF-β signal transduction. The antibody or fragment for use according to claim 35, wherein the patient has cancer. 38. The use according to claim 36, wherein the cancer is selected from the group consisting of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma. Antibodies or fragments. 38. The antibody or fragment for use according to claim 36 or 37, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. The antibody or fragment for use according to any one of claims 36 to 38, wherein the cancer is a mesenchymal tumor. 40. The antibody or fragment for use according to any one of claims 36 to 39, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. The antibody or fragment according to any one of claims 1 to 6 for use in treating cancer in a patient in combination with an inhibitor of an immune checkpoint protein. The antibody or fragment for use of claim 41, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2. The antibody or fragment for use according to claim 42, wherein the inhibitor of the immune checkpoint protein is an anti-PD-1 antibody. The antibody or fragment for use according to claim 43, wherein the anti-PD-1 antibody comprises heavy chain CDR1-3 of SEQ ID NO: 5 and light chain CDR1-3 of SEQ ID NO: 6. The anti-PD-1 antibody of claim 43, wherein the anti-PD-1 antibody comprises a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO: 5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO: 6 Antibodies or fragments for use in the art. The antibody or fragment for use according to claim 43, wherein the anti-PD-1 antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 5 and a light chain amino acid sequence as shown in SEQ ID NO: 6. The antibody for use according to any one of claims 41 to 46, wherein the anti-TGF-β antibody comprises a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2. snippet. 48. The antibody or fragment for use according to any one of claims 41 to 47, wherein the cancer is refractory to anti-PD-1 antibody treatment. 49. The antibody or fragment for use according to any one of claims 41 to 48, wherein the cancer is advanced or metastatic melanoma or cutaneous squamous cell carcinoma. The antibody or fragment according to any one of claims 41 to 49, wherein the cancer is a solid tumor of the mesenchymal subtype. 51. The antibody or fragment for use according to any one of claims 41 to 50, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. 52. The method of claims 41-51, wherein the cancer is selected from the group consisting of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma. An antibody or fragment for use in any one of claims. The antibody or fragment for use according to any one of claims 41 to 52, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. The antibody or fragment for use according to any one of claims 43 to 53, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient the same day. The antibody or fragment for use of any one of claims 43-54, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient once every two weeks. The antibody or fragment for use according to any one of claims 43 to 55, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered at a dose of 0.05-20 mg / kg body weight, respectively. The antibody or fragment according to any one of claims 1 to 6 for use in increasing the immune response in a patient in need of an increase in the immune response in combination with an inhibitor of the immune checkpoint protein. The antibody or fragment for use of claim 57, wherein the inhibitor of immune checkpoint protein is an anti-PD-1 antibody. The antibody or fragment for use according to claim 58, wherein the anti-PD-1 antibody comprises:
a) HCDR1-3 of SEQ ID NO: 5 and LCDR1-3 of SEQ ID NO: 6;
b) a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO: 5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO: 6; or
c) the heavy chain amino acid sequence shown in SEQ ID NO: 5 and the light chain amino acid sequence shown in SEQ ID NO: 6. The antibody for use according to any one of claims 57 to 59, wherein the anti-TGF-β antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2. snippet. Use of the antibody or fragment of any one of claims 1 to 6 for the manufacture of a medicament for inhibiting TGF-β signal transduction in a patient in need of inhibition of TGF-β signal transduction. The use of claim 61, wherein the patient has cancer.  63. The use of claim 62, wherein the cancer is selected from the group consisting of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma. 64. The use of claim 62 or 63, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. The use according to any one of claims 62 to 64, wherein the cancer is a mesenchymal tumor. 66. The use of any of claims 62-65, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. Use of the antibody or fragment of any one of claims 1 to 6 for use in the manufacture of a medicament for treating cancer in a patient in combination with an inhibitor of an immune checkpoint protein. The use of claim 67, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2. The use of claim 68, wherein the inhibitor of immune checkpoint protein is an anti-PD-1 antibody. The use of claim 69, wherein the anti-PD-1 antibody comprises heavy chain CDR1-3 of SEQ ID NO: 5 and light chain CDR1-3 of SEQ ID NO: 6. 70. The antibody of claim 69, wherein the anti-PD-1 antibody comprises a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO: 5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO: 6 Use. The use of claim 69, wherein the anti-PD-1 antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 5 and a light chain amino acid sequence as shown in SEQ ID NO: 6. 69. The use of any one of claims 67-72, wherein the anti-TGF-β antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2. 73. The use according to any one of claims 67 to 73, wherein the cancer is refractory to anti-PD-1 antibody treatment. 75. The use of any one of claims 67 to 74, wherein the cancer is advanced or metastatic melanoma, or cutaneous squamous cell carcinoma. 76. The use of any one of claims 67 to 75, wherein the cancer is a solid tumor of the mesenchymal subtype. The use of any one of claims 67-76, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. 78. The cancer according to any one of claims 67 to 77, wherein the cancer consists of melanoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urinary epithelial cancer, and renal cell carcinoma. Use selected from the group which becomes. 79. The use of any one of claims 67-78, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. The use of any one of claims 69-79, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient the same day. 81. The use of any one of claims 69-80, wherein the anti-TGF-β antibody and anti-PD-1 antibody are administered to the patient once every two weeks. The use of any one of claims 69-81, wherein the anti-TGF-β antibody and anti-PD-1 antibody are each administered at a dose of 0.05-20 mg / kg body weight. Use of an antibody or fragment of any one of claims 1 to 6 in combination with an inhibitor of an immune checkpoint protein for use in the manufacture of a medicament for increasing an immune response in a patient in need thereof. . 84. The use of claim 83, wherein the inhibitor of immune checkpoint protein is an anti-PD-1 antibody. 85. The use of claim 84, wherein the anti-PD-1 antibody comprises:
a) HCDR1-3 of SEQ ID NO: 5 and LCDR1-3 of SEQ ID NO: 6;
b) a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO: 5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO: 6; or
c) the heavy chain amino acid sequence shown in SEQ ID NO: 5 and the light chain amino acid sequence shown in SEQ ID NO: 6.  86. The use of any one of claims 83-85, wherein the anti-TGF-β antibody comprises a heavy chain amino acid sequence shown in SEQ ID NO: 1 and a light chain amino acid sequence shown in SEQ ID NO: 2.

Images