Anti-TGF beta antibodies and their use

Abstract

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

Description

[Technical Field]

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

[0002] Sequence List This application includes a sequence listing, which was filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, prepared on January 11, 2018, is named 022548_WO011_SL.txt and has a size of 30,458 It's a part-time job. [Background technology]

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

[0004] TGF-β has three known isoforms—TGF-β1, 2, and 3. All three isoforms are initially transcribed as precursor peptides. After cleavage, the mature C-terminus remains bound to the N-terminus (called the latent associated peptide or LAP), forming a small latent complex (SLC) secreted from the cell. Receptor engagement is hindered because the SLC cannot bind to the TGF-β receptor II (TGFβRII). Activation by dissociation of the N- and C-terminuses occurs through one of several mechanisms, including protein cleavage, acidic pH, or integrin structural changes (Non-Patent Literature 1).

[0005] TGF-β1, 2, and 3 have multifaceted functions and are expressed in different patterns across cell and tissue types. While their in vitro activity is similar, individual knockouts in specific cell types suggest non-identical roles in vivo, despite sharing the ability to bind to the same receptor (Non-Patent Literature 2). When TGF-β binds to TGFβRII, the receptor's constitutive kinase activity phosphorylates and activates TGF-βRII, phosphorylating SMAD2 / 3, enabling binding to SMAD4, nuclear localization, and transcription of TGF-β response genes (ibid.). In addition to this classical signaling cascade, non-classical pathways transmit signals via other factors including p38 MAPK, PI3K, AKT, JUN, JNK, and NF-κB. TGF-β signaling is also regulated by other pathways including WNT, Hedgehog, Notch, INF, TNF, and RAS. Therefore, the ultimate outcome of TGF-β signaling is the crosstalk of all these signaling pathways integrating the cellular state and environment. Same document. [Prior art documents] [Non-patent literature]

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

[0007] Given the diverse functions of TGF-β, there is a need for a pan-TGF-β specific therapeutic antibody that is safe for human patients (Non-Patent Literature 3). However, TGF-β is highly conserved across species. As a result, producing antibodies against human TGF-β in animals such as mice is a challenging task.

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

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

[0010] In one embodiment, the present invention provides an isolated monoclonal antibody comprising heavy chain complementarity determining regions (CDRs) 1-3 in SEQ ID NO: 1 and light chain CDRs 1-3 in SEQ ID NO: 2, which specifically binds to human TGF-β1, TGF-β2, and TGF-β3, wherein the antibody comprises a human IgG4 constant region having a mutation at position 228 (EU numbering). In some embodiments, the mutation is a serine-to-proline mutation (S228P). In some embodiments, the antibody comprises a heavy chain variable domain (V) corresponding to residues 1-120 of SEQ ID NO: 1. H ) Amino acid sequence and light chain variable domain (V) corresponding to residues 1-108 of SEQ ID NO: 2 L ) comprises an amino acid sequence. In further embodiments, the antibody comprises the heavy chain amino acid sequence described in SEQ ID NO: 1 (with or without C-terminal lysine) and the light chain amino acid sequence described in SEQ ID NO: 2. The present invention also features an F(ab')2 antigen-binding fragment of the above antibody.

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

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

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

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

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

[0016] In some regimens, anti-TGF-β antibodies and anti-PD-1 antibodies are administered to the patient every two or three weeks. In some regimens, the two drugs are administered at doses of 0.01–40 mg / kg body weight (e.g., 0.02–20, 0.05–15, or 0.05–20).

[0017] The present invention also provides a method for increasing the immune response in a patient in need, comprising the step of administering an immune checkpoint inhibitor and the antibody or fragment of the present invention to the patient. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody that includes (1) HCDR1-3 in SEQ ID NO: 5 and LCDR1-3 in 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.

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

[0019] In some embodiments, cancers, including mesenchymal tumors, are characterized by the overexpression of one or more of the following biomarkers: ACTA2 (smooth muscle α2 actin), VIM (vimentin), MGP (matrix Gla protein), ZWINT (ZW10 interacting kinetochore protein), and ZEB2 (zinc finger E-box binding homeobox 2). The expression levels of such biomarkers are determined, for example, at the mRNA or protein level in a biological sample from a patient, such as a tumor biopsy or circulating tumor cells.

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

[0021] The present invention also includes a nucleic acid expression vector encoding the heavy chain, light chain, or both of the antibody of the present invention; a host cell containing the heavy chain and light chain coding sequences of the antibody; and a method for producing an antibody using a host cell, comprising the steps of culturing the host cell in a suitable culture medium, expressing the antibody gene, and then recovering the antibody. [Brief explanation of the drawing]

[0022] [Figure 1A] This graph shows the effects of Ab1, fresolimmab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E) at 1 ng / ml. Antibody concentrations are in μg / ml. [Figure 1B] This graph shows the effects of Ab1, fresolimmab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E) at 1 ng / ml. Antibody concentrations are in μg / ml. [Figure 1C] This graph shows the effects of Ab1, fresolimmab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E) at 1 ng / ml. Antibody concentrations are in μg / ml. [Figure 1D]This graph shows the effects of Ab1, fresolimmab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E) at 1 ng / ml. Antibody concentrations are in μg / ml. [Figure 1E] This graph shows the effects of Ab1, fresolimmab, and 1D11 on the proliferation of mink lung (Mv 1Lu) cells treated with human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E) at 1 ng / ml. Antibody concentrations are in μg / ml. [Figure 2] This bar graph shows the effect of Ab1 50 μg / ml on the differentiation of human inducible regulatory T cells (iTreg). The stimulation provided to the T cells was anti-CD3 and anti-CD28 antibodies plus IL-2. [Figure 3] This bar graph shows the effect of Ab1 on the differentiation of human inducible regulatory T cells (iTreg) in human CD4+ T cell cultures treated with human TGF-β1 2 ng / ml. The stimuli provided to the T cells were anti-CD3 and anti-CD28 antibodies plus IL-2. [Figure 4] This bar graph shows the effects of Ab1 (30 μg / ml) and human TGF-β1 (18 ng / ml) on NFATC-driven luciferase expression in Jurkat T cells after T cell stimulation and anti-PD-1 treatment. [Figure 5] This graph shows the median tumor volume value by median absolute deviation (MAD) in the treatment groups using the C57BL / 6 MC38 colon mouse model. Vehicle: PBS. "Anti-PD-1": x-anti-mPD-1 Mab (see the embodiments for carrying out the invention below). "Isotype control of Ab1": anti-HEL hlgG4. [Figure 6] This is a scatter plot showing the change in tumor volume from baseline at day 27 of the treatment used in the C57BL / 6 MC38 colon mouse model. Control: PBS. "Anti-PD-1 RPM114mlgG1": x-anti-mPD-1 Mab. [Figure 7A] This graph shows the time course of tumor volume for each 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 hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7B] This graph shows the time course of tumor volume for each 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 hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7C] This graph shows the time course of tumor volume for each 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 hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7D] This graph shows the time course of tumor volume for each 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 hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7E] This graph shows the time course of tumor volume for each 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 hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 7F] This graph shows the time course of tumor volume for each 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 hlgG4. αPD1: x-anti-mPD-1 Mab. [Figure 8] This graph shows the effect of Ab1 on the active TGF-β1 concentration in LoVo tumor lysates. [Figure 9A] This graph shows the time-course serum concentrations of Ab1 and fresolimmab in five groups of rats given a single dose of one of the antibodies at 5 mg / kg. Groups (Gr.) 1-3 were given three different batches of fresolimmab (B1, B2, and B3). Groups 4 and 5 were given two different batches of Ab1 (B1 and B2). [Figure 9B] This graph shows the time course of serum concentrations of Ab1 and fresolimmab in monkeys given a single dose of either antibody at 1 mg / kg. [Figure 9C] This graph shows the time course of serum concentrations of Ab1 and fresolimmab in monkeys given five weekly doses of Ab1 at 1 mg / kg per dose or for the indicated study period, or every other week at 1 mg / kg per dose. [Figure 9D] This graph shows the time course of serum concentrations of Ab1 and fresolimmab in monkeys given a single dose of either antibody at 10 mg / kg. [Figure 9E] This graph shows the time course of serum concentrations of Ab1 and fresolimmab in monkeys given five weekly doses of Ab1 at 10 mg / kg per dose or every other week for the indicated study period. [Figure 10A] This graph shows the changes in TGF-β1 levels in MC38 tumors after treatment with Ab1 (+ / - anti-PD1). [Figure 10B] This graph shows the change in MIP-2 levels in MC38 tumors after treatment with Ab1 (+ / - anti-PD1). [Figure 10C] This graph shows the change in KC / GRO levels in MC38 tumors after treatment with Ab1 (+ / - anti-PD1). [Figure 11A] This graph quantifies CellTrace Violet staining and IFN-γ staining in CD8pos cells. [Figure 11B] This graph shows that Ab1 restored both proliferation and IFN-γ production in TGFβ-treated CD8+ T cells. [Figure 12A] This graph shows the relative abundance (log2 transformed) of CD8+ T cells across a list of syngeneic mouse tumor models for colorectal cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma, and renal cancer. [Figure 12B] This graph shows the activation of the TGFβ pathway across a list of syngeneic mouse tumor models for colorectal cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma, and renal cancer. [Modes for carrying out the invention]

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

[0024] The antibody Ab1 has an estimated molecular weight of 144 kD when not glycosylated. Its heavy and light chain amino acid sequences are sequence numbers 1 and 2, respectively. These two sequences are shown below. The variable domain is italicized. The CDR is shown in a square. The glycosylation site within the constant domain of the heavy chain is in bold (N297).

[0025] [ka]

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

[0027] Ab1 and related antibodies specifically bind to human TGF-β1, -β2, and -β3. By "specifically", the inventors mean that the binding is, for example, determined by surface plasmon resonance (see, e.g., Example 1 below) or Bio-Layer interferometry, 10 -8 M (e.g., 1 - 5 nM) or less, such as less than 10 -7 M, having a K D . Ab1 and related antibodies may also have strong TGF-β neutralizing ability when assayed in a mink lung epithelial cell assay (see, e.g., Example 2 below) or an EC50 of about 0.05 to 1 μg / ml determined in an A549 cell IL-11 induction assay (see, e.g., Example 6 of PCT International Publication No. WO 2006 / 086469, the disclosure of which is incorporated herein by reference in its entirety).

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

[0029] During manufacturing, fresolimmab can form up to 6-18% half-antibodies under non-reducing denaturation conditions (i.e., a dimer with one heavy chain and one light chain, rather than a tetramer with two heavy chains complexed with two light chains). In contrast, Ab1 produces substantially fewer half-antibodies (<1%). Therefore, Ab1 and the associated antibody result in a purer drug product during manufacturing.

[0030] Furthermore, Ab1 and related antibodies can have improved pharmacokinetic (PK) profiles compared to fresolimmab. They can exhibit linear PK behavior with much longer half-lives and lower efflux rates than fresolimmab, resulting in approximately 1.7 times higher in vivo exposure than fresolimmab. For example, in rats, Ab1 has been shown to have an average half-life of 7.1 days compared to 4.3 days for fresolimmab, and an efflux rate (CL) of 0.30 ml / hr / kg compared to 0.51 ml / hr / kg for fresolimmab (Example 7, hereafter). In cynomolgus monkeys, Ab1 exhibits improved efflux compared to fresolimmab. It has been shown to have an average half-life of 13 days compared to 4.5 days, and an efflux rate (CL) of 0.40 ml / hr / kg compared to 0.66 ml / hr / kg for fresolimmab. (Same literature.) These improved PK properties suggest that Ab1 and related antibodies can be administered to patients at lower doses and / or less frequently than fresolimmab, achieving the same or better clinical efficacy, causing fewer adverse side effects and fewer anti-drug antibody reactions, and thus allowing for longer treatment periods if necessary.

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

[0032] While not bound by theory, the inventors hypothesize that a 228-residue mutation in the heavy chain of Ab1 and related antibodies results in increased stability as well as improved PK and toxicity profiles.

[0033] The constant domains of Ab1 and related antibodies may be further modified as needed, for example at the Kabat residue L248 (e.g., by introducing the L248E mutation), to reduce any undesirable effector function of the molecule.

[0034] As used herein, the terms “antibody” (Ab) or “immunoglobulin” (Ig) refer to a tetrameric protein comprising two heavy (H) chains (approximately 50–70 kDa) and two light (L) chains (approximately 25 kDa) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (V H ) and heavy chain constant region (C H ) includes. Each light chain contains a light chain variable domain (V L ) and light chain constant region (C L ) consists of V H and V L The domain is further subdivided into highly variable regions called "complementarity-determining regions" (CDRs), which are scattered with more conserved regions called "framework regions" (FRs). H or V L It consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each region is as defined by IMGT® (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or as defined by 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 follow Chothia et al., Nature 342:878-883 (1989).

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

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

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

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

[0039] I. Use of Ab1 and related antibodies TGF-β receptors are widely expressed on immune cells, leading to the broad effects of TGF-β in both the innate and adaptive immune systems. TGF-β is associated with many disease conditions, such as birth defects, cancer, chronic inflammation, autoimmune diseases, and fibrotic disorders. Therapeutic doses of Ab1 or related antibodies are used to treat these conditions. The “therapeutic” dose refers to the amount of Ab1, related antibody, or other therapeutic agent mentioned herein that alleviates one or more symptoms of the treated condition. This dose varies based on the treated condition or patient and is determined by a healthcare professional using well-established principles.

[0040] In some embodiments, Ab1 or related antibodies are administered at doses of 40, 20, or 15 mg / kg or less (such as 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / kg). In some further embodiments, doses 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. Dosage frequency may be, for example, daily, every two days, every three days, every four days, or every five days, weekly, every two weeks, or every three weeks, monthly, or every two months. Antibodies are administered intravenously (e.g., intravenous infusion over 0.5 to 8 hours), subcutaneously, topically, or by any other route of administration, as appropriate for the condition and formulation.

[0041] Ab1 and related antibodies are derived from human antibody genes and therefore have low immunogenicity in humans. Toxicity studies of Ab1 are detailed in Example 8 below. In rats, specific cardiac and pulmonary side effects were observed. Therefore, when patients are treated with Ab1 or related antibodies, patients should be monitored for adverse events.

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

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

[0044] They are also useful in treating, preventing, and reducing the risk of developing renal failure, including, but not limited to, focal segmental glomerulosclerosis (FSGS), diabetic (type I and type II) nephropathy, radiation nephropathy, obstructive nephropathy, diffuse systemic sclerosis, congenital kidney diseases (e.g., polycystic kidney disease, cavernous kidney, horseshoe kidney), glomerulonephritis, nephrosclerosis, nephrocalcinosis, systemic or glomerular hypertension, tubulointerstitial nephropathy, tubular acidosis, renal tuberculosis, and renal infarction. They are particularly useful when used in combination with renin-angiotensin-aldosterone antagonists, including, but not limited to, renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, AngII receptor antagonists (also known as "AngII receptor blockers"), and aldosterone antagonists. For example, see International Publication No. 2004 / 098637, the disclosure of which is incorporated herein by reference in its entirety.

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

[0046] Ab1 and related antibodies are even more useful in conditions where promoting re-epithelialization is beneficial. Such conditions include, but are not limited to, venous ulcers, ischemic ulcers (pressure ulcers), diabetic ulcers, skin conditions such as transplant sites, transplant donor sites, abrasions and burns, bronchial epithelial diseases such as asthma and ARDS, mucositis associated with cytotoxic treatment, esophageal ulcers (reflex disease), gastroesophageal reflux disease, gastric ulcers, and intestinal epithelial diseases such as small and large intestinal lesions (inflammatory bowel disease).

[0047] Further use of Ab1 and related antibodies is desirable in conditions where endothelial cell proliferation is desirable, such as stabilization of atherosclerosis, promotion of vascular anastomosis healing, or inhibition of smooth muscle cell proliferation in arterial diseases, restenosis, and asthma.

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

[0049] Ab1 and related antibodies are also useful for the prevention and / or treatment of ophthalmic conditions such as glaucoma and scarring after fibrous zoectomy.

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

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

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

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

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

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

[0056] Ab1 and related antibodies are also useful in inhibiting cyclosporine-mediated malignancies or cancer progression (e.g., metastasis).

[0057] In the context of cancer treatment, it is naturally understood that “treatment” includes any medical actions that result in partial remission of cancer, which slows cancer growth, delays cancer progression or recurrence, or reduces cancer metastasis, as well as extending the patient’s life expectancy.

[0058] C. Combination Therapies in Oncology The level of cytotoxic T cell infiltration in cancer has been observed to be associated with favorable clinical outcomes (Fridman et al., Nat Rev Cancer (2012) 12(4):298-306; and Galon et al., Immunity (2013) 39(1):11-26). Furthermore, cytotoxic T cells (CD4 + T H1 Helper T cells that support T cells and the cytokines they produce (e.g., 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, above).

[0059] TGF-β suppresses almost all aspects of the antitumor immune response. Cytokines promote iTreg differentiation and cytotoxicity (CD8 + ) Reduces cell proliferation and invasion. Inhibition of TGF-β by Ab1 or related antibodies alleviates the immunosuppressive tumor microenvironment as described above, leading to positive outcomes for cancer patients.

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

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

[0062] Cancers treated with a combination of Ab1 or related antibodies and immune checkpoint inhibitors such as anti-PD-1 antibodies include the cancers listed in the subsection above.

[0063] In some embodiments, cancers such as advanced or metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and Hodgkin lymphoma are refractory to the aforementioned anti-PD-1, anti-PD-L1, or anti-PD-L2 therapies. Refractory patients are those whose disease is progressing, for example, radiologically, within 12 weeks of the initiation of treatment, with no evidence of response.

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

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

[0066] For example, CDRs and Vs disclosed in the PCT release. H and V L The sequence, or heavy and light chain sequences, and antibodies that bind to the same PD-1 epitope as the antibodies disclosed in the PCT publication. Antibodies having antigen-binding fragments and antibodies and related antibodies disclosed in International Publication No. 2015 / 112800 containing antigen-binding fragments are used in conjunction with Ab1 or related antibodies of the present invention to treat cancer. In relevant embodiments, useful anti-PD-1 antibodies are the heavy and light chain amino acid sequences shown below as SEQ ID NOs. 5 and 6, respectively; V of SEQ ID NOs. 5 and 6 H and V LArrays (indicated in italics); or one or more (e.g., all six) of sequence numbers 5 and 6 may contain CDRs (indicated in squares).

[0067] [ka]

[0068] In some embodiments, antibodies of the present invention, such as anti-PD-1 antibodies, do not have C-terminal lysine in their heavy chain. The C-terminal lysine is removed during manufacturing or by recombinant technology (i.e., the coding sequence of the heavy chain does not contain the C-terminal lysine codon). Therefore, antibodies containing the heavy chain amino acid sequence of SEQ ID NO: 5 without C-terminal lysine are also considered within the present invention.

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

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

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

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

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

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

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

[0076] Furthermore, antibody expression can be enhanced using many known techniques. For example, the glutamine synthase gene expression system (GS system) is a common method for enhancing expression under specific conditions.

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

[0078] Mai. Pharmaceutical composition The antibodies of the present invention are formulated for suitable storage stability. For example, the antibodies are lyophilized, stored, or restored for use with pharmaceutically acceptable excipients. For combination therapy, two or more antibodies or other therapeutic agents are formulated simultaneously and provided, for example, in mixed and single compositions.

[0079] The terms “excipient” or “carrier” are used herein to describe any component other than the compounds of the present invention. The choice of excipient largely depends on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. “Pharmacologically acceptable excipients” include any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic and absorption retarders, etc. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffer, dextrose, glycerin, ethanol, etc., and combinations thereof. In some cases, isotonic agents, such as sugars, mannitol, sorbitol, or polyalcohols such as sodium chloride, are included in the composition. Further examples of pharmaceutically acceptable substances are auxiliaries, preservatives or buffers such as humectants or small amounts of humectants or emulsifiers that enhance the shelf life or efficacy of antibodies.

[0080] The pharmaceutical compositions of the present invention are prepared, packaged, or sold in bulk as single unit doses or as multiple single unit doses. As used herein, “unit dose” refers to an individual amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is typically equal to the dose of the active ingredient administered to a subject or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0081] The pharmaceutical compositions of the present invention are typically preferred for parenteral administration. As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical bleaching of the target tissue and administration of the pharmaceutical composition via tissue bleaching, and thus usually resulting in direct administration into the bloodstream, muscle, or viscera. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, application of the composition by surgical incision, application of the composition by tissue penetration nonsurgical wounds, etc. In particular, parenteral administration is considered to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, subarachnoid, intraventricular, intraurethral, ​​intracranial, intratumoral, and intraarticular synovial bursa injection or infusion; as well as renal dialysis infusion techniques. Local perfusion is also possible. Preferred embodiments include intravenous and subcutaneous routes.

[0082] Formulations of pharmaceutical compositions suitable for parenteral administration typically contain an active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations are prepared, packaged, or sold in forms suitable for bolus or serial administration. Injectable formulations are prepared, packaged, or sold in unit-dosage forms such as ampoules or multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, oily or aqueous vehicles, pastes, etc. Such formulations may further contain, but are not limited to, one or more additional components including suspensions, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (e.g., pH 3 to 9), but in some applications they are more preferably formulated as sterile non-aqueous solutions or in dry form, used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include liquids or suspensions in sterile aqueous solutions, e.g., aqueous solutions of propylene glycol or dextrose. Such dosage forms are preferably buffered if desired. Other useful orally administered formulations include those containing the active ingredient in microcrystalline form or liposomal formulations. Parenteral formulations are formulated to be immediate and / or regulated release. Regulated release formulations include delayed-, maintained-, pulse-, controlled-, targeted-, and programmed-release.

[0083] IV. Exemplary Embodiments Further specific embodiments of the present invention are described below: 1. An isolated monoclonal antibody that specifically binds to human TGF-β1, TGF-β2, and TGF-β3, comprising heavy chain complementarity determining regions (CDRs) 1-3 in SEQ ID NO: 1 and light chain CDRs 1-3 in SEQ ID NO: 2, wherein the antibody comprises a human IgG4 constant region having proline at position 228 (EU numbering). 2. Heavy chain variable domain (V) corresponding to residues 1-120 of SEQ ID NO: 1 H ) Amino acid sequence and light chain variable domain (V) corresponding to residues 1-108 of SEQ ID NO: 2 L The antibody according to Embodiment 1, comprising the amino acid sequence. 3. The antibody according to Embodiment 2, comprising the heavy chain amino acid sequence described in SEQ ID NO: 1 (with or without lysine at the C-terminus) and the light chain amino acid sequence described in SEQ ID NO: 2. 4. An antigen-binding fragment of the antibody described in Embodiment 3, which is F(ab')2. 5. An antibody or fragment according to any one of Embodiments 1 to 4, which exhibits an extended half-life or increased exposure compared to fresolimmab. 6. The following: a) CD4 + Inhibits the differentiation of T cells into inducible regulatory T cells (iTreg). b) CD8 + Increases T cell proliferation; c) Increase clustering of natural killer (NK) cells; d) Increase the level of MIP-2; and e) Increase the level of KC / GRO An antibody or fragment according to any one of Embodiments 1 to 5, having one or more of the characteristics. A composition comprising an antibody or fragment according to any one of Embodiments 1 to 6, containing less than 7.1% half-antibody. 8. An antibody or fragment described in any one of Embodiments 1 to 6, used as a drug. 9. A method for inhibiting TGF-β signaling in a patient in need thereof, comprising the step of administering to the patient an antibody or fragment according to any one of embodiments 1 to 6 in a therapeutic dose. 10. The method according to Embodiment 9, wherein the patient has cancer. 11. Cancers include melanoma, lung cancer, squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, The method according to Embodiment 10, selected from the group consisting of head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma. 12. The method according to Embodiment 10 or 11, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. 13. The method according to any one of embodiments 10 to 12, wherein the cancer is a mesenchymal tumor. 14. The method according to any one of embodiments 10 to 13, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. 15. A method for treating a patient's cancer, (1) The antibody or fragment described in any one of Embodiments 1 to 6, and (2) Inhibitors of immune checkpoint proteins The method comprising the step of administering to a patient. 16. The method according to Embodiment 15, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2. 17. The method according to Embodiment 16, wherein the inhibitor of the immune checkpoint protein is an anti-PD-1 antibody. 18. The method according to Embodiment 17, wherein the anti-PD-1 antibody comprises the heavy chain CDR1-3 in SEQ ID NO: 5 and the light chain CDR1-3 in SEQ ID NO: 6. 19. The anti-PD-1 antibody corresponds to residues 1-117 of SEQ ID NO: V H The amino acid sequence and V corresponding to residues 1-107 of SEQ ID NO: 6 L The method according to Embodiment 17, comprising an amino acid sequence. 20. The method according to Embodiment 17, wherein the anti-PD-1 antibody comprises the heavy chain amino acid sequence (with or without C-terminal lysine) described in SEQ ID NO: 5 and the light chain amino acid sequence described in SEQ ID NO: 6. 21. The method according to any one of Embodiments 15 to 20, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence described in SEQ ID NO: 1 (with or without C-terminal lysine) and the light chain amino acid sequence described in SEQ ID NO: 2. 22. The method according to any one of embodiments 15 to 21, wherein the cancer is refractory to anti-PD-1 antibody treatment. 23. The method according to any one of Embodiments 15 to 22, wherein the cancer is advanced or metastatic melanoma or squamous cell carcinoma. 24. The method according to any one of embodiments 15 to 23, wherein cancer is a mesenchymal subtype of a solid tumor. 25. The method according to any one of embodiments 15 to 24, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. 26. The method according to any one of Embodiments 15 to 25, wherein the cancer is selected from the group consisting of melanoma, lung cancer, squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma. 27. The method according to any one of embodiments 15 to 26, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. 28. The method according to any one of Embodiments 15 to 27, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient on the same day. 29. The method according to any one of Embodiments 15 to 28, wherein the patient is administered an anti-TGF-β antibody and an anti-PD-1 antibody every other week. 30. The method according to any one of Embodiments 15 to 29, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered at a dose of 0.05 to 20 mg / kg body weight, respectively. 31. A method for increasing an immune response in a patient in need thereof, comprising the step of administering to the patient an immune checkpoint inhibitor and an antibody or fragment according to any one of Embodiments 1 to 6. 32. The method according to Embodiment 31, wherein the checkpoint inhibitor is an anti-PD-1 antibody. 33. Anti-PD-1 antibodies are: a) HCDR1-3 of SEQ ID NO: 5 and LCDR1-3 of SEQ ID NO: 6; b) V corresponding to residues 1-117 of SEQ ID NO: 5 and residues 1-107 of SEQ ID NO: 6, respectively. H and V L ;or c) Heavy chain having the amino acid sequence described in SEQ ID NO: 5 (with or without C-terminal lysine) and light chain having the amino acid sequence described in SEQ ID NO: 6 The method according to embodiment 32, including the method described in embodiment 32. 34. The method according to any one of Embodiments 31 to 33, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence described in SEQ ID NO: 1 (with or without C-terminal lysine) and the light chain amino acid sequence described in SEQ ID NO: 2. 35. The method according to any one of embodiments 31 to 34, wherein the patient has cancer. 36. The method according to Embodiment 35, wherein the patient is refractory to prior treatment with immune checkpoint inhibitors and / or has a mesenchymal subtype of a solid tumor. 37. The method according to Embodiment 35 or 36, wherein the cancer is selected from the group consisting of melanoma, lung cancer, squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma. 38. The method according to any one of embodiments 35 to 37, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP, and ZWINT. 39. The method according to any one of embodiments 35 to 38, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment. 40. An antibody or fragment according to any one of Embodiments 1 to 6 for use in treating a patient by any of the methods described above. 41. Use of an antibody or fragment according to any one of Embodiments 1 to 6 for the manufacture of a drug for treating a patient by any of the methods described above. 42. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain, light chain, or both of an antibody or fragment from any one of Embodiments 1 to 6. 43. An expression vector containing an isolated nucleic acid molecule of Embodiment 42. 44. Host cells containing the expression vector of Embodiment 43. 45. A step of providing a host cell containing first and second nucleotide sequences encoding the heavy chain and light chain of an antibody or antigen-binding fragment, respectively. A step of growing host cells under conditions that enable the production of antibodies or antigen-binding fragments, A step to recover the antibody or antigen-binding fragment and A method for producing an antibody or antigen-binding fragment according to any one of Embodiments 1 to 6, including the above. 46. A method for producing a pharmaceutical composition, A step of providing an antibody or antigen-binding fragment according to any one of Embodiments 1 to 6, The process involves mixing an antibody or antigen-binding fragment with a pharmaceutically acceptable excipient. The method comprising the above. 47. A product or kit comprising an antibody or antigen-binding fragment and another therapeutic agent as described in any one of Embodiments 1 to 6. 48. The product or kit according to Embodiment 47, wherein the other therapeutic agent is an immune checkpoint inhibitor as described herein.

[0084] The present invention will be further described in the following examples, but without limiting the scope of the invention as described in the claims.

[0085] [Examples] To better understand this invention, the following embodiments are provided. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Examples]

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

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

[0088] [Table 1]

[0089] Since human TGF-β3 and mouse TGF-β3 have identical amino acid sequences, no different affinity measurements were calculated for these two proteins. Similarly, mouse and rat TGF-β1 and β2 have identical amino acid sequences, and no different affinity measurements were calculated for them.

[0090] The k of Ab1 determined by the method described above.a , k d , and K D The values ​​are shown in Table 2 below. K of Ab1 to human TGF-β1, β2, and β3 D The values ​​were determined to be 1.48, 3.00, and 1.65 nM, respectively. K of Ab1 to mouse / rat TGF-β1 and β2 D The values ​​were determined to be 2.80 and 1.88 nM, respectively. These binding properties were similar to those of fresolimmab.

[0091] [Table 2]

[0092] The data above demonstrate that Ab1 is a potent and selective pan-TGF-β inhibitor. Measurements using surface plasmon resonance demonstrated that Ab1 has affinities between 1 and 5 nM for all human and mouse TGF-β isoforms. High levels of specificity were confirmed by GLP immunohistochemistry (IHC) tissue cross-reactivity testing using normal rat, cynomolgus monkey, and human tissues. [Examples]

[0093] Ab1's TGF-β neutralizing ability The in vitro ability of Ab1 to neutralize TGF-β activity was measured using a cell-based assay. This assay measured the ability of TGF-β to inhibit the proliferation of untransformed mink lung epithelial cells (Mv 1Lu cells). See, for example, International Publication No. 2006 / 086469 and Mazzieri, et al., Eds, “Methods in Molecular Biology,” Vol. 142, “Transforming Growth Factor-β Protocols.” The ability of Ab1, fresolimmab, and 1D11 (mouse anti-TGF-β antibody, its heavy and light chain sequences disclosed herein as SEQ ID NOs. 9 and 10) to neutralize human TGF-β1, 2, 3 and mouse TGF-β1 and 2 was evaluated. Recombinant TGF-β proteins were either manufactured internally (human TGF-β1, 2, and 3) or obtained from R&D Systems, Inc. (mouse TGF-β1 and 2).

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

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

[0096] To investigate the antagonist activity of Ab1 to endogenously produced TGF-β in cultured cells, we used a total CD4 sample without the addition of exogenous TGF-β. + T cells were treated for 6 days with 50 μg / ml isotype control (human IgG4, kappa anti-egg lysozyme (HEL) antibody, Crown Biosience), Ab1, or fresolimmab, with or without stimulation (anti-CD-3, anti-CD-28, and IL-2), and then analyzed by flow cytometry. CD25 + FOXP3 + The mean percentage and standard deviation of the population are taken from the parent population (lymphocytes / liver / single cell / CD4 + CD127 - ) was calculated three times. Anti-CD3, anti-CD28, and whole human CD4 by IL-2 + T cell stimulation is performed in cultured FOXP3 + CD25 + The percentage of iTregs was increased from 0% to 15%. Treatment with Ab1 50 μg / ml or fresolimmab 50 μg / ml reduced the percentage of iTregs to a similar extent (8% and 7%, respectively; Figure 2). In contrast, treatment with human IgG4 (hIgG4) isotype control had the least effect on iTreg differentiation (20% iTregs) (Figure 2). CD4 isolated from a second healthy volunteer + T cells produced similar results.

[0097] To investigate the antagonist activity of Ab1 against exogenous TGF-β, all CD4 cells were incubated with human TGF-β1 at 2 ng / ml. + T cells were treated for 6 days with various antibody concentrations of isotype control, Ab1, or fresolimmab, either in the presence or absence of stimulation (anti-CD3, anti-CD28, and IL-2), and then analyzed by flow cytometry. CD25 + FOXP3 +The mean percentage and standard deviation of the population are taken from the parent population (lymphocytes / liver / single cell / CD4 + CD127 - ) was calculated three times unless otherwise noted. Whole human CD4 stimulated with exogenous TGF-β1 (2 ng / ml) + Addition of Ab1 to T cells increased the percentage of iTregs in culture from 15% to 55%. Treatment with increasing concentrations of Ab1 followed suit, decreasing the percentage of iTregs from 55% to 15% at 200 μg / ml and by 43% at 6.25 μg / ml. Treatment with fresolimmab reduced the percentage of iTregs to a similar extent as Ab1 (from 55% to 16% at 200 μg / ml and 32% at 6.25 μg / ml). Treatment with various concentrations of isotype control antibodies did not have an effect on the 60% iTreg percentage at 200 μg / ml and 6.25 μg / ml. See Figure 3. CD4 isolated from the second healthy volunteer. + T cells produced similar results.

[0098] This study demonstrated that Ab1 may offer clinical benefits by inhibiting TGF-β-induced iTreg differentiation and thus alleviating the immunosuppressive tumor microenvironment. [Examples]

[0099] Efficacy of Ab1 and anti-PD-1 antibody combination in vitro In this study, the inventors investigated whether TGF-β prevents maximal T cell stimulation in vitro after anti-PD-1 treatment, and if so, whether Ab1 can counteract this prevention. The level of T cell activation was measured using luciferase expression from expression constructs under transcriptional regulation of NFATc (activated T cell nuclear factor, cytoplasmic 1) regulatory sequences.

[0100] The inventors used a cell assay system purchased from Promega for this study. This system includes two cell types: 1) Jurkat T cells expressing a luciferase reporter driven by human PD-1 and an NFAT response element, and 2) CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate the allogeneic T cell receptor in an antigen-independent manner. When co-cultured, Jurkat T cells interacted with CHO-K1 cells, resulting in the translocation of NFATc to the nucleus, which drives T cell receptor stimulation and luciferase expression. However, PD-1 / PD-L1 engagement recruited non-receptor type 11 protein tyrosine phosphatase (SHP2) to the T cell receptor complex, inhibiting NFATC nuclear translocation and subsequent luciferase expression. Blocking PD-1 signaling mitigated SHP2-dependent repression and thus enabled maximal luciferase expression. Therefore, the system provided a functional method for determining the effects of TGF-β on T cell signaling and the effects of Ab1 on anti-PD-1 treatment of T cells.

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

[0102] The results showed that the addition of anti-hPD-1 antibody to Jurkat T cells co-cultured with CHO-K1 cells for 24 hours induced greater luciferase activity (865794 relative luminescence units [RLU]) than when an isotype control was added (234963 RLU, multiplicative change = 3.685, p < 0.0001) or when no antibody was added (206043 RLU, multiplicative change = 4.202, p < 0.0001). Pretreatment of Jurkat T cells with TGF-β1 18 ng / ml for 12 days induced lower luciferase activity (638866 RLU) in CHO-K1 cell co-culture in the presence of anti-hPD-1 antibody compared to Jurkat T cells not treated with TGF-β1 (865794 RLU, multiplicity change = -1.355, p < 0.0001) (Figure 4).

[0103] To evaluate the antagonist capacity of Ab1, Jurkat T cells were pre-treated with TGF-β1 18 ng / ml for 12 days in the presence of Ab1, isotype control Ab, or Ab, and then co-cultured with CHO-K1 cells in the presence of anti-PD-1 for 24 hours. The presence of Ab1 (924186 RLU) reduced the TGF-β-dependent suppression of luciferase activity compared to isotype control Ab (639440 RLU, multiplicity change = 1.445, p < 0.0001) and Ab control-free (638866 RLU, multiplicity change = 1.447, p < 0.0001). Jurkat T cells co-cultured with CHO-K1 cells in the presence of anti-PD-1 Ab without TGF-β1 pretreatment, and then supplemented with Ab1 (975654 RLU) or isotype control (955717 RLU), showed a statistically increased luciferase activity compared to the Ab-free control, but with the smallest multiplier change (865794 RLU, multiplier changes = 1.127 and 1.104, respectively, p-values ​​= 0.0023 and 0.001284) (Figure 4). The RLU values ​​are also shown in Table 3 below.

[0104] [Table 3]

[0105] To rule out the possibility that TGF-β1 pretreatment of Jurkat T cells would lead to decreased proliferation or viability, and therefore decreased luciferase activity during 24-hour co-culture with CHO-K1 cells, we incubated Jurkat T cells with TGF-β1 18 ng / ml or PBS for 7 days in the presence of Ab1, anti-HEL hlgG4, or without antibody (vehicle). Every 2-3 days, equal volumes of each group were seeded into new flasks, at which point both TGF-β1 and antibody were refreshed (a total of two re-seeding events occurred). Assessment of the final culture demonstrated that viability was altered in all treatment groups (ranging from 94% to 96%). Furthermore, the total number of Jurkat T cells in the final culture of each treatment group was very similar (ranging from 25 million to 29 million).

[0106] The above study demonstrated that the increase in downstream signaling of T cell receptors after anti-PD-1 treatment is suppressed by TGF-β, resulting in suboptimal T cell stimulation. Our data suggest that inhibition of TGF-β alleviates the immunosuppressive tumor microenvironment, allowing checkpoint modulators such as anti-PD-1 agents to induce a better immune response and thus increase the proportion of patients who benefit from immuno-oncological treatment. [Examples]

[0107] Efficacy of Ab1 and anti-PD1 antibody combination in vivo The inventors then studied the effects of combined anti-TGF-β and anti-PD-1 treatment in a C57BL / 6 mouse cancer model.

[0108] Tolerability / Preliminary safety material and method The tolerability of Ab1 and anti-mouse PD-1 (mPD-1) monoclonal antibodies (mAbs) as monotherapy and in combination was evaluated in female C57BL / 6 mice. Ab1 (10, 20, and 50 mg / kg) or isotype control Ab (anti-HEL hlgG4, purchased from Crown Bioscience; used at 10 and 20 mg / kg) was administered intravenously every 3 days (Q3D) or intravenously twice weekly in combination with anti-PD1 Mab 5 mg / kg. The anti-PD-1 Ab used in this study is referred to as "anti-mPD1_hyb_RMP114_mIgG1LCfullrat" (or x-anti-mPD-1 Mab). It is rat IgG 2a The chimeric rat anti-mPD-1 antibody was generated by replacing the rat Fc region of clone RMP1-14 (BioXcell, Cat.#BE0146) with the mouse IgG1Fc region. The heavy and light chain amino acid sequences of this chimeric antibody are shown in SEQ ID NOs. 7 and 8. Tolerability was assessed by measuring animal body weight and clinical observation. At the end of the 3-week treatment, 4 hours after the last treatment, final sample collection was performed, and tissues (heart, kidney, liver, lung, and spleen) were fixed with formaldehyde and sent for histopathological analysis.

[0109] The dosage was considered highly toxic if individual mice experienced a 15% weight loss over three consecutive days, a 20% weight loss in one day, or 10% or more drug-related deaths, unless tumor-induced cachexia resulting in weight loss was observed in the control vehicle treatment group. Animal body weight included tumor weight.

[0110] Toxicity / safety results Tolerance studies in C57BL / 6 mice showed that all tested dose levels of Ab1 and x-anti-mPD-1 Mab, both as single agents and in combination, were well tolerable. No significant changes in body weight were observed at any dose tested in any treatment group. No serious or significant clinical observations were observed during the study. Histopathological analysis identified an increase in lymphocyte count in the spleen (white marrow) in all treatment groups, including the isotype control antibody treatment group, regardless of dose relationship, in any combination. No other significant microscopic findings were observed. Two mice in the combination group of isotype control Ab (10 mg / kg) and anti-PD-1 Mab (5 mg / kg) were confirmed dead after the final dose on the last day of the study. Histopathological analysis did not identify any drug associated with the cause of death.

[0111] Efficacy test The efficacy of combined anti-TGF-β and anti-PD-1 treatment in C57BL / 6 mice carrying subcutaneous MC38 syngeneic colorectal tumors was evaluated. Mice were administered either Ab1 25 mg / kg, x-anti-mPD-1 Mab 5 mg / kg, or both for 3 weeks in Q3D. This study demonstrated that the combination of Ab1 and anti-mPD-1 Mab exhibited significantly greater antitumor activity than the single agents alone. Materials, methods, and dates of this study are described in detail below.

[0112] material and method animal Female C57BL / 6 mice were obtained from Charles River Labs (Wilmington, MA, USA). The animals were allowed to acclimate for at least 3 days before being introduced to the study. The mice were 11 weeks old at the start of the study and weighed between 17.0 and 20.9 g. They were given free access to food (Harlan2916 rodent diet, Massachusetts, USA) and sterile water and were housed in a 12-hour light / dark cycle.

[0113] tumor cells MC38 is a colorectal adenocarcinoma cell lineage. Cells were obtained from the National Cancer Institute (Bethesda, MD, USA) and cultured in complete medium (CM) containing L-glutamine-containing Roswell Park Memorial Institute medium (RPMI)-1640 (Gibco, Cat#11875) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) (Gibco, Cat#10438026) at 37°C under 5% CO2. Cells were harvested, resuspended in Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Cat#14190), and cultured at a rate of 1 × 10⁶ cells per mouse. 6 200 μl of cells were subcutaneously transplanted (SC) into the right flank of female C57BL / 6 mice.

[0114] compound Ab1 was administered to animals in aqueous solution. It was filtered through a 0.22 μm filter using PES and stored in sterile water at 2-10°C. The antibody was administered to animals intraperitoneally (IP) at a dose of 10 ml / kg and 25 mg / kg.

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

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

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

[0118] Mice were euthanized if a disease rate of ≥20% or weight loss was observed. Tumors were measured with calipers twice a week until the final sacrifice. Tumor size was approximately 2000 mm 3 When reached, or if there were health problems in the animals (20% of the tumor area was ulcerated), the animals were euthanized and the date of death was recorded. Solid tumor volume was estimated from two-dimensional tumor measurements using the following formula: Tumor volume (mm 3 ) = [length (mm) × width 2 (mm 2 )] / 2 was calculated according to.

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

[0120] Efficacy The primary efficacy endpoints were changes in tumor volume from baseline, as indicated by ΔT / ΔC, median percent regression, partial regression, and complete regression. The change in tumor volume in each treatment (T) and control (C) group was calculated daily for each animal by subtracting the tumor volume on the day of the first treatment (staging day) from the tumor volume on a specific observation day. Median ΔT was calculated for the treatment group and median ΔC was calculated for the control group. The ΔT / ΔC ratio was calculated and expressed as a percentage. ΔT / ΔC = (Median Delta T / Median Delta C) × 100

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

[0122] Percent tumor regression was defined as the percentage of decreased tumor volume in the treatment group at a specific observation day compared to the tumor volume at the start of the study (t0). At a specific time point (t), for each animal, the percent regression was calculated using the following formula: Regression %(t) = [(Volume t0 - Volume t ) / Volume t0 × 100 and was calculated using this formula.

[0123] The median percent regression for a group on a given day was then calculated by taking the median of the individual regression % values calculated for each animal in the group. The day of calculation was determined by the day on which ΔT / ΔC was calculated, except in cases where the median percent regression was not representative of the group's activity. In this case, if the median percent regression was maximum, the day was determined by the first day.

[0124] Statistical Analysis A two-way analysis of variance with treatment and day (replication) as factors was performed on the change in tumor volume from baseline. Treatments with significant interaction or treatment effects *For the 27-day period, comparative analysis was performed using multiple Bonferroni-Holm correlations, followed by comparison of all treatment groups with the control group for each day from 8 to 27. The change in tumor volume from baseline was calculated for each animal and each day by subtracting the tumor volume on the first treatment day (day 8) from the tumor volume on the identified observation day.

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

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

[0127] As single agents, Ab1 (25 mg / kg Q3D) and anti-PD-1 Mab (5 mg / kg Q3D) did not disrupt tumor growth compared to animals treated with the Ab1 isotype control (anti-HEL hlgG4). The ΔT / ΔC ratios at day 27 of treatment were 93% and 109%, respectively (Table 4). The combination of anti-PD-1 Mab and anti-HEL hlgG4 demonstrated minimal antitumor activity with a ΔT / ΔC of 31% at day 27 of treatment (statistically indistinguishable from the control group), and complete regression was observed in only 2 out of 10 mice. However, the combination of anti-PD-1 Mab and Ab1 demonstrated superior antitumor activity from day 15 to 27 with a ΔT / ΔC of -1 at day 27 of treatment (statistically different from the control group), and complete regression was observed in 6 out of 10 mice (Table 4).

[0128] [Table 4]

[0129] Tables 5 and 6, and Figures 5–7, show further data illustrating the activity of antibodies alone or in combination on tumor volume in mouse models.

[0130] [Table 5]

[0131] [Table 6]

[0132] The data in the table and figures are for Ab1 25 mg / kg Q3D and x-anti-mPD-1 Mab The combination with 5 mg / kg Q3D demonstrated a greater antitumor effect than either antibody at those doses. Compared to the combination with Ab1 as a single agent, this difference was statistically significant, with p-values ​​of 0.0007, <0.0001, and <0.0001 on days 19, 23, and 27, respectively. Compared to the combination with x-anti-mPD-1 Mab as a single agent, this difference was also statistically significant, with p-values ​​of 0.0276, 0.0004, and 0.0024 on days 19, 23, and 27 (Table 6). For the combination group of anti-HEL hlgG 425 mg / kg Q3D and x-anti-mPD-1 Mab 5 mg / kg Q3D, the treatment effect on tumor volume change from baseline was not significantly different from the effect of either agent alone on any given day of measurement.

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

[0134] In another study, the inventors evaluated the antitumor activity of combinations of Ab1 at doses of 1, 10, or 25 mg / kg and mouse PD-1 antibody at doses of 5 mg / kg against a subcutaneous MC38 mouse colorectal cancer model in C57BL / 6J mice. Exponentially growing MC38 colorectal adenocarcinoma cells (NCl, Frederick, MD) were cultured in RPMI-1640 supplemented with 10% FBS in an incubator humidified with 5% CO2, and then subcutaneously transplanted into the flanks of female C57 / Bl6J mice (Jackson Laboratory, Bar Harbor, ME) (1 × 10⁻¹⁶). 6 (Individual cells). Tumors average size 50-75 mm. 3Upon reaching a certain stage, the mice were pooled and randomly divided into a control group and a treatment group (10 mice per group). Tumor-bearing mice were then treated intraperitoneally three times a week with PBS, IgG4 isotype control antibody (25 mg / kg), or Ab1 (1, 10, and 25 mg / kg) until each animal received a total of 6–7 doses. Tumors were measured twice a week using digital calipers, and tumor volume was calculated (mm²). 3 (=L×W×H), graphed using GraphPad Prism. Tumors >2000mm 3 If the tumor grew to a certain size, or if it showed ulceration on >20% of the tumor surface, the mice were euthanized with CO2 at the end of the study.

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

[0136] [Table 7]

[0137] In summary, these preclinical data demonstrate that the combination of PD-1 inhibition and TGF-β inhibition can inhibit tumor growth more effectively than checkpoint inhibitor blockade alone. [Examples]

[0138] Intratumor TGF-β1 levels Intratumoral TGF-β1 levels were studied in a subcutaneous xenograft-grafted BALB / c mouse model of LoVo colorectal cancer. Mice were given either Ab1 or isotype control Mab 10, 25, or 50 mg / kg, with tumor volume of 100 mm². 3 The treatment was started when the patient was small, and consisted of a total of eight IV doses, administered intravenously every three days.

[0139] Tumor samples stored at -80°C in 2.8mm ceramic balls (MoBio 13114-50) and 2ml plastic tubes 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 tissue, and then homogenized using a Precellys® 24 Dual homogenizer (Bertin Instruments) at 4°C for two cycles at 6500 rpm for 20 seconds each. The lysate was purified by centrifugation at 20,000 × g for 10 minutes in an Eppendorf 5417C centrifuge at 4°C. The supernatant was transferred to a clean, chilled Eppendorf tube and further purified by centrifugation for another 20 minutes as described above. The supernatant was then transferred to a 96-well plastic storage block, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0140] The following day, the samples were thawed at room temperature and placed on ice. The protein concentration of the lysates was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo 23225) according to the manufacturer's instructions. The 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 distributed into plastic microcentrifuge tubes.

[0141] TGF-β1 concentrations in normalized tumor lysates were measured using a human TGF-β1 kit (MSD, K151IUC-2) with electrochemiluminescence 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 calibrator. Samples were loaded twice onto plates. The electrochemiluminescence signal was measured using MESO. Measurements were performed using a SECTOR S 600 plate reader (MSD), and TGF-β1 concentrations in the samples were quantified based on standard curves using MSD Discovery Workbench software v4.0.

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

[0143] The results showed that in mice injected with the isotype control, the median intratumoral TGF-β1 level was 21.4 pg / mg of total protein, while no equivalent level was detected in mice injected with Ab1 (Figure 8).

[0144] To demonstrate the relevance of the above findings in humans, we tested 10 human colorectal tumor samples and 10 human melanoma tumor samples with respect to their intratumoral TGF-β1 levels as described above, using the method described above. In the human CRC samples, TGF-β1 levels ranged from approximately 7 to 25 pg / mg. In the human melanoma samples, TGF-β1 levels ranged from approximately 1 pg / ml to at most 43 pg / ml. These data further support the use of anti-TGF-β1 therapeutic agents such as Ab1 in tumors being treated, either alone or in combination with other immune checkpoint inhibitors such as anti-PD-1 antibodies. [Examples]

[0145] Pharmacokinetic studies of Ab1 This example describes a study that characterized the pharmacokinetic (PK) profile of Ab1 and compared it with that of fresolimmab. In one study, five groups of cannula-treated Sprague Dolly rats were intravenously administered a single dose of either Ab1 or fresolimmab 5 mg / kg. Each group consisted of five females and five males. Blood samples were collected from the rats at 0.25, 6, 24, 48, 72, 144, 192, and 240 hours after administration. Serum concentrations of Ab1 and fresolimmab were determined by ELISA. Comparability was determined when the 90% confidence interval of the AUC ratio (of the test substance relative to the reference) was within the range of 80% to 125%.

[0146] The time-course serum antibody concentrations from the five rat groups are shown in Figure 9A. The PK parameters from groups 2, 4, and 5 (see legend in Figure 9A) are shown in Table 7 below. This study highlights the significantly longer half-life (7.1 days vs. 4.3 days average T) of fresolimmab compared to phrezolimumab. 1 / 2 The study showed that Ab1 exhibited linear PK behavior at slower elimination rates (0.30 ml / hr / kg vs. 0.51 ml / hr / kg CL). The data indicated that Ab1 resulted in 1.7 times higher exposure than fresolimmab in rats.

[0147] [Table 8]

[0148] Further PK studies in Ab1 (Study 2) were conducted in groups of cynomolgus monkeys. Each group consisted of 5 females and 5 males, and Ab1 was administered intravenously as a single dose of 1 mg / kg (Figure 9B) or 10 mg / kg (Figure 9D), or as five weekly doses of 1 mg / kg (Figure 9C) or 10 mg / kg (Figure 9E) per dose. Serum concentrations of Ab1 over time in the monkeys are shown in Figures 9B-E. For comparison, serum concentrations of fresolimmab administered to monkeys in single or repeated Q2W (bi-weekly) doses in the previous study are also shown in the figures. These data showed that Ab1 also exhibited linear PK behavior in monkeys, showing higher exposure after single or repeated administration than fresolimmab at both 1 mg / kg and 10 mg / kg doses. In a single dose of 10 mg / kg, Ab1 had a half-life of 13 days, while fresolimmab had a half-life of 4.5 days; Ab1 had a CL of approximately 0.40 ml / hr / kg, while fresolimmab had a CL of 0.66 ml / hr / kg. Similar to the rat studies, the monkey studies also showed that Ab1 resulted in approximately 1.7 times higher exposure than fresolimmab.

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

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

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

[0152] [Table 9]

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

[0154] Toxicity study of Ab1 Toxicity studies of Ab1 were conducted in rats and cynomolgus monkeys. Drug safety assurance endpoints were evaluated using GLP (Good Laboratory Practice) guidelines for weekly repeated doses over 5 weeks. No Ab1-related histopathological findings were observed at injection sites at doses 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. No Ab1-related effects on body temperature, respiratory rate, blood pressure, and ECG parameters were recorded in neurological investigations at any dose level tested.

[0155] The NOAEL (No Observed Adverse Effect Level) in rats was found to be 3 mg / kg / dose with repeated weekly administration for 5 weeks, and the STD10 (Severe Toxicity Dose, which causes death or irreversible severe toxicity in 10% of animals) was found to be between 3 and 10 mg / kg / dose in rats. Toxicity included cardiac valve dilation characterized by multiple thickening microneuromas; as well as abnormal pulmonary conditions such as mixed-cell alveolar exudate, mixed-cell perivascular infiltration, muscular arterial hypertrophy, hemorrhage, and / or increased lung weight.

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

[0157] [Table 10]

[0158] Based on the above toxicity data, Ab1 is expected to be safely administered to human patients at a dosage level of about 0.05 mg / kg to 0.5 mg / kg per week, or at a less frequent dosage level such as once every two weeks.

Example

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

[0160] To investigate the role of the immune response, mice deficient in the β2 microglobulin gene and thus lacking a cytotoxic T cell response were transplanted with B16-F10 in the plantar surface and treated as described above. In contrast to the results seen in immunocompetent mice, 1D11 had no effect on the number of metastases in the draining regional lymph nodes of these mice. These results suggest that the mechanism of TGF-β inhibition is by adaptive cellular immunity. + +

Example

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

[0162] Furthermore, the inventors found not only that there was a correlation between anti-PD-1 response and activation of TGF-β signaling, but also that the correlation was strong (R = 0.59, p-value by t-test < 9E-4). Thus, the inventors arrived at the following, their gateway indication 1: TGF-β-mediated immunosuppression in melanoma (e.g., metastatic melanoma) can contribute to innate resistance. Furthermore, the inventors found that TGF-β-induced gene expression changes are quenchable by 1D11 treatment, confirming the specificity of the signature of TGF-β activation. These results supported the advantage of using anti-TGF-β and anti-PD-1 therapeutic agents in combination to treat cancer patients who do not respond to anti-PD-1 monotherapy.

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

[0164] To study biomarkers in the tumor microenvironment, the immunostructure of patient tumors was evaluated in CRC and melanoma using the MultiOmyx, multiplex IHC assay. Multiplexing was performed with 12 biomarkers in one FFPE section from each tumor sample (totaling 22 immune cell types). The study included the extent of inflammation and assessed how well the analysis evaluated each tumor type and correlated with possible treatment effects. Statistical methods were developed to assess differences at the cell population level, including replication agreement, volcanic type sequencing for variance analysis, and correlation matrices. The MultiOmyx assay demonstrated excellent technical reproducibility and accuracy, a favorable dynamic range, and differences in inflammatory status in selected immune cells and areas of interest, including both positive and negative correlations between cell populations. [Examples]

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

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

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

[0168] 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) along with anti-PD-1 (5 mg / kg) showed a reduction in tumor-activated TGF-β levels, demonstrating Ab1 engagement with its target in vivo (Figure 10A). The reduction in activated TGF-β1 levels was observed within 1 hour and persisted for at least 168 hours.

[0169] MIP-2 (CXCL2) and KC / GRO (CXCL1) are chemotactic chemokines for granulocytes, including neutrophils. Levels of MIP-2 and KC / GRO were also evaluated in these same samples. After treatment with Ab1 in combination with anti-PD-1, intratumoral levels of MIP-2 increased at least fourfold in animals treated with Ab1 in combination with anti-PD-1 compared to animals treated with PBS or anti-PD-1 alone; the increase in MIP-2 levels was shown to last for at least 168 hours (Figure 10B). Similarly, KC / GRO levels were also shown to increase, but at later time points of 72 and 168 hours compared to those of MIP-2 (Figure 10C). Thus, the combination of Ab1 and anti-PD-1 mAb induced a decrease in 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 within the tumor microenvironment. Furthermore, the observed increases in MIP-2 and KC / GRO levels indicated that these are cytokines affected by TGF-β neutralization and therefore could contribute as potentially useful biomarkers in patients treated with Ab1. [Examples]

[0170] Repair of NK cell clustering by Ab1 treatment TGF-β is known to influence 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 are a dense class of mechanisms that enhance their activity. It has recently been reported that IL-2 forms tertiaries and is activated through the localization of IL-2 within these densely packed clusters (Kim et al., Scientific Reports (2017) 7:40623). Purified human NK cells clustered in vitro in the presence of IL-2 are these It was shown that they form densely packed clusters.

[0171] In this study, the inventors evaluated the effect of TGF-β on NK cell "clustering" in the absence and presence of Ab1. NK cells were newly isolated from healthy donor blood by negative selection using the NK cell RosetteSep reagent (Stem Cell Technologies) according to the manufacturer's protocol. NK cells were measured in round-bottom assay plates (Costar) in IL-2 (100 IU / mL) supplemented with Myelocult (Stem Cell Technologies), at a concentration of 1.2 × 10⁶ cells. 5 Cells were cultured in individual cells / well. TGF-β1 was added at final concentrations of 0.1, 1, or 10 ng / mL in the presence of either unrelated IgG4 or Ab1 at 100 μg / mL, as shown. Cells were cultured for 72 hours, and NK cell clustering was visualized by capturing images with a Nikon microscope.

[0172] The addition of gradually increasing doses of TGF-β1 inhibited NK cell clustering. Adding Ab1, instead of the IgG4 control antibody, to NK cell cultures resulted in NK cell clustering. These results demonstrate that TGF-β neutralization affects NK cell activation, leading to increased activation and NK cell proliferation, thus supporting the immune system's antitumor response. [Examples]

[0173] Ab1 treatment for proliferative CD8 + Restoration of TGF-β-mediated suppression of IFN-γ production in T cells In addition to the innate immune system, TGF-β is CD8 + It has been reported to inhibit T cell activity (Flavell et al., Nature Reviews Immunology (2010) 10:554-567). CD 8+ To explore the roles of TGF-β and Ab1 in T cell activity, purified human CD3 + A mixed lymphocyte (MLR) assay system was established in which cells were mixed with BLCL cells. CD8 + Cell proliferation and IFN-γ production were first evaluated in the presence of TGF-β. In particular, CD3 + The cells were isolated from PBMCs fractionated from healthy donors after Ficoll gradient isolation, using the EasySep T Cell enrichment kit (StemCell Technologies). CD3 + The cells were then labeled with CellTrace Violet (ThermoFisher) according to the manufacturer's protocol. The MLR assay was performed in RPMI supplemented with 10% FBS, and the labeled CD3 + cells (2×10 5 BLCL cells (Astarte) irradiated with individual cells Bio) (2 x 10 4 The procedure was carried out by mixing with individual cells (2 mins). As shown, TGF-β1, IgG4 control antibody and / or Ab1 were added to the culture, and the culture was incubated for 4 days at 5% CO2, 37°C. The cells were then stimulated for 4 hours in the presence of a PMA cell stimulation cocktail (eBioscience) and a protein transporter inhibitor cocktail (eBioscience). Live cells were identified by staining with Zombie NIR viability dye (BioLegend) on ice and washed with FACs buffer. The cells were fixed with True-Nuclear buffer (BioLegend), washed, pelletized, and resuspended in FACs buffer. The 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 using BD Canto, and the results were analyzed with FlowJo software, including live cells, single cells, and CD3 cells. + The cells were gated. IFNγ + CD8 +The percentage of T cells decreased in CellTrace Based on Violet staining, the CD8 has undergone proliferation and is positive for INF-γ staining. + Cells were quantified by gating. FMO was performed as a control for all antibody staining.

[0174] Inclusion of TGF-β in the MLR assay is approximately 4-fold in IFN-γ-positive CD8 + It was shown that the percentage of T cells was reduced (Figure 11A). Inclusion of Ab or control Ab was shown to reduce these INFγ in the absence of TGFβ. + It did not show any effect on the development of proliferating CD8+ cells (Figure 11B). However, the inclusion of Ab1, rather than the control antibody, showed a dose-dependent effect on IFNγ + CD8 + Cell proliferation can be restored. These results suggest that TGF-β neutralization is an effector that expresses INF-γ CD8. + We demonstrated that the adaptive immune system can be influenced by blocking the immunosuppressive effect of TGF-β on cell proliferation. + CD8 + T cells were suggested to play an important role in antitumor immunity (Ikeda et al., Cytokine Growth Factor Rev (2002) 13:95-109). [Examples]

[0175] Response of syngeneic mouse models to anti-TGF-β therapy In this study, the inventors investigated which syngeneic mouse models could be used to predict the response to treatment with anti-TGF-β antibody Ab1 and anti-PD-1. To classify the mouse models, the inventors used CD8 to tumors in mice. + T cell infiltration and TGF-β pathway activation were evaluated. CD8 + T cell infiltration was identified from data obtained from RNASeq, specifically CD8 +Assays were performed based on T cell characteristics. Seventeen different mouse syngeneic models with tumor cells arising from several indications (shown below the X-axis in FIGS. 12A and 12B) were transcriptionally profiled using whole transcriptome RNAseq. The "list" of these syngeneic models was constructed with the common background strain C57 / BL6 with 5 to 7 biological replicates per model. After Illumina 2000 sequencing, gene expression profiles expressed in transcripts per million reads (TPM) were generated by standard processing of raw sequence reads using the STAR aligner and Cufflinks transcript estimators. The resulting multi-sample data matrix was ultimately quantile normalized.

[0176] FIG. 12A shows the relative abundance of CD8 + T cells across the list (log2 transformed). The relative CD8 + T cell abundance was estimated using the specific marker gene CD8B, which has been shown 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 plot summarizes the range of values across biological replicates. The MC38 model showed approximately twice as much CD8 + T cell infiltration as the EMT6 model (left and right boxes, respectively). The A20 and EL4 lymphoma models showed the highest and lowest levels of CD8 + T cell infiltration overall, and CD8 + T cells in EL4 were negligible.

[0177] The MC38, MC38.ova, CT26, and L1210 mouse cell lines had the highest levels The CD8 gene characteristics of the EMT-6 tumor were shown. Furthermore, the EMT-6 breast cancer cell lineage was shown to exhibit near-baseline T cell infiltration, which is consistent with recent reports that EMT6 tumors have an immunoexclusion phenotype (S. Mariathasan et al. 2017, ESMO Immuno-Oncology Congress, Geneva, Geneva Switzerland).

[0178] Figure 12B shows the TGF-β pathway activation across the list. Using transcriptional features of 170 genes involved in TGF-β pathway activation, derived from in vitro stimulation of MCF7 cells with TGFβ and confirmed by comparison with several other TGF-β features, a pathway activation score was assigned to each profile in the list. The scores were calculated using Regulatory Gene Set Enrichment Analysis (rGSEA, Theilhaber et al. 2014) and based on the gene background. The enrichment of characteristic genes for und was expressed as log2. The MC38 model showed average activation, while the EMT6 model showed very high TGF-β pathway activation (left and right boxes, respectively). [Examples]

[0179] Efficacy of Ab1 and anti-PD1 antibody combination in a mouse breast cancer model In this study, the inventors investigated the therapeutic effect of Ab1 with and without anti-PD-1. Exponentially growing EMT-6 mammary cells (CRL-2755, ATCC) were cultured in RPMI-1640 supplemented with 10% FBS in an incubator humidified with 5% CO2, and then subcutaneously transplanted into the flanks of female BALB / c mice (Shanghai Lingchang Bio-Technology Co. Ltd, Shanghai, China) (0.5 × 10⁻¹⁴). 6 Individual cells / mouse). Tumors average size 68-116 mm. 3Upon reaching a certain stage, the mice were pooled and randomly divided into control and treatment groups (10 mice per group). Tumor-bearing mice were then treated intraperitoneally with PBS, Ab1 (10 and 25 mg / kg) three times a week for a total of six doses. Tumors were measured twice a week using digital calipers, and tumor volume was calculated (mm²). 3 (L×W×H), graphed using Graph Pad Prism. Tumors >3000mm 3 If the tumor grew to a certain size, or if it showed ulceration on >20% of the tumor surface, the mice were euthanized with CO2 at the end of the study.

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

[0181] [Table 11]

[0182] Unless otherwise specified herein, scientific and technical terms used in relation to the present invention have meanings that will be commonly understood by those skilled in the art. Illustrative methods and materials are described below, but the present invention Methods and materials similar to or equivalent to those described in the substantive may also be used in the implementation or testing of the present invention. All literature and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification, including definitions, shall be controlled. Many sentences are cited herein, but this citation does not acknowledge that any of these sentences constitute part of the common general knowledge of the art. Furthermore, unless otherwise required by context, singular terms include plural forms, and plural terms include singular forms. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, pharmaceutical and pharmacochemical chemistry, as well as protein and nucleic acid chemistry, and hybridization, as described herein, are well known and commonly used in the art. Enzyme reactions and purification techniques were carried out according to the manufacturer's instructions, as commonly achieved in the art or as described herein. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” are understood to mean the inclusion of the integer or group of integers mentioned, but not the exclusion of any other integer or group of integers.

[0183] The sequences described herein are listed below.

[0184] [Sequence Listing] Sequence ID 1 (Ab1 heavy chain) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SNVISWVRQA PGQGLEWMGG VIPIVDIANY AQRFKGRVTI TADESTSTTY MELSSLRSED TAVYYCASTL GLVLDAMDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK Sequence ID 2 (Ab1 light chain) ETVLTQSPGT LSLSPGERAT LSCRASQSLG SSYLAWYQQK PGQAPRLLIY GASSRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYADSPITFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC Sequence ID 3 (Fresolimmab heavy chain, including leader sequence residues 1-19) MGWSCIILFL VATATGVHSQ VQLVQSGAEV KKPGSSVKVS CKASGYTFSS NVISWVRQAP GQGLEWMGGV IPIVDIANYA QRFKGRVTIT ADESTSTTYM ELSSLRSEDT AVYYCASTLG LVLDAMDYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK YGPPCPSCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK Sequence ID 4 (Fresolimmab light chain, including leader sequence residues 1-19) MGWSCIILFL VATATGVHSE TVLTQSPGTL SLSPGERATL SCRASQSLGS SYLAWYQQKP GQAPRLLIYG ASSRAPGIPD RFSGSGSGTD FTLTISRLEP EDFAVYYCQQ YADSPITFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Sequence ID 5 (Anti-PD-1Mab heavy chain) EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA PGKGLEWVSG ISGGGRDTYF ADSVKGRFTI SRDNSKNTLY LQMNSLKGED TAVYYCVKWG NIYFDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK Sequence ID 6 (Anti-PD-1Mab light chain) DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP GKAPNLLIYA ASSLHGGVPS RFSGSGSGTD FTLTIRTLQP EDFATYYCQQ SSNTPFTFGP GTVVDFRRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Sequence ID 7 (x-anti-mPD-1Mab heavy chain) EVQLQESGPG LVKPSQSLSL TCSVTGYSIT SSYRWNWIRK FPGNRLEWMG YINSAGISNY NPSLKRRISI TRDTSKNQFF LQVNSVTTED AATYYCARSD NMGTTPFTYW GQGTLVTVSS AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV PSSTWPSETV TCNVAHPASS TKVDKKIVPR DCGCKPCICT VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEV QFSWFVDDVE VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW NGQPAENYKN TQPIMDTDGS YFVYSKLNVQ KSNWEAGNTF TCSVLHEGLH NHHTEKSLSH SPG Sequence ID 8 (x-anti-mPD-1Mab light chain) DIVMTQGTLP NPPSGESVS ITCRSSKSLL YSDGKTYLNW YLQRPGQSPQ LLIYWMSTRA SGVSDRFSGS GSGTDFTLKI SGVEAEDVGI YYCQQGLEFP TFGGGTKLEL KRADAAPTVS IFPPSTEQLA TGGASVVCLM NNFYPRDISV KWKIDGTERR DGVLDSVTDQ DSKDSTYSMS STLSLTKADY ESHNLYTCEV VHKTSSSPVV KSFNRNEC Sequence ID 9 (1D11 heavy chain) HVQLQQSGPE LVRPGASVKL SCKASGYIFI TYWMNWVKQR PGQGLEWIGQ IFPASGSTNY NEMFEGKATL TVDTSSSTAY MQLSSLTSED SAVYYCARGD GNYALDAMDY WGQGTSVTVS SAKTTPPSVY PLAPGSAAQT NSMVTLGCLV KGYFPEPVTV TWNSGSLSSG VHTFPAVLQS DLYTLSSSVT VPSSTWPSQT VTCNVAHPAS STKVDKKIVP RDCGCKPCIC TVPEVSSVFI FPPKPKDVLT ITLTPKVTCV VVDISKDDPE VQFSWFVDDV EVHTAQTKPR EEQFNSTFRS VSELPIMHQD WLNGKEFKCR VNSAAFPAPI EKTISKTKGR PKAPQVYTIP PPKEQMAKDK VSLTCMITDF FPEDITVEWQ WNGQPAENYK NTQPIMDTDG SYFVYSKLNV QKSNWEAGNT FTCSVLHEGL HNHHTEKSLS HSPGK Sequence ID 10 (1D11 light chain) NIVLTQSPAS LAVSLGQRAT ISCRASESVD SYGNSFMHWY QQKSGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YCQQNNEDPL TFGAGTKLEL KRADAAPTVS IFPPSEQLT SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC

Claims

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

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

1. H ) Amino acid sequence and light chain variable domain (V) corresponding to residues 1-108 of SEQ ID NO: 2 L ) The use according to claim 1, comprising an amino acid sequence.

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

2.

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

Images