TGF-beta antibodies

Abstract

The present invention relates to antibodies capable of activating TGF-[beta] 1, in particular antibodies capable of binding to potential TGF-[beta] 1 and releasing mature TGF-[beta] 1 cytokines, and uses thereof.

Description

Field of the Invention

[0001] The present invention relates to antibodies and antigen-binding fragments thereof that bind to latent TGF-β31 and activate TGF-β31. Since TGF-β1 is an important immunosuppressive cytokine, the antibodies and antigen-binding fragments described herein can be used in methods for inhibiting pathological immune responses, such as for preventing and treating autoimmune and alloimmune diseases. The antibodies and antigen-binding fragments described herein can also be used in methods for preventing and treating tumor diseases. Background of the Invention

[0003] TGF-β is a pleiotropic cytokine that regulates a variety of biological processes, including cell growth, differentiation, tissue homeostasis, and extracellular matrix remodeling. It plays an important and complex role in regulating immune system function (such as Travis and Sheppard, Annu. Rev. Immunol. 2014; 32: 51-82 and Sanjabi et al., Cold Spring Harb Perspect Biol. 2017; 9: a022236). Mice with TGF-β1 deletion die after birth, but suffer from excessive inflammation, highlighting the importance of this cytokine in regulating immune responses (Shull et al., Nature, 1992; 359: 693-99).

[0004] Three isoforms of TGF-β are found in mammals: TGF-β1; TGF-β2; and TGF-β3. The main isoform expressed in the immune system is TGF-β1. All TGF-β isoforms are initially synthesized as precursor proteins, consisting of a short signal peptide (guiding TGF-β to the endoplasmic reticulum), a large N-terminal region called a latent associated peptide (LAP), and a shorter C-terminal fragment (which is a mature TGF-β cytokine). TGF-β is secreted from cells in the form of homodimers (including three interchain disulfide bonds), and before or after secretion, LAP is separated from mature cytokines via furin cleavage. The N-terminal LAP remains non-covalently associated with mature cytokines to form a latent TGF-β complex. In some cases, latent TGF-β is associated with a family of regulatory proteins known as "latent TGF-β binding proteins" or "LTBPs". The complex of latent TGF-β with one or more LTBPs is sometimes referred to as a "large latent complex".

[0005] The crystal structure of latent TGF-β has been solved, which reveals how TGF-β is converted from its latent form to its active form (Shi et al., Nature 2011; 474: 343-49). The crystal structure shows that in the latent form, the mature cytokine region is surrounded by LAP, so that the site on the mature cytokine that can contact the TGF-β receptor complex is blocked. The mature cytokine is fixed in place by multiple contacts with a flexible loop structure called the "latency lasso" within the LAP. TGF-β activation requires the release of LAP-mediated inhibition.

[0006] TGF-β is activated in vivo via a variety of mechanisms. An important mode of activation is through the binding of integrins to the linear tripeptide motif "RGD" within the LAP region of TGF-β. This binding of LAP to integrins exerts a deforming physical force on the bound latent TGF-β complex, leading to the release of mature cytokines. Integrins that are particularly important in the activation of latent TGF-β include αvβ6 (usually expressed by epithelial cells) and αvβ8 (usually expressed by dendritic cells). Other mechanisms by which latent TGF-β is activated in vivo may include binding to thrombospondin-1 (TSP-1) or by enzymatic activation, such as proteases (such as serine proteases and metalloproteases) that cleave the LAP region.

[0007] Once activated, TGF-β mediates its action by binding to the TGF-β receptor complex on the cell surface. This complex consists of two transmembrane type I TGF-β receptors (TGF-βRI) and two transmembrane type II TGF-β receptors (TGF-βRII). Both receptors have a cytoplasmic region with serine / threonine kinase activity, and the binding of TGF-β to the receptor complex causes TGF-βRII to phosphorylate TGF-βR1. Phosphorylation at the receptor level triggers an intracellular signal cascade, whereby R-Smad proteins (Smad-2 and Smad-3) are recruited to the receptor complex and subsequently phosphorylated. Phosphorylated Smad-2 and Smad-3 form a complex with Smad-4, which translocates to the nucleus to activate or inhibit gene expression by binding to the Smad response regulatory region in the promoter fragment of the TGF-β regulated gene. TGF-β binding to the TGF-β receptor complex can also trigger signaling through other well-characterized intracellular pathways and mediators, including the MAP kinase cascade, the PI3K pathway, and the RhoGTPase family. In most cell types, including normal and precancerous or malignant epithelial cells, TGF-β-induced signals are cytostatic. Therefore, TGF-β1 exerts tumor suppressor activity on epithelial cells in the early stages of cancer.

[0008] As mentioned above, TGF-β1 is also an important cytokine that regulates immune responses. TGF-β1 is produced by a variety of cells within the immune system and has effects at the level of multiple cellular targets, especially T cells. In this regard, TGF-β1 plays an important role in regulating the differentiation of T cells into effector and regulatory cell subsets.

[0009] Among its roles as a key immunosuppressive cytokine, TGF-β1 is a potent inhibitor of T helper 1 (Th1) and T helper 2 (Th2) effector T cell differentiation and a potent inhibitor of CD8+ T cell proliferation. In contrast, TGF-β1 actively promotes the generation of regulatory T cells (Tregs), including natural or thymic Tregs generated in the thymus early in life and induced or peripheral Tregs generated by naive CD4+ T cells in the periphery. In the thymus, TGF-β1 protects Tregs from apoptosis during developmental selection; in the periphery, TGF-β1 promotes the conversion of naive CD4+ T cells into Tregs by upregulating the expression of the transcription factor Foxp3.

[0010] The activation of TGF-β1 on the surface of Tregs (and some other cell types) is regulated by binding to a transmembrane protein called "GARP" (glycoprotein A repeat major protein). In fact, studies have shown that GARP is required for the expression of latent TGF-β on the surface of activated Tregs, platelets, and endothelial cells, and plays an important role in regulating the bioavailability of active TGF-β in the vicinity of Tregs (Tran et al., Proc Natl Acad Sci USA. 2009; 106: 13445-50; Wang et al., Mol Biol Cell. 2012; 23: 1129-39; Vermeersch et al., PLoS One. 2017; 12(3): e0173329).

[0011] Given the importance of TGF-β1 signaling in regulating immune responses, attempts have been made to generate antibodies that bind to TGF-β and modulate its biological activity. International patent applications WO2015 / 015003 and WO2016 / 125017 describe antibodies that can bind to GARP and latent TGF-β1 complexes formed on the surface of Treg cells. These antibodies inhibit TGF-β1 activation, thereby attenuating the immunosuppressive activity of the Treg population. Therefore, it is proposed that these antibodies be used as immunostimulatory antibodies to treat conditions or diseases that require upregulation of immune responses, such as immunotherapy for cancer.

[0012] However, there is still an unmet need for antibodies that bind to and activate TGF-β1 (or promote TGF-β1 activation) to increase the immunosuppressive activity of Treg populations. As an object of the present invention, it is proposed that these antibodies be used as immunosuppressive antibodies to treat conditions or diseases that require downregulation of immune responses, such as for the treatment of autoimmune and alloimmune diseases or conditions. They are also proposed for the prevention or treatment of conditions or diseases that require increased tumor suppression, cell growth inhibition activity of TGF-β1, such as for the precancerous, primary or secondary metastatic stages of cancer. Summary of the invention

[0013] The present invention provides antibodies capable of activating TGF-β1, ie, antibodies capable of binding to latent TGF-β and releasing mature TGF-β cytokine thereby freeing from the inhibition exerted by the LAP region of the full-length TGF-β1 protein.

[0014] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that binds to latent TGF-β1, wherein the antibody or antigen-binding fragment thereof activates TGF-β1.

[0015] In certain embodiments, the antibody or antigen-binding fragment binds to an epitope comprising one or more amino acids from a region of human or murine latent TGF-β1 (consisting of amino acids 87-222), wherein human latent TGF-β1 is represented by SEQ ID NO:1 and murine latent TGF-β1 is represented by SEQ ID NO:5.

[0016] In certain embodiments, the antibody or antigen-binding fragment binds to an epitope comprising one or more amino acids from a region of human or murine latent TGF-β1 (consisting of amino acids 87-135), wherein human latent TGF-β1 is represented by SEQ ID NO:1 and murine latent TGF-β1 is represented by SEQ ID NO:5.

[0017] The antibody or antigen-binding fragment of claim 1 or claim 2, which binds to an epitope comprising one or more amino acids from a region of human or murine latent TGF-β1 (consisting of amino acids 136-200), wherein human latent TGF-β1 is represented by SEQ ID NO: 1 and murine latent TGF-β1 is represented by SEQ ID NO: 5.

[0018] In certain embodiments, the antibody or antigen-binding fragment binds to an epitope comprising one or more amino acids from a region of human or murine latent TGF-β1 (consisting of amino acids 201-222), wherein human latent TGF-β1 is represented by SEQ ID NO:1 and murine latent TGF-β1 is represented by SEQ ID NO:5.

[0019] For embodiments in which the antibody or antigen-binding fragment binds to human latent TGF-β1, the antibody or antigen-binding fragment may bind to an epitope comprising amino acid residues E119, K123, S138, R141, S209, R210, G212, E213, I214 and / or E215 of human latent TGF-β1.

[0020] For embodiments in which the antibody or antigen-binding fragment binds to human latent TGF-β1, the antibody or antigen-binding fragment may bind to an epitope comprising amino acid residues G212 and E215 of human latent TGF-β1.

[0021] For embodiments in which the antibody or antigen-binding fragment binds to murine latent TGF-β1, the antibody or antigen-binding fragment may bind to an epitope comprising amino acid residues S138, R141, P145, E146, S175, L208, N209 and / or D212 of murine latent TGF-β1.

[0022] For embodiments in which the antibody or antigen-binding fragment binds to murine latent TGF-β1, the antibody or antigen-binding fragment may bind to an epitope comprising amino acid residues L208 and D212 of murine latent TGF-β1. For antibodies or antigen-binding fragments that bind to murine latent TGF-β1 via an epitope comprising amino acid residues L208 and D212, the epitope may not comprise another amino acid residue from within the region of murine latent TGF-β1 (consisting of amino acids 201-222).

[0023] In certain embodiments, the antibody or antigen binding fragment activates TGF-β1 when latent TGF-β1 is complexed with GARP. Specifically, the antibody or antigen binding fragment can activate TGF-β1 when latent TGF-β1 binds to the surface of one or more cell types selected from the following: regulatory T cells (Treg), megakaryocytes, platelets, B lymphocytes, endothelial cells, fibroblasts, mesenchymal cells and hepatic stellate cells. In a preferred embodiment, the antibody or antigen binding fragment activates TGF-β1 when latent TGF-β1 binds to the surface of regulatory T cells (Treg).

[0024] In certain embodiments, the antibody or antigen-binding fragment activates TGF-β1 when latent TGF-β1 is associated with one or more cell types selected from: myeloid cells; lymphoid cells; hematopoietic cells; and cancer cells at a precancerous stage.

[0025] In certain embodiments, the antibody or antigen-binding fragment is bivalent, preferably an IgG antibody, more preferably an IgG1 antibody. In certain embodiments, the antibody or antigen-binding fragment comprises a CH1 domain, a hinge region, a CH2 domain and / or a CH3 domain of human IgG, preferably human IgG1.

[0026] In certain embodiments, the antigen-binding fragment is selected from: an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH or VHH); a single-chain antibody (scFv); a (scFv)2 fragment, a F(ab')2 fragment; a Fab fragment; a Fd fragment; a Fv fragment; a one-armed (monovalent) antibody; a diabody; a triabody; a tetrabody; or any antigen-binding molecule formed by combination, assembly or conjugation of such antigen-binding fragments.

[0027] In certain embodiments, the antibodies or antigen-binding fragments are defined by reference to specific CDR, VH and / or VL sequences described herein. For embodiments in which the domains of the antibodies or antigen-binding fragments are defined by a specific percentage sequence identity to a reference sequence, the VH and / or VL domains may retain the same CDR sequences as those present in the reference sequence, such that the variations are present only in the framework regions. The present invention also provides antibodies or antigen-binding fragments that bind to the same epitope as the antibodies or antigen-binding fragments defined herein with reference to a specific SEQ ID NO.

[0028] Also provided is an isolated nucleic acid encoding the antibody or antigen-binding fragment, including a polynucleotide encoding the VH, VHH and / or VL domains of the antibody or antigen-binding fragment described herein. The present invention also provides an expression vector comprising the above nucleic acid operably linked to a regulatory sequence, which allows the antibody, antigen-binding fragment, variable heavy chain domain (VH or VHH) or variable light chain domain (VL) to be expressed in a host cell or a cell-free expression system. A host cell or a cell-free expression system containing the above expression vector is also provided.

[0029] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment according to the first or second aspect of the present invention and at least one pharmaceutically acceptable carrier or excipient.

[0030] There is also provided an antibody or antigen-binding fragment according to the first or second aspect of the invention or a pharmaceutical composition according to the invention for use as a medicament.

[0031] In another aspect, the present invention provides a method for preventing or treating an autoimmune disease and / or alloimmune disease in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to the foregoing aspects of the present invention or a pharmaceutical composition according to the present invention. In certain embodiments, the disease or condition is selected from the group consisting of: inflammatory bowel disease (IBD), multiple sclerosis (MS), graft-versus-host disease (GVHD), allogeneic transplant rejection, antibody-mediated allograft rejection (AMR), allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), ANCA vasculitis, autoimmune diseases of the adrenal glands, gland), autoimmune encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, immune thrombocytopenia,ITP or idiopathic thrombocytopenic purpura or idiopathic thrombocytopenia purpura or immune mediated thrombocytopenia), autoimmune urticaria, Behcet's disease, bullous pemphigoid (BP), cardiomyopathy, Castleman's syndrome, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy,CIDP, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, COVID-19mediated postural orthostatic tachycardia syndrome (POTS), Crohn's disease, delayed graft function after kidney transplant, dilated cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, factor VIII deficiency VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome (GBS), Goodpasture's syndrome, Hashimoto's thyroiditis, hemophilia A, hemolytic disease of the fetus and newborn,HDFN、idiopathic membranous neuropathy、idiopathic pulmonary fibrosis、IgA neuropathy、IgM polyneuropathies、juvenile arthritis、Kawasaki's disease、lichen plantus、lichen sclerosus、lupus erthematosis、systemic lupus erythematosus (SLE)、lupus nephritis、membranous neuropathy、membranous nephropathy、Méniere's disease、mixed connective tissue disease、mucous membrane pemphigoid、graft type 1 diabetes 1diabetes mellitus), multifocal motor neuropathy (MMN), myelinoligodendrocyte glycoprotein antibody-associated disease (MOGAD), myasthenia gravis (MG), generalized myasthenia gravis (gMG), ocular myasthenia gravis (OMG), myositis, neuromyelitis optica (NMO), paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris (PV), pemphigus foliaceus (PEMPHIGUS GRACEUS,PF), pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis, dermatomyositis (DM), necrotizing autoimmune myopathy (NAM), Antisynthetase Syndrome (ASyS), primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis arthritis, RA), sarcoidosis, scleroderma, Sjögren's syndrome ( syndrome, solid organ transplant rejection, stiff-man syndrome, takayasu arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenia purpura, thyroid eye disease, ulcerative colitis, uveitis, warm autoimmune hemolytic anemia (WAIHA), dermatitis herpetiformis vasculitis, anti-neutrophil cytoplasmic antibody-associated vasculitis, vasculitides), vitiligo, and Wegner's granulomatosis.

[0032] In another aspect, the present invention provides a method for preventing or treating a tumor disease in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to the aforementioned aspects of the present invention or a pharmaceutical composition according to the present invention.

[0033] The present invention also provides a kit comprising the antibody or antigen-binding fragment according to the first or second aspect of the present invention, and optionally instructions for use.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Fourteen monoclonal antibodies that activate latent TGF-β1.

[0036] A. Schematic representation of the results obtained by screening mAbs against GARP or latent TGF-β1 to search for TGF-β1 activating antibodies.

[0037] B. Representative results of TGF-β1 reporter gene assay in HEK-293 cells. HEK-293T cells were transfected to express human (upper figure) or murine (lower figure) GARP: TGF-β1 complex, and firefly luciferase under the control of TGF-β1 responsive promoter (pCAGA-12), and Renilla luciferase under the control of constitutive promoter, to control transfection efficiency. Transfected cells were incubated with mAb (20 μg / ml) or recombinant human TGF-β1 (rhTGF-β1) at the concentration shown. The bar graph represents the ratio of firefly luciferase activity standardized to Renilla luciferase activity (mean + SD of three replicates). The shaded histogram represents antibodies that show that the firefly / renilla ratio is higher than the ratio measured when there is no rhTGF-β1 or mAb in at least 3 independent experiments. These mAbs are identified as TGF-β1 activation antibodies.

[0038] C. EC50 concentrations of the indicated TGF-β1 activating antibodies.

[0039] Figure 2 .Requirement for bivalent monospecific antibodies and GARP expression.

[0040] A. The antibody activated mouse or human TGF-β1 when used as a bivalent, monospecific full-length mAb or Fab'2 fragment, but not when used as a monovalent Fab fragment. HEK-293T cells were cultured according to Figure 1 Transfection was performed as shown. The transfected cells were incubated with 20 μg / ml of full-length mAb, Fab or Fab'2 fragment, or rhTGF-β1 at the concentrations shown. The bar graphs represent the ratio of firefly luciferase activity standardized to Renilla luciferase activity (mean + SD of three replicates). The shaded histograms represent antibodies or antibody fragments with enhanced signals compared to incubations without rhTGF-β1.

[0041] B. TGF-β1 activating antibodies activate latent TGF-β1 regardless of whether latent TGF-β1 is presented by GARP on the cell surface. Cells transfected and incubated with antibodies or rhTGF-β1 were as shown in A.

[0042] C. Western Blot analysis of TGF-β1 content in 25 μg of the indicated mAb preparations or the indicated amounts of rhTGF-β1 using ECL visualization with anti-mature TGF-β1 antibody.

[0043] Figure 3 The epitopes bound by the activating LMT and LHT mAbs are unique.

[0044] A. Sequence alignment of human and mouse TGF-β1 proteins. Signal peptide: amino acids 1 to 29; LAP: amino acids 30 to 278; mature TGF-β1: amino acids 279 to 391.

[0045] B. Binding of LMT and LHT mAbs to HEK-293T cells transfected to express GARP:latent TGF-β1 complexes containing HA-tagged chimeric forms of TGF-β1 illustrated on the left. Values ​​on the right represent % binding to a given chimera compared to binding to wild-type (WT) mouse TGF-β1 (100%).

[0046] C. Ala scan analysis of antibodies binding to Ala mutants in region 201-222 of potential TGF-β1. Bar graph represents % binding to a given Ala mutant compared to WT mouse TGF-β1 (100%). Binding below 50% is represented by white bars. Mean + SD of 2 to 3 experiments.

[0047] D. Ala scanning analysis identifying residues in the potential TGF-β1 region 201-222 necessary for cytokine activation by LHT-22 and LMT-12. The bar graph shows the % activation of the corresponding single A mutants compared to WT TGF-β1 (expression levels of the mutants were normalized to WT TGF-β1). Binding below 50% is indicated by white bars.

[0048] Figure 4 LMT-10 and LMT-12 improved survival in a graft-versus-host disease model.

[0049] A. Schematic diagram of experimental design. On day 0, fresh spleen cells (70-90 million) from C57BL / 6 mice were injected intraperitoneally into B6D2 F1 mice. From day -1, 400 μg mAb was injected intraperitoneally once a week. From day 0, mice were scored twice a week, and during the acute phase of the disease (day 13 to day 20), mice were scored every day. Scores were determined based on weight loss (1 or 2 points), posture (1 or 2 points), mobility (1 or 2 points), and fluffy hair (1 or 2 points). When the score of the mouse reached 6, it was killed.

[0050] B. Kaplan Meier plots showing the proportion of mice surviving the indicated days after spleen cell transplantation. P values ​​were calculated using the Gehan-Breslow Wilcoxon test (Graphpad Prism). Data were pooled from three independent experiments.

[0051] C. Evolution of GvHD scores of individual mice measured in 3 independent experiments. The ratios represent the proportion of mice that died (above the dashed line) or survived (below the dashed line) due to GvHD 30-50 days after allogeneic splenocyte transplantation.

[0052] Figure 5 In a graft-versus-host disease model, LMT-12 activates TGF-β1 from a GARP-independent source.

[0053] A. Activation of latent TGF-β1 by activating mAb was inhibited in the presence of blocking anti-GARP mAb. HEK-293T cells were transfected and incubated with antibodies or rhTGF-β1 at the indicated concentrations, as Figure 1 The bar graphs represent the ratio of firefly luciferase activity normalized to Renilla luciferase activity (mean + SD of three replicates). The shaded histograms represent cases where the firefly / Renilla ratio was superior to the ratio measured in the absence of rhTGF-β1 or mAb.

[0054] B. LMT-12 exerted the same therapeutic activity in GvHD whether administered alone or in combination with the blocking anti-GARP potential TGF-β1 antibody 58A2. Figure 4 The induction and scoring of GvHD were performed as described. The upper figure - Kaplan Meier graph represents the proportion of mice surviving during the experiment, wherein the P value is calculated using the Gehan-Breslow Wilcoxon test (Graphpad Prism). The lower figure - the evolution of the GvHD score of individual mice. The ratio represents the proportion of mice that died (above the dotted line) or survived (below the dotted line) due to GvHD 30-50 days after transplantation.

[0055] Figure 6 . Specificity test of LMT-12 for latent TGF-β1 in mice

[0056] The binding specificity of mAb LMT-12 was tested by flow cytometry as described in Example 9. mAb LMT-12 was found to bind specifically to murine latent TGF-β, but not to murine latent TGF-β2 or murine latent TGF-β3.

[0057] Detailed Description

[0058] A. Definition

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0060] "Transforming Growth Factor Beta" or "TGF-β" - The term "TGF-β" as used herein refers to the cytokine "transforming growth factor beta". As described elsewhere herein, TGF-β is a pleiotropic cytokine that regulates a variety of effects, particularly within the immune system. The term TGF-β as used herein is broad enough to encompass human isoforms of this protein and homologs of any species. There are three TGF-β isoforms in humans: TGF-β1, TGF-β2, and TGF-β3. The amino acid sequences of full-length human TGF-β1 and full-length murine TGF-β1 are shown in the following table as SEQ ID NOs: 1 and 5, respectively. These sequences correspond to sequences stored in the Uniprot database: human TGF-β1-P01137-1; and mouse TGF-β1–P04202-1.

[0061] The full-length TGF-β1 protein can be divided into: (i) a signal peptide (amino acid residues 1-29); (ii) a potential associated peptide or "LAP" (amino acid residues 30-278); and (iii) a mature cytokine region (amino acid residues 279-390). These three regions are shown below as SEQ ID NOs: 2, 3, and 4 for human TGF-β1 and SEQ ID NOs: 6, 7, and 8 for murine TGF-β1, respectively.

[0062] Table 1: Human and mouse TGF-β1 proteins

[0063]

[0064]

[0065]

[0066] "GARP" - The term "GARP" as used herein refers to the protein "glycoprotein A repeat-based", also known in the literature as "leucine-rich repeat protein 32" or "LRRC32". GARP is an 80 kDa type I transmembrane protein and acts as a receptor for latent TGF-β on the surface of regulatory T cells, among others (Stockis et al., Eur J Immunol. 2009 Dec; 39(12): 3315-22).

[0067] The amino acid sequence of full-length human GARP is shown below as SEQ ID NO: 67. This sequence corresponds to the sequence deposited in the Uniprot database as human GARP-Q14392-1.

[0068] MRPQILLLLALLTLGLAAQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLSGNQLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNL SRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLN LDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSG CVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSL LPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVSAILLTTLAACCCVRRQKFNQQYKA(SEQ ID NO:67)

[0069] "Antibody" or "immunoglobulin" - The term "immunoglobulin" as used herein includes polypeptides having a combination of two heavy chains and two light chains, whether or not they have any associated specific immunoreactivity. "Antibody" refers to an assembly that has significant known specific immunoreactivity to the target antigen (herein latent TGF-β1). The term "latent TGF-β1 antibody" or alternatively "TGF-β1 antibody" is used herein to refer to antibodies that exhibit immunospecificity for latent TGF-β1 (including human latent TGF-β1) and (if indicated) its species homologs. Antibodies and immunoglobulins contain light chains and heavy chains, with or without interchain covalent bonds between them. The basic immunoglobulin structure in vertebrate systems is relatively easy to understand.

[0070] The general term "immunoglobulin" encompasses five different classes of antibodies that can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention. The following discussion is generally directed to the IgG class of immunoglobulin molecules. In the case of IgG, the immunoglobulin comprises two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons, and two identical heavy chains with a molecular weight of 53,000-70,000. The four chains are connected by disulfide bonds in a "Y"-shaped structure, with the light chains extending from the mouth of the "Y" to the variable region, bracketing the heavy chains in the middle.

[0071] The light chain of an antibody is divided into kappa or lambda (κ, λ). Each heavy chain category can be combined with a κ or λ light chain. In general, when an immunoglobulin is produced by a hybridoma, a B cell or a genetically engineered host cell, the light chain and the heavy chain are covalently bound to each other, and the "tail" parts of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y-shaped structure to the C-terminus at the bottom of each chain. It will be understood by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), and there are also some subclasses (such as γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, respectively, IgG, IgM, IgA, IgD or IgE. Immunoglobulin subclasses (subtypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, etc. have been fully characterized and are known to have functional specialization. Modified versions of each of these classes and subtypes may be readily discerned by those skilled in the art in light of the present disclosure and, therefore, are within the scope of the present invention.

[0072] As described above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and the VH domain of an antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site located at the end of each arm of the Y-shape. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each chain of the VH and VL chains.

[0073] The term "antibody" as used herein is also intended to cover "VHH antibodies" or "heavy chain only antibodies".

[0074] "VHH Antibody" - As used herein, the term "VHH antibody" or "heavy chain only antibody" refers to a type of antibody produced only by species of the Camelidae family, which includes camels, llamas, and alpacas. Heavy chain only antibodies or VHH antibodies are composed of two heavy chains and lack light chains. Each heavy chain has a variable domain at the N-terminus, and these variable domains are referred to as "VHH domains" to distinguish them from the variable domains of the heavy chains of the conventional heterotetrameric antibodies described above, i.e., VH domains.

[0075] "Epitope" - As used herein, the term "epitope" refers to a region of a TGF-β1 protein to which an antibody binds. An antibody will typically bind to its respective TGF-β1 epitope via complementary binding sites on the antibody. The epitope to which the antibody binds will typically comprise multiple amino acids from the TGF-β1 protein. An epitope may include contiguous amino acids in the TGF-β1 protein, i.e., a linear epitope, or may include discontinuous amino acids in the TGF-β1 protein, i.e., a conformational epitope.

[0076] "Binding site" - As used herein, the term "binding site" includes the region of an antibody or antigen binding fragment that is responsible for selective binding to a target antigen of interest (e.g., TGF-β1). A binding domain comprises at least one binding site. Exemplary binding domains include antibody variable domains. The antibodies of the invention may comprise a single binding site or multiple (e.g., two, three, or four) binding sites.

[0077] "Variable region" or "variable domain" - The terms "variable region" and "variable domain" are used interchangeably herein and are intended to have the same meaning. The term "variable" refers to certain portions of the variable domains, VH and VL, that differ greatly in sequence between antibodies and are used for the binding and specificity of each particular antibody for its target antigen. However, this variability is not evenly distributed among the variable domains of an antibody. It is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains, which form part of the antigen binding site. The first, second and third hypervariable loops of the Vλ light chain domain are referred to herein as L1 (λ), L2 (λ) and L3 (λ), and can be defined as comprising residues 24-33 (L1 (λ), consisting of 9, 10 or 11 amino acid residues), 49-53 (L2 (λ), consisting of 3 residues) and 90-96 (L3 (λ), consisting of 5 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the Vκ light chain domain are referred to herein as L1 (κ), L2 (κ) and L3 (κ), and can be defined as comprising residues 25-33 (L1 (κ), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2 (κ), consisting of 3 residues) and 90-97 (L3 (κ), consisting of 6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VH domain are referred to herein as H1, H2 and H3, and can be defined as comprising residues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et al., Methods, 2000; 20:267-279).

[0078] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops of the VL domain, respectively, and encompass hypervariable loops obtained from Vκ and Vλ subtypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops of the VH domain, respectively, and encompass hypervariable loops obtained from any known heavy chain subtype, including γ, ε, δ, α, or μ.

[0079] Hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise a portion of a "complementarity determining region" or "CDR" as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous, because hypervariable loops (HV) are defined based on structure, while complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983), and the extent of HV and CDR may differ in some VH and VL domains.

[0080] The CDRs of VL and VH domains can generally be defined as comprising the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Thus, HV can be contained within the corresponding CDRs, and references herein to the "hypervariable loops" of VH and VL domains should be interpreted as also including the corresponding CDRs, and vice versa, unless otherwise indicated.

[0081] As defined below, the more conservative part of the variable domain is called the framework region (FR). The variable domains of natural heavy and light chains each contain four FRs (FR1, FR2, FR3 and FR4, respectively), and most of them adopt a β-folded structure connected by three hypervariable loops. The hypervariable loops in each chain are closely held together by FRs and contribute to the formation of the antigen binding site of the antibody together with the hypervariable loops in the other chain. The structural analysis of antibodies reveals the relationship between the sequence and shape of the binding site formed by the complementary determining region (Chothia et al., J.Mol.Biol.227:799-817 (1992)); Tramontano et al., J.Mol.Biol, 215:175-182 (1990)). Although its sequence variability is high, five of the six loops only adopt a small part of the main chain conformation, which is called "canonical structure". These conformations are determined firstly by the length of the loop and secondly by the presence of key residues at certain positions within the loop and in the framework regions that dictate the conformation by their ability to pack, hydrogen bond, or assume unusual main-chain conformations.

[0082] "CDR" - As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen binding sites found within the variable regions of the heavy and light chain polypeptides. These specific regions have been described in the following literature: Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), as well as Chothia et al., J. Mol. Biol. 196: 901-917 (1987) and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), where the definitions include overlaps or subsets of amino acid residues when compared to each other. For comparison, the amino acid residues that comprise the CDRs defined in each of the references cited above are listed. Preferably, the term "CDR" is a CDR defined by Kabat based on sequence comparison.

[0083] Table 2: CDR Definition

[0084]

[0085] 1 The residue numbering follows the nomenclature of Kabat et al. (see above).

[0086] 2 The residue numbering follows the nomenclature of Chothia et al. (see above).

[0087] 3The residue numbering follows the nomenclature of MacCallum et al. (see above).

[0088] "Framework Region" - As used herein, the term "framework region" or "FR region" includes amino acid residues that are part of the variable region but not part of the CDR (e.g., using the CDR definition of Kabat). Thus, the variable region framework is approximately 100-120 amino acids in length, but includes only those amino acids outside of the CDR. For the specific example of a heavy chain variable domain and the CDRs defined by Kabat et al., framework region 1 corresponds to the variable region domain comprising amino acids 1-30; framework region 2 corresponds to the variable region domain comprising amino acids 36-49; framework region 3 corresponds to the variable region domain comprising amino acids 66-94, and framework region 4 corresponds to the variable region domain from amino acid 103 to the end of the variable region. The framework region of the light chain is similarly separated by each light chain variable region CDR. Similarly, using the CDR definitions of Chothia et al. or McCallum et al., the framework region boundaries are separated by the corresponding CDR ends described above. In a preferred embodiment, the CDRs are as defined by Kabat.

[0089] In naturally occurring antibodies, the six CDRs present on each monomer antibody are short, discontinuous amino acid sequences that are specifically positioned to form an antigen binding site when the antibody presents its three-dimensional structure in an aqueous environment. The rest of the heavy and light chain variable domains show less intermolecular variability in the amino acid sequence and are referred to as framework regions. The framework region primarily adopts a β-folded conformation, and the CDRs form loops that connect and, in some cases, form part of the β-folded structure. Therefore, these framework regions serve to form a scaffold that positions the six CDRs in the correct direction through interchain non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. The complementary surface promotes the non-covalent binding of antibodies to immunoreactive antigen epitopes. One of ordinary skill in the art can easily determine the position of the CDRs.

[0090] "Constant region" - As used herein, the term "constant region" refers to the portion of an antibody molecule other than the variable domain or variable region. An immunoglobulin light chain has a single domain "constant region", commonly referred to as a "CL or CL1 domain". This domain is located at the C-terminus of the VL domain. Depending on the class of immunoglobulins (γ, μ, α, δ, ε), the immunoglobulin heavy chain is different in its constant region. Heavy chains γ, α and δ have a constant region consisting of three immunoglobulin domains (called CH1, CH2 and CH3) and a flexible hinge region separating the CH1 and CH2 domains. Heavy chains μ and ε have a constant region consisting of four domains (CH1-CH4). The constant region of the heavy chain is located at the C-terminus of the VH domain.

[0091] The amino acid numbering of the immunoglobulin heavy and light chains is from the N-terminus at the end of the bifurcation of the Y-shaped structure to the C-terminus at the bottom of each chain. Different numbering schemes are used to define the constant domains of the immunoglobulin heavy and light chains. According to the EU numbering scheme, the heavy chain constant domains of the IgG molecule are identified as follows: CH1 - amino acid residues 118-215; CH2 - amino acid residues 231-340; CH3 - amino acid residues 341-446. According to the Kabat numbering scheme, the heavy chain constant domains of the IgG molecule are identified as follows: CH1 - amino acid residues 114-223; CH2 - amino acid residues 244-360; CH3 - amino acid residues 361-477. "Fc domain" or "Fc region" generally defines the portion of the heavy chain constant region, including the CH2 and CH3 domains. The Fc region may also include some residues from the hinge region. The "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain and the CH2 domain. The hinge region comprises about 25 residues and is flexible, thereby allowing two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: an upper hinge domain, a middle hinge domain, and a lower hinge domain (Roux KH et al., J. Immunol. 161: 4083-90 1998). An antibody of the invention comprising a "fully human" hinge region may contain one of the hinge region sequences shown in Table 3 below.

[0092] Table 3: Human hinge sequences

[0093]

[0094] "Antigen binding fragment" - The term "antigen binding fragment" or "fragment" as used herein refers to a part of an antibody or antibody chain that contains fewer amino acid residues than a complete or complete antibody or antibody chain. The term "antigen binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with a complete antibody (i.e., a complete antibody derived therefrom) for antigen binding (i.e., specific binding to TGF-β1). The term "antigen binding fragment" as used herein includes, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, a Fd fragment, a Fv fragment, a one-arm (monovalent) antibody, a double antibody, a three-antibody, a four-antibody, or any antigen binding molecule formed by a combination, assembly or conjugation of such antigen binding fragments. The term "antigen binding fragment" as used herein is also intended to encompass antibody fragments selected from single antibodies, domain antibodies, and nanobodies. Fragments can be obtained, for example, via chemical or enzymatic treatment of a complete or complete antibody or antibody chain or by recombinant means.

[0095] "Fab" - "Fab" or "Fab fragment" refers to a molecule consisting of a heavy chain and a light chain, wherein the light chain consists of a VL domain and a constant domain (CL, Cκ or Cλ), and the heavy chain consists of only a VH domain and a CH1 domain. A Fab fragment is usually one arm of a conventional Y-shaped immunoglobulin molecule. Under the action of papain, an immunoglobulin molecule can produce a Fab fragment. Papain cleaves the immunoglobulin molecule in the hinge region, thereby producing two Fab fragments and a separate Fc region.

[0096] "scFv" or "scFv fragment" - "scFv" or "scFv fragment" means single chain variable fragment. scFv is a fusion protein of the VH domain and VL domain of an antibody connected via a linker.

[0097] "Valency" - As used herein, the term "valency" refers to the number of potential target or antigen binding sites in an antibody. Each target or antigen binding site specifically binds to one antigen or a specific site on a target antigen. When an antibody comprises more than one target binding site, each target binding site may specifically bind to the same or a different molecule (e.g., may bind to a different ligand or a different antigen, or a different epitope on the same antigen).

[0098] "Specificity" and "multispecific antibodies" - As used herein, the term "specificity" refers to the ability to bind (e.g., immunoreact) to a given target, such as TGF-β1. Preferably, the antibodies described herein "specifically bind" to their target antigens, wherein the term "specific binding" refers to the ability of any antibody to preferentially immunoreact with a given target. The antibodies described herein may be monospecific and contain one or more binding sites that specifically bind to a particular target. The antibodies described herein may also take the form of "multispecific antibodies", such as bispecific antibodies, in which the multispecific antibodies bind to two or more target antigens.

[0099] "Derived from" - The term "derived from" as used herein, a designated protein (e.g., a camel antibody or an antigen-binding fragment thereof) refers to the origin of a polypeptide or amino acid sequence. In one embodiment, a polypeptide or amino acid sequence derived from a specific starting polypeptide is a CDR sequence or a sequence associated therewith. In one embodiment, the amino acid sequence derived from a specific starting polypeptide is discontinuous. For example, in one embodiment, one, two, three, four, five or six CDRs are derived from a starting antibody. In one embodiment, a polypeptide or amino acid sequence derived from a specific starting polypeptide or amino acid sequence has an amino acid sequence substantially identical to the starting sequence or a portion thereof, wherein the portion is composed of at least 3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or a person of ordinary skill in the art can otherwise determine its origin in the starting sequence. In one embodiment, one or more CDR sequences derived from a starting antibody are changed to produce a variant CDR sequence, such as an affinity variant, wherein the variant CDR sequence retains target antigen binding activity.

[0100] "Camelid-derived" - In certain embodiments, the antibodies of the present invention comprise framework amino acid sequences and / or CDR amino acid sequences of conventional antibodies derived from camelids or VHH antibodies produced by active immunization of camelids. However, the antibodies of the present invention comprising amino acid sequences of camelid origin can be engineered to comprise framework and / or constant region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelid mammalian species. For example, human or non-human primate framework regions, heavy chain portions, constant regions and / or hinge portions may be included in TGF-β1 antibodies. In one embodiment, one or more non-camelid amino acids may be present in the framework region of the "camelid-derived" antibody, for example, the camelid framework amino acid sequence may contain one or more amino acid mutations in which corresponding human or non-human primate amino acid residues are present. In addition, camelid-derived VH and VL domains or humanized variants thereof can be linked to the constant domains of human antibodies to produce chimeric molecules, as described elsewhere herein.

[0101] "Conservative amino acid substitution" - A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide may be replaced by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids may be replaced by a string that is structurally similar but differs in order and / or composition of the side chain family members.

[0102] "Heavy chain portion" - As used herein, the term "heavy chain portion" includes an amino acid sequence derived from an immunoglobulin heavy chain constant domain. The polypeptide comprising the heavy chain portion includes at least one of the following: a CH1 domain, a hinge (e.g., upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment of the present invention may include the Fc portion of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, the antibody or antigen-binding fragment of the present invention may lack at least a portion of a constant domain (e.g., all or part of a CH2 domain). In certain embodiments, at least one, preferably all constant domains are derived from a human immunoglobulin heavy chain. For example, in a preferred embodiment, the heavy chain portion includes a fully human hinge domain. In other preferred embodiments, the heavy chain portion includes a fully human Fc portion (e.g., hinge, CH2, and CH3 domain sequences from a human immunoglobulin).

[0103] In certain embodiments, the constitutive constant domains of the heavy chain portion are from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide may include a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain comprising portions of different immunoglobulin molecules. For example, a hinge may include a first portion from an IgG1 molecule and a second portion from an IgG3 or IgG4 molecule. As described above, it is understood by those of ordinary skill in the art that the constant domains of the heavy chain portion may be modified so that their amino acid sequences are different from naturally occurring (wild-type) immunoglobulin molecules. That is, the polypeptides of the present invention disclosed herein may include changes or modifications to one or more heavy chain constant domains (CH1, hinge, CH2 or CH3) and / or light chain constant region domains (CL). Exemplary modifications include adding, deleting or replacing one or more amino acids in one or more domains.

[0104] "Chimeric" - A "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are bound together in the fusion polypeptide, or they may normally exist in the same protein but are arranged in a new arrangement in the fusion polypeptide. Chimeric proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in a desired relationship. Exemplary chimeric antibodies of the invention include fusion proteins comprising camelid-derived VH and VL domains, or humanized variants thereof, fused to the constant domains of a human antibody (e.g., human IgG1, IgG2, IgG3, or IgG4).

[0105] "Synthetic" - The term "synthetic" as used herein with respect to polypeptides includes polypeptides that include an amino acid sequence that does not occur naturally. For example, a non-naturally occurring polypeptide is a modified form of a naturally occurring polypeptide (e.g., includes a mutation, such as an addition, substitution, or deletion) or includes a first amino acid sequence (which may or may not occur naturally) linked in a linear amino acid sequence to a second amino acid sequence (which may or may not occur naturally) to which it is not naturally linked in nature.

[0106] "Engineered" - As used herein, the term "engineered" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or some combination of these techniques). Preferably, the antibodies of the invention are engineered, including, for example, humanized and / or chimeric antibodies, as well as antibodies engineered to improve one or more properties (e.g., antigen binding, stability / half-life, or effector function).

[0107] "Modified antibody" - As used herein, the term "modified antibody" includes synthetic forms of antibodies that have been altered so as not to exist naturally, for example, antibodies comprising at least two heavy chain portions but not two complete heavy chains (e.g., domain-deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been altered to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules linked to scFv molecules, etc. scFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. In addition, the term "modified antibody" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen). In another embodiment, the modified antibody of the present invention is a fusion protein comprising at least one heavy chain portion lacking a CH2 domain and comprising a binding domain of a polypeptide comprising a binding portion of one member of a receptor ligand pair.

[0108] The term "modified antibody" may also be used herein to refer to amino acid sequence variants of the antibodies of the invention as defined structurally herein. One of ordinary skill in the art will appreciate that antibodies may be modified to produce variant antibodies having altered amino acid sequences compared to the antibody from which they were derived. For example, nucleotide or amino acid substitutions may be made resulting in conservative substitutions or changes in "non-essential" amino acid residues (e.g., in CDRs and / or framework residues). Amino acid substitutions may include replacement of one or more amino acids with naturally occurring or non-natural amino acids.

[0109] "Humanizing substitutions" - As used herein, the term "humanizing substitutions" refers to amino acid substitutions in which the amino acid residue at a specific position in the VH or VL domain of an antibody (e.g., a TGF-β1 antibody of camelid origin) is replaced with an amino acid residue at the equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be a VH or VL domain encoded by a human germline. Humanizing substitutions may be made in the framework regions and / or CDRs of an antibody as defined herein.

[0110] "Humanized variant" - The term "humanized variant" as used herein refers to a variant antibody that contains one or more "humanizing substitutions" compared to a reference antibody, wherein a portion of the reference antibody (e.g., a VH domain and / or a VL domain containing at least one CDR, or a portion thereof) has an amino acid derived from a non-human species, and the "humanizing substitutions" occur within the amino acid sequence derived from the non-human species.

[0111] "Germline variant" - The term "germline variant" is used herein to specifically refer to "humanized variants", wherein "humanizing substitutions" result in one or more amino acid residues present at a specific position in a non-human antibody VH or VL domain being replaced by an amino acid residue at the equivalent position in a reference human VH or VL domain encoded by a human germline. Typically, for any given "germline variant", the replacement amino acid residues substituted into the germline variant are all or predominantly from a single human germline-encoded VH or VL domain. The terms "humanized variant" and "germline variant" are often used interchangeably herein. The introduction of one or more "humanizing substitutions" into a camelid-derived (e.g., llama-derived) VH or VL domain results in the generation of a "humanized variant" of a camelid (llama)-derived VH or VL domain. If the substituted amino acid residues are predominantly or entirely derived from a single human germline-encoded VH or VL domain sequence, the result may be a "human germline variant" of a camelid (llama)-derived VH or VL domain.

[0112] "Affinity variant" - The term "affinity variant" as used herein refers to a variant antibody that exhibits one or more changes in an amino acid sequence compared to a reference antibody, wherein the affinity variant exhibits an altered affinity for a target antigen compared to the reference antibody. For example, compared to a reference TGF-β antibody, an affinity variant will exhibit an altered affinity for TGF-β1. Preferably, the affinity variant will exhibit an improved affinity for the target antigen compared to the reference antibody. Affinity variants typically exhibit one or more changes in the amino acid sequence in the CDR compared to the reference antibody. Such substitutions may result in the original amino acid present at a given position in the CDR being replaced by a different amino acid residue, which may be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue. Amino acid substitutions may be conservative or non-conservative.

[0113] "High human homology" - An antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) can be considered to have high human homology if the VH domain and VL domain taken together exhibit at least 90% amino acid sequence identity with the closest matching human germline VH and VL sequences. Antibodies with high human homology can include antibodies comprising VH and VL domains of natural non-human antibodies that exhibit sufficiently high % sequence identity with human germline sequences, including, for example, antibodies comprising VH and VL domains of conventional antibodies of camelids, as well as engineered variants of such antibodies, particularly humanized or germlined variants, and "fully human" antibodies.

[0114] In one embodiment, the VH domain of an antibody with high human homology may exhibit 80% or more amino acid sequence identity or sequence homology with one or more human VH domains across the framework regions FR1, FR2, FR3 and FR4. In other embodiments, the amino acid sequence identity or sequence homology between the VH domain of a polypeptide of the invention and the closest matching human germline VH domain sequence may be 85% or more, 90% or more, 95% or more, 97% or more, or up to 99% or even 100%. VH domain variants with high human homology may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to the original non-human VH domain sequence from which they are derived.

[0115] In one embodiment, a VH domain of an antibody with high human homology may contain one or more (eg, 1 to 10) amino acid sequence mismatches across the framework regions FR1, FR2, FR3, and FR4 compared to the closest matching human VH sequence.

[0116] In another embodiment, the VL domain of an antibody with high human homology can exhibit 80% or more sequence identity or sequence homology with one or more human VL domains across framework regions FR1, FR2, FR3 and FR4. In other embodiments, the amino acid sequence identity or sequence homology between the VL domain of a polypeptide of the invention and the closest matching human germline VL domain sequence can be 85% or more, 90% or more, 95% or more, 97% or more, or up to 99% or even 100%. VL domain variants with high human homology can have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to the original non-human VL domain sequence from which they are derived.

[0117] In one embodiment, a VL domain of an antibody with high human homology may contain one or more (eg, 1 to 10) amino acid sequence mismatches across the framework regions FR1, FR2, FR3, and FR4 compared to the closest matching human VL sequence.

[0118] B. TGF-β1 activating antibodies that bind latent TGF-β1

[0119] Activation of TGF-β1

[0120] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to latent TGF-β1, wherein the antibody or antigen-binding fragment thereof activates TGF-β1. Therefore, the antibodies and antigen-binding fragments described herein may be referred to as any of the following: "activating TGF-β1 antibodies"; "TGF-β1 activating antibodies"; "anti-latent TGF-β1 activating antibodies"; or "anti-latent TGF-β1 antibodies that activate TGF-β1 release".

[0121] As explained elsewhere herein, latent TGF-β1 consists of a TGF-β1 homodimer in which two LAP regions of the TGF-β protein have been cleaved from the mature TGF-β1 cytokine region at the C-terminus. Two N-terminal LAP polypeptides remain covalently associated with the C-terminal mature TGF-β cytokine, thereby preventing TGF-β1 from binding to its receptor complex and initiating downstream signaling cascades. The antibodies and antigen-binding fragments described herein are capable of binding to the latent TGF-β1 complex and releasing the mature TGF-β1 cytokine from the inhibition mediated by the LAP region. This may involve the physical dissociation of the LAP polypeptide from the mature TGF-β1 cytokine. Alternatively, the antibodies or antigen-binding fragments described herein can bind to the latent TGF-β1, causing the latent complex to deform, so that the mature TGF-β1 cytokine is exposed and can bind to the TGF-β receptor complex. The mature TGF-β1 form that is not inhibited by LAP and can bind to the TGF-β receptor complex is referred to herein as "active TGF-β1".

[0122] As used herein, "activation of TGF-β1" means the conversion of latent TGF-β1 into active TGF-β1. This activation can be measured by a variety of methods. These methods include, but are not limited to, in vitro methods such as Western blotting, ELISA, TGF-β1 reporter gene assays, and TGF-β1 activity assays.

[0123] In certain embodiments, TGF-β1 activation is measured by exposing cells expressing the TGF-β receptor complex to a combination of latent TGF-β1 and an antibody or antigen-binding fragment described herein and detecting an increase in the phosphorylation state of the TGF-β receptor complex. Alternatively or additionally, TGF-β1 activation can be measured by detecting an increase in phosphorylation of an R-Smad protein (e.g., Smad-2). The phosphorylation state of the TGF-β receptor complex and / or the R-Smad protein (e.g., Smad-2) can be measured using any suitable technique, including but not limited to Western blot and ELISA. The phosphorylation level observed when the cell is exposed to a combination of latent TGF-β1 and an antibody or antigen-binding fragment of the present invention can be compared with one or more controls. For example, cells expressing the TGF-β receptor complex can be exposed to a range of active TGF-β1 concentrations, so that the phosphorylation state of the TGF-β receptor complex and / or the R-Smad protein (e.g., Smad-2) can be measured in the presence of a known concentration of active TGF-β1. Thus, the level of phosphorylation seen when an antibody or antigen-binding fragment of the invention binds to latent TGF-β1 can be compared to the activation seen in the presence of a standard concentration of active TGF-β1.

[0124] In certain embodiments, TGF-β1 activation can be measured using an in vitro assay in which changes in gene expression serve as a readout of TGF-β1 activation. Such assays include, but are not limited to, TGF-β1 reporter gene assays, such as the reporter gene assays described in the Examples below. TGF-β1 reporter gene assays typically involve the use of a TGF-β1-responsive reporter gene construct in which a TGF-β1-responsive element (e.g., found in the promoter region of a TGF-β1-responsive gene) is linked to a reporter gene such that activation of TGF-β1 results in increased reporter gene expression. The TGF-β1-responsive element can be a Smad complex binding element, such as one comprising (CAGA) 12 or by (CAGA) 12 The nucleotide sequence consisting of (as described in Dennler et al., EMBO J. 1998; 17: 3091-3100).

[0125] The reporter gene can be any protein encoding gene, and its expression can be measured and / or quantified. Preferred reporter genes include, but are not limited to, reporter genes encoding GFP and luciferase. The TGF-β1 reporter gene construct exemplified herein combines a luciferase reporter gene, so that an increase in luminescence can be used as a measure of TGF-β1 activation.

[0126] A complete TGF-β1 reporter gene assay may include the following steps: (i) engineering cells to carry a TGF-β1 reporter gene construct as described herein; (ii) exposing the cells to a combination of latent TGF-β1 and one or more TGF-β1 activating antibodies or antigen-binding fragments; (iii) measuring the expression of the reporter gene. As exemplified herein, cells carrying the TGF-β1 reporter gene may also be engineered to express latent TGF-β1 protein. In certain embodiments, the cells used in the TGF-β1 reporter gene assay are engineered to express both latent TGF-β1 and GARP, so that the latent TGF-β1 is bound to the cell surface. Controls may be included in an assay (e.g., as described herein) to control the transfection efficiency relative to the TGF-β1 reporter gene construct.

[0127] As described above for assays based on measuring phosphorylation levels, the activity levels seen in a TGF-β1 reporter assay can be compared to one or more controls. For example, cells carrying a TGF-β1 reporter construct can be exposed to one or more concentrations of active TGF-β1, so that the readout of the TGF-β1 reporter construct can be normalized for one or more known concentrations of active TGF-β1. Thus, the activation of the TGF-β1 reporter construct seen when the antibody or antigen-binding fragment of the invention binds to latent TGF-β1 can be compared to the activation seen in the presence of a standard concentration of active TGF-β1.

[0128] Alternatively or additionally to the above, activation mediated by the antibodies and antigen-binding fragments described herein can be measured relative to activation mediated under standard or physiological conditions found in vitro or in vivo. As described elsewhere herein, latent TGF-β1 can be activated via a variety of mechanisms, including binding of the "RGD" motif within LAP to integrins, binding to thrombospondin, and by cleavage by proteases. Given that binding of latent TGF-β1 to αVβ6 and αVβ8 integrins is well established as a physiological mechanism by which latent TGF-β1 is activated in vivo, activation in the presence of the antibodies or antigen-binding fragments described herein can be measured in vitro relative to activation mediated by αVβ6 and / or αVβ8 integrins. Activation levels equal to or greater than that seen with integrins can classify antibodies as "TGF-β1 activating antibodies".

[0129] The antibodies or antigen-binding fragments of the present invention can activate TGF-β1 by binding to an uncomplexed form of latent TGF-β1 (i.e., when latent TGF-β1 is not bound to a regulatory protein such as LTBP or GARP). Alternatively or additionally, the antibodies or antigen-binding fragments of the present invention can activate TGF-β1 by binding to latent TGF-β1 when latent TGF-β1 is complexed with one or more other proteins. In certain embodiments, the antibodies or antigen-binding fragments of the present invention activate TGF-β1 when latent TGF-β1 is complexed with GARP. As described elsewhere herein, GARP is a transmembrane protein expressed by certain cell types, including regulatory T cells (Tregs), B lymphocytes, megakaryocytes, platelets, endothelial cells, and fibroblasts. Antibodies or antigen-binding fragments capable of activating TGF-β1 by binding to latent TGF-β1 complexed with GARP can result in local activation of TGF-β1 in vivo, i.e., near cell types expressing GARP. In certain preferred embodiments, when latent TGF-β1 binds to the surface of Treg, the antibody or antigen-binding fragment described herein activates TGF-β1. In certain embodiments, when latent TGF-β1 binds to the surface of megakaryocytes, platelets and / or endothelial cells, the antibody or antigen-binding fragment described herein activates TGF-β1. In certain embodiments, when latent TGF-β1 binds to the surface of B lymphocytes, fibroblasts, mesenchymal cells and / or hepatic stellate cells, the antibody or antigen-binding fragment described herein activates TGF-β1.

[0130] Potential TGF-β1 may be associated or bound to the cell surface via proteins other than GARP, for example by binding to LTBP or other non-peptide molecules. In certain embodiments, the antibodies or antigen-binding fragments described herein activate TGF-β1 associated or bound to the surface of one or more cell types selected from the group consisting of myeloid cells, lymphoid cells, hematopoietic cells, and cancer cells at precancerous stages.

[0131] The activating TGF-β1 antibody or antigen-binding fragment of the present invention can bind to human latent TGF-β1 and / or murine latent TGF-β1. The sequence of human latent TGF-β1 is represented by SEQ ID NO:1, and the sequence of murine latent TGF-β1 is represented by SEQ ID NO:5. The activating TGF-β1 antibodies and antigen-binding fragments described herein generally bind to human latent TGF-β1 or murine latent TGF-β1, that is, they do not cross-react between these two species. The activating TGF-β1 antibody or antigen-binding fragment of the present invention can exhibit binding specificity to TGF-β1, that is, the antibody or antigen-binding fragment binds to TGF-β1, but does not bind to other isoforms (specifically TGF-β2 and TGF-β3).

[0132] Epitope

[0133] The activating TGF-β1 antibodies or antigen-binding fragments of the present invention can be characterized by their unique binding epitopes within latent TGF-β1.

[0134] In some embodiments, the binding of amino acids of a TGF-β1 antibody or antigen-binding fragment to an epitope within a potential TGF-β1 refers to physical interactions known in the art, including covalent bonding or hydrogen bonding.

[0135] In some embodiments, the interfacial engagement of amino acids of a TGF-β1 antibody or antigen-binding fragment with an epitope within latent TGF-β1 refers to non-physical interactions that contribute to activation of latent TGF-β1.

[0136] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope comprising one or more amino acids from a region of TGF-β1 consisting of amino acids 87-222, wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins represented by SEQ ID NOs: 1 and 5, respectively. In certain embodiments, the epitope comprises two amino acids from within this region.

[0137] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope comprising one or more amino acids from a region of TGF-β1 consisting of amino acids 87-135, wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins represented by SEQ ID NOs: 1 and 5, respectively. In certain embodiments, the epitope comprises two amino acids from within this region.

[0138] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope comprising one or more amino acids from a region of TGF-β1 consisting of amino acids 136-200, wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins represented by SEQ ID NOs: 1 and 5, respectively. In certain embodiments, the epitope comprises two amino acids from within this region.

[0139] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to an epitope comprising one or more amino acids from a region of TGF-β1 consisting of amino acids 201-222, wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins represented by SEQ ID NOs: 1 and 5, respectively. In certain embodiments, the epitope comprises two amino acids from within this region.

[0140] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from a region of TGF-β1 consisting of amino acids 87-222, wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins represented by SEQ ID NOs: 1 and 5, respectively. In certain embodiments, the conformational epitope requires two amino acids from within this region.

[0141] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from a region of TGF-β1 consisting of amino acids 87-135 (wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins as shown in SEQ ID NOs: 1 and 5, respectively). In certain embodiments, the conformational epitope requires two amino acids from within this region.

[0142] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from a region of TGF-β1 consisting of amino acids 136-200, wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins represented by SEQ ID NOs: 1 and 5, respectively. In certain embodiments, the conformational epitope requires two amino acids from within this region.

[0143] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that requires one or more amino acids from a region of TGF-β1 consisting of amino acids 201-222, wherein these amino acid positions are defined according to the full-length sequences of human and mouse TGF-β1 proteins represented by SEQ ID NOs: 1 and 5, respectively. In certain embodiments, the conformational epitope requires two amino acids from within this region.

[0144] The region of TGF-β1 consisting of amino acids 87-222 differs between human and mouse TGF-β1 proteins as follows:

[0145] Human latent TGF-β1: (aa 87-222)

[0146] RVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMF FNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNR LLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAH(SEQ IDNO:82)

[0147] Murine Potential TGF-β1: (aa 87-222)

[0148] RVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMF FNTSDLREAVPEPVLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNR LLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAH (SEQ ID NO: 83).

[0149] The region of TGF-β1 consisting of amino acids 87-135 differs between human and mouse TGF-β1 proteins as follows:

[0150] Human latent TGF-β1: (aa 87-135)

[0151] RVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMF F(SEQ ID NO:84)

[0152] Murine Potential TGF-β1: (aa 87-135)

[0153] RVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMF F (SEQ ID NO:85).

[0154] The region of TGF-β1 consisting of amino acids 136-200 differs between human and mouse TGF-β1 proteins as follows:

[0155] Human latent TGF-β1: (aa 136-200)

[0156] NTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDV(SEQ ID NO:86)

[0157] Murine Potential TGF-β1: (aa 136-200)

[0158] NTSDLREAVPEPVLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDV (SEQ ID NO:87).

[0159] The region of TGF-β1 consisting of amino acids 201-222 is located within the LAP region; more specifically, it corresponds to amino acids 172-193 of the LAP region (represented by SEQ ID NOs: 3 and 7, respectively - human and murine). This region is located near the dimerization interface of the latent TGF-β1 protein (see Shi et al., Nature 2011; 474: 343-49). The "latency loop" or "latency lasso," i.e., the LAP region that is most tightly associated with mature TGF-β1 within the latent complex (see Shi et al., supra) is located at amino acid positions 59-73 of the full-length TGF-β1 protein (see SEQ ID NOs: 1 and 5) or at amino acid positions 30-44 of the LAP region (see SEQ ID NOs: 3 and 7). It is therefore noteworthy that in certain embodiments, the TGF-β1 activating antibodies or antigen-binding fragments described herein bind to an epitope comprising amino acids from the LAP region distal to the latency loop or latency lasso. However, these antibodies or antigen-binding fragments are able to activate TGF-β1 by alleviating LAP-mediated inhibition.

[0160] The region of TGF-β1 consisting of amino acids 201-222 differs between the human and mouse TGF-β1 proteins as follows:

[0161] Human Potential TGF-β1: (aa 201-222)TGVVRQWLSRGGEIEGFRLSAH (SEQ ID NO: 80)

[0162] Murine potential TGF-β1: (aa 201-222)TGVVRQWLNQGDGIQGFRFSAH (SEQ ID NO:81).

[0163] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope comprising amino acid residues or a conformational epitope that requires amino acid residues E119, K123, S138, R141, S209, R210, G212, E213, I214 and / or E215 of the full-length TGF-β1 protein represented by SEQ ID NO:1.

[0164] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 forms an interface with a region or conformational epitope comprising amino acid residues, which requires amino acid residues D122, F124, F134, E142, P145, E146, P147, K173, Y174, S175, N176 and / or N177 of the full-length TGF-β1 protein represented by SEQ ID NO:1.

[0165] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope comprising amino acids E119 and / or K123 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. For antibodies or antigen-binding fragments that bind to an epitope comprising E119 and / or K123 of human latent TGF-β1, in certain embodiments, the epitope does not comprise another amino acid residue from the region consisting of amino acids 87-135 of human latent TGF-β1.

[0166] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to a conformational epitope that requires amino acid residues E119 and / or K123 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. For antibodies or antigen-binding fragments that bind to a conformational epitope that requires E119 and / or K123 of human latent TGF-β1, in certain embodiments, the epitope does not require another amino acid residue from the region consisting of amino acids 87-135 of human latent TGF-β1.

[0167] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 forms an interface with a region comprising amino acid residues or a conformational epitope within the region consisting of amino acids 87-135 of human latent TGF-β1, wherein the conformational epitope requires amino acid residues D122, F124 and / or F134 of the full-length TGF-β1 protein represented by SEQ ID NO:1.

[0168] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope comprising amino acids S138 and / or R141 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. For antibodies or antigen-binding fragments that bind to an epitope comprising S138 and / or R141 of human latent TGF-β1, in certain embodiments, the epitope does not comprise another amino acid residue from the region consisting of amino acids 136-200 of human latent TGF-β1.

[0169] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to a conformational epitope that requires amino acid residues S138 and / or R141 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. For antibodies or antigen-binding fragments that bind to a conformational epitope that requires S138 and / or R141 of human latent TGF-β1, in certain embodiments, the epitope does not require another amino acid residue from the region consisting of amino acids 136-200 of human latent TGF-β1.

[0170] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 forms an interface with a region comprising amino acid residues or a conformational epitope requiring amino acid residues E142, P145, E146, P147, K173, Y174, S175, N176 and / or N177 of the full-length TGF-β1 protein represented by SEQ ID NO:1 within the region consisting of amino acids 136-200 of human latent TGF-β1.

[0171] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to an epitope comprising amino acid residues S209, R210, G212, E213, I214 and / or E215 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. For antibodies or antigen-binding fragments that bind to an epitope comprising S209, R210, G212, E213, I214 and / or E215 of human latent TGF-β1, in certain embodiments, the epitope does not comprise another amino acid residue from the region consisting of amino acids 201-222 of human latent TGF-β1. In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment does not bind to R205 and / or R210 and / or E213.

[0172] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 binds to a conformational epitope that requires amino acid residues S209, R210, G212, E213, I214, and / or E215 of the full-length TGF-β1 protein represented by SEQ ID NO: 1. For antibodies or antigen-binding fragments that bind to a conformational epitope that requires S209, R210, G212, E213, I214, and / or E215 of human latent TGF-β1, in certain embodiments, the epitope does not require another amino acid residue from the region consisting of amino acids 201-222 of human latent TGF-β1. In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that does not require R205 and / or R210 and / or E213.

[0173] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to human latent TGF-β1 forms an interface with a region comprising amino acid residues or with a conformational epitope requiring amino acid residues L208 and / or G211 of the full-length TGF-β1 protein represented by SEQ ID NO: 1 within the region consisting of amino acids 201-222 of human latent TGF-β1. In one embodiment, the region comprising amino acid residues or the conformational epitope requiring amino acid residues L208 and / or G211 of the full-length TGF-β1 protein represented by SEQ ID NO: 1 is essential for the activity of the activating TGF-β1 antibody or antigen-binding fragment thereof.

[0174] In certain embodiments, the activating amino acid residues of the human latent TGF-β1 protein represented by SEQ ID NO: 1 required for activating the activity of the TGF-β1 antibody or antigen-binding fragment include G212 and E215. In certain embodiments, the amino acid residues of the human latent TGF-β1 protein represented by SEQ ID NO: 1 required for activating the activity of the TGF-β1 antibody or antigen-binding fragment include W207, L208, G211, G212, I214, E215 and F217.

[0175] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to murine latent TGF-β1 binds to an epitope comprising amino acid residues or to a conformational epitope requiring amino acid residues S138, R141, P145, E146, S175, L208, N209 and / or D212 of the full-length TGF-β1 protein represented by SEQ ID NO:5.

[0176] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to murine latent TGF-β1 binds to an epitope comprising amino acid residues S138, R141, P145, E146 and / or S175 of the full-length TGF-β1 protein represented by SEQ ID NO: 5. For antibodies or antigen-binding fragments that bind to an epitope comprising S138, R141, P145, E146 and / or S175 of murine latent TGF-β1, in certain embodiments, the epitope does not comprise another amino acid residue from the region consisting of amino acids 136-200 of murine latent TGF-β1. In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment does not bind to G211 and / or F217.

[0177] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to murine latent TGF-β1 binds to a conformational epitope that requires amino acids S138, R141, P145, E146, and / or S175 of the full-length TGF-β1 protein represented by SEQ ID NO: 5. For antibodies or antigen-binding fragments that bind to conformational epitopes S138, R141, P145, E146, and / or S175 of murine latent TGF-β1, in certain embodiments, the epitope does not require another amino acid residue from the region consisting of amino acids 136-200 of murine latent TGF-β1. In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that does not require G211 and / or F217.

[0178] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to murine latent TGF-β1 forms an interface with a region comprising amino acid residues or a conformational epitope requiring amino acid residues E142, P147, P148, L149, Y174 and / or N176 of the full-length TGF-β1 protein represented by SEQ ID NO:5 within the region consisting of amino acids 136-200 of murine latent TGF-β1.

[0179] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to murine latent TGF-β1 binds to an epitope comprising amino acid residues L208, N209, and D212 of the full-length TGF-β1 protein represented by SEQ ID NO: 5. For antibodies or antigen-binding fragments that bind to an epitope of L208, N209, and D212 of murine latent TGF-β1, in certain embodiments, the epitope does not comprise another amino acid residue from the region consisting of amino acids 201-222 of murine latent TGF-β1. In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment does not bind to G211 and / or F217.

[0180] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to murine latent TGF-β1 binds to a conformational epitope that requires amino acid residues L208, N209, and D212 of the full-length TGF-β1 protein represented by SEQ ID NO: 5. For antibodies or antigen-binding fragments that bind to a conformational epitope that requires L208, N209, and D212 of murine latent TGF-β1, in certain embodiments, the epitope does not require another amino acid residue from the region consisting of amino acids 201-222 of murine latent TGF-β1. In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment binds to a conformational epitope that does not require G211 and / or F217.

[0181] In certain embodiments, the activating TGF-β1 antibody or antigen-binding fragment that binds to murine latent TGF-β1 forms an interface with a region comprising amino acid residues or a conformational epitope requiring amino acid residues L208 and G211 of the full-length TGF-β1 protein represented by SEQ ID NO:5 within the region consisting of amino acids 201-222 of murine latent TGF-β1.

[0182] In certain embodiments, the amino acid residues of the murine latent TGF-β1 protein represented by SEQ ID NO:5 required for activating the activity of the TGF-β1 antibody or antigen-binding fragment include L208 and / or D212. In certain embodiments, the amino acid residues of the murine latent TGF-β1 protein represented by SEQ ID NO:5 required for activating the activity of the TGF-β1 antibody or antigen-binding fragment include L208 and / or D212.

[0183] Antibody and antigen-binding fragment formats

[0184] The TGF-β1 antibodies and antigen-binding fragments of the present invention may be in any suitable form. The term "antibody" herein is used in the broadest sense, including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), VHH antibodies, as long as they exhibit appropriate immunospecificity for potential TGF-β1.

[0185] The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the individual antibodies constituting the group are identical except for possible natural mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed to a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations (which typically include different antibodies directed to different determinants (epitopes) on an antigen), each monoclonal antibody is directed only to a single determinant or epitope on an antigen." Antibody fragment" or "antigen binding fragment" comprises a part of a full-length antibody, typically its antigen binding or variable domains. Antibody fragments are described elsewhere herein, and examples of antibody fragments include Fab, Fab', F(ab')2, bispecific Fab and Fv fragments, double antibodies, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv) and multispecific antibodies formed by antibody fragments (see Holliger and Hudson, Nature Biotechnol.23:1126-36 (2005), the contents of which are incorporated herein by reference).

[0186] In certain embodiments, the TGF-β1 antibody or antigen-binding fragment of the present invention is multivalent, preferably bivalent. In certain embodiments, the TGF-β1 antibody or antigen-binding fragment is bivalent, has two identical antigen-binding sites, and immunoreacts with the same epitope on the potential TGF-β1. Such bivalent antibodies and antigen-binding fragments include, but are not limited to, conventional heterotetrameric Y-shaped antibodies, F(ab')2 fragments, and (scFv)2 fragments.

[0187] The TGF-β1 antibodies and antigen-binding fragments described herein are intended for human therapeutic use and are therefore typically immunoglobulins of the IgA, IgD, IgE, IgG, IgM type, often of the IgG type, in which case they may belong to any of the four subclasses IgG1, IgG2a and b, IgG3 or IgG4. In a preferred embodiment, the TGF-β1 antibody is an IgG antibody. Particularly preferred are IgG1 antibodies. Monoclonal antibodies are preferred because they are highly specific, being directed against a single antigenic site.

[0188] TGF-β1 antibodies and antigen-binding fragments may exhibit a high degree of human homology as defined elsewhere herein. Such antibody molecules with a high degree of human homology may include antibodies comprising the VH and VL domains of natural non-human antibodies, which exhibit sufficiently high % sequence identity with human germline sequences. In certain embodiments, the antibody or antigen-binding fragment thereof is a humanized or germline variant of a non-human antibody.

[0189] In certain embodiments, the TGF-β1 antibodies and antigen-binding fragments described herein are of camelid origin. The camelid-derived antibodies may be heavy chain-only antibodies, i.e., VHH antibodies, or may be conventional heterotetrameric antibodies. In a preferred embodiment, the TGF-β1 and antigen-binding fragments are derived from heterotetrameric antibodies of camelids. In a further preferred embodiment, the TGF-β1 antibody is a VHH antibody or is derived from a VHH antibody.

[0190] For example, TGF-β1 antibodies and antigen-binding fragments can be selected from immune libraries obtained by a method comprising the step of immunizing a camelid with a target of interest. Camelids are immunized with a target protein or polypeptide fragment thereof, or with an mRNA molecule or cDNA molecule expressing the protein or polypeptide fragment thereof. Methods for generating antibodies in camelid species and selecting antibodies against preferred targets from camelid immune libraries are described, for example, in International Patent Application No. WO2010 / 001251 (which is incorporated herein by reference).

[0191] In certain embodiments, the TGF-β1 antibodies and antigen-binding fragments may be of camelid origin, in that they comprise at least one hypervariable (HV) loop or complementarity determining region obtained from a VH domain or VL domain of a species of the family Camelidae. In particular, the TGF-β1 antibodies and antigen-binding fragments may comprise VH and / or VL domains or CDRs thereof obtained by active immunization of hybrid camelids (e.g., llamas) with latent TGF-β1.

[0192] In this context, the term "derived from" implies a structural relationship, i.e., the HV or CDR of the antibody comprises an amino acid sequence originally encoded by a camelid immunoglobulin gene (or a minor variant thereof). However, this does not necessarily imply a specific relationship in terms of the production process used to prepare the antibody or antigen-binding fragment thereof.

[0193] The camelid-derived antibody or antigen-binding fragment thereof may be derived from any species of Camelidae, including but not limited to llama, dromedary, alpaca, vicuna, guanaco or camel.

[0194] Antibody molecules comprising VH and VL domains or their CDRs of camelid origin are typically recombinantly expressed polypeptides and may be chimeric polypeptides. The term "chimeric polypeptide" refers to an artificial (non-naturally occurring) polypeptide produced by juxtaposing two or more peptide fragments that would otherwise not be contiguous. This definition includes "species" chimeric polypeptides produced by juxtaposing peptide fragments encoded by two or more species (e.g., camelid and human).

[0195] In certain embodiments, the entire VH domain and / or the entire VL domain may be obtained from a species of the Camelidae family. The Camelid-derived VH domain and / or the Camelid-derived VL domain may then be subjected to protein engineering, wherein one or more amino acid substitutions, insertions or deletions are introduced into the Camelid amino acid sequence. These engineered changes preferably include amino acid substitutions relative to the Camelid sequence. Such changes include "humanization" or "germlining", wherein one or more amino acid residues in a Camelid-encoded VH or VL domain are replaced by equivalent residues from a homologous human-encoded VH or VL domain.

[0196] Isolated camelid VH and VL domains obtained by active immunization of camelids (e.g., llamas) with latent TGF-β1 can be used as the basis for engineering TGF-β1 antibodies and antigen-binding fragments according to the present invention. Starting with intact camelid VH and VL domains, one or more amino acid substitutions, insertions or deletions can be engineered that deviate from the starting camelid sequence. In certain embodiments, such substitutions, insertions or deletions may be present in the framework regions of the VH domain and / or the VL domain.

[0197] In other embodiments, a "chimeric" antibody molecule is provided, comprising VH and VL domains of camelid origin (or engineered variants thereof) and one or more constant domains from a non-camelid antibody, such as a human-encoded constant domain (or an engineered variant thereof). In such embodiments, it is preferred that both the VH domain and the VL domain are from the same camelid species, for example both the VH and VL may be from Lama glama, or both the VH and VL may be from Lama pacos (before the introduction of engineered amino acid sequence variations). In such embodiments, both the VH and VL domains may be derived from a single animal, particularly a single animal that has been actively immunized with the antigen of interest.

[0198] As an alternative to engineering changes to the primary amino acid sequence of the camelid VH and / or VL domains, individual camelid-derived hypervariable loops or CDRs, or combinations thereof, may be isolated from the camelid VH / VL domains and transferred into an alternative (i.e. non-camelid) framework (e.g. a human VH / VL framework) by CDR grafting.

[0199] In non-limiting embodiments, the TGF-β1 antibody may comprise a CH1 domain and / or a CL domain (from the heavy chain and the light chain, respectively), the amino acid sequence of which is completely or substantially human. For antibody molecules intended for human therapeutic use, the entire constant region of the antibody or at least a portion thereof generally has a completely or substantially human amino acid sequence. Therefore, one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may be completely or substantially human in terms of their amino acid sequence. The CH1 domain, hinge region, CH2 domain, CH3 domain and / or CL domain (and / or CH4 domain, if present) may be derived from a human antibody, preferably a human IgG antibody, more preferably a human IgG1 antibody of subtype IgG1, IgG2, IgG3 or IgG4.

[0200] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may all have completely or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% with a human constant region. The term "human amino acid sequence" in this context refers to an amino acid sequence encoded by a human immunoglobulin gene, including germline, rearranged, and somatically mutated genes. The present invention also contemplates polypeptides comprising a constant domain of a "human" sequence that has been altered by one or more amino acid additions, deletions, or substitutions relative to a human sequence, except for those embodiments that explicitly require the presence of a "fully human" hinge region.

[0201] The TGF-β1 antibody may have one or more amino acid substitutions, insertions or deletions in the constant region of the heavy chain and / or light chain, particularly in the Fc region. Amino acid substitutions may result in the replacement of the substituted amino acid by a different naturally occurring amino acid or a non-natural or modified amino acid. Other structural modifications are also allowed, such as changes in glycosylation patterns (e.g., by adding or deleting N or O-linked glycosylation sites).

[0202] TGF-β1 antibody can be modified in the Fc region to increase the binding affinity to neonatal receptor FcRn. The increased binding affinity is measurable at acidic pH (e.g., from about pH 5.5 to about pH 6.0). The increased binding affinity is also measurable at neutral pH (e.g., from about pH 6.9 to about pH 7.4). "Increased binding affinity" refers to an increase in binding affinity to FcRn relative to an unmodified Fc region. Typically, an unmodified Fc region will have the wild-type amino acid sequence of human IgG1, IgG2, IgG3 or IgG4. In such embodiments, the increased FcRn binding affinity of an antibody molecule with a modified Fc region is measured relative to the binding affinity of wild-type IgG1, IgG2, IgG3 or IgG4 to FcRn.

[0203] In certain embodiments, one or more amino acid residues in the Fc region may be substituted with a different amino acid to increase binding to FcRn. Several Fc substitutions have been reported that increase FcRn binding, thereby improving the pharmacokinetics of antibodies. For example, such substitutions are reported in the following literature: Zalevsky et al. (2010) Nat. Biotechnol. 28 (2): 157-9; Hinton et al. (2006) J Immunol. 176: 346-356; Yeung et al. (2009) J Immunol. 182: 7663-7671; Presta LG. (2008) Curr. Op. Immunol. 20: 460-470; and Vaccaro et al. (2005) Nat. Biotechnol. 23 (10): 1283-88, the contents of which are incorporated herein in their entirety.

[0204] In certain embodiments, the TGF-β1 antibody comprises a modified human IgG Fc domain comprising or consisting of amino acid substitutions H433K and N434F, wherein the Fc domain numbering is consistent with EU numbering. In further embodiments, the TGF-β1 antibody described herein comprises a modified human IgG Fc domain comprising or consisting of amino acid substitutions M252Y, S254T, T256E, H433K and N434F, wherein the Fc domain numbering is consistent with EU numbering.

[0205] In certain embodiments, the TGF-β1 antibody comprises a modified human IgG Fc domain consisting of up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, or up to 20 substitutions relative to the corresponding wild-type IgG sequence.

[0206] In certain embodiments, the Fc region can be engineered to have no effector function. In certain embodiments, the antibodies of the present invention can have an Fc region derived from a naturally occurring IgG subtype (e.g., IgG4) with reduced effector function. The Fc region derived from IgG4 can be further modified to increase therapeutic utility, such as by introducing modifications that minimize the exchange of arms between IgG4 molecules in vivo. The Fc region derived from IgG4 can be modified to include an S228P substitution.

[0207] In certain embodiments, one, two or more amino acid substitutions are introduced into the IgG constant region Fc region to change the effector function of the polypeptide. For example, one or more of the following mutations can be made in the constant region of an antibody described herein: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; L235A substitution; C236 deletion; P238A substitution; S239D substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution, numbered according to the EU numbering system.

[0208] In certain embodiments, one or more of the following mutations are introduced into the constant region of an antibody described herein: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; L235A substitution; C236 deletion; P238A substitution; S239D substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution, and such antibodies with altered, reduced or even abolished effector function are attractive in the context of the present invention.

[0209] In certain embodiments, antibodies and antigen-binding fragments are modified in terms of glycosylation. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of the antibody for the target antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. Such aglycosylation can increase the affinity of the antibody for the antigen.

[0210] C. Exemplary Activating TGF-β1 Antibodies Binding Latent TGF-β1

[0211] The present invention provides exemplary TGF-β1 antibodies and antigen-binding fragments thereof. The exemplary TGF-β1 antibodies and antigen-binding fragments of the present invention can be specifically defined according to their structural features, as described below.

[0212] In some embodiments, the present invention includes antibodies or antigen-binding fragments thereof, wherein the CDRs have an amino acid sequence that is at least about 90% identical to a SEQ ID NO described herein, or an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97%, at least 99%, at least 99.5%, or at least 99.9% identical.

[0213] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to human latent TGF-β1 is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein

[0214] The variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 11 [GGSIDLTYGMDY] or a sequence variant thereof;

[0215] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 10 [AIRWNGVTTYYAESMKG] or a sequence variant thereof;

[0216] The variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 9 [DYTMN] or a sequence variant thereof;

[0217] The variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 26 [ALYMYTGSNNGRV] or a sequence variant thereof;

[0218] The variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 25 [NTVARHS] or a sequence variant thereof;

[0219] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 24 [GLSSGSVTRNNYPD] or a sequence variant thereof; and

[0220] wherein the sequence variant comprises one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in said sequence.

[0221] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to human latent TGF-β1 is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein

[0222] The variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 14 [SRYGVVITTTYFGGVDY] or a sequence variant thereof;

[0223] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 13 [AINTGGGITYYTDSVKG] or a sequence variant thereof;

[0224] The variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 12 [HYWMY] or a sequence variant thereof;

[0225] The variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 29 [LSYESSGYAV] or a sequence variant thereof;

[0226] The variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 28 [GNSNRPS] or a sequence variant thereof;

[0227] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 27 [QGGNFGSYYAS] or a sequence variant thereof; and

[0228] Wherein the sequence variant comprises one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in said sequence.

[0229] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to human latent TGF-β1 is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein

[0230] The variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 57 [YITRAQLMGAWDY] or a sequence variant thereof;

[0231] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 56 [RITNSGGTAFYTDSVEG] or a sequence variant thereof;

[0232] The variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 55 [SYWMD] or a sequence variant thereof;

[0233] The variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 60 [AQSTYYPYD] or a sequence variant thereof;

[0234] The variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 59 [QVSNRAS] or a sequence variant thereof;

[0235] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 58 [KASQSLVHTDGKTYLS] or a sequence variant thereof; and

[0236] wherein the sequence variant comprises one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in said sequence.

[0237] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to human latent TGF-β1 is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein:

[0238] The variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 11 [GGSIDLTYGMDY] or a sequence variant thereof, or SEQ ID NO: 14 [SRYGVVITTTYFGGVDY] or a sequence variant thereof, or SEQ ID NO: 57 [YITRAQLMGAWDY] or a sequence variant thereof;

[0239] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 10 [AIRWNGVTTYYAESMKG] or a sequence variant thereof, or SEQ ID NO: 13 [AINTGGGITYYTDSVKG] or a sequence variant thereof, or SEQ ID NO: 56 [RITNSGGTAFYTDSVEG] or a sequence variant thereof;

[0240] The variable heavy chain CDR1 sequence comprises or consists of: SEQ ID NO: 9 [DYTMN] or a sequence variant thereof, or SEQ ID NO: 12 [HYWMY] or a sequence variant thereof, or SEQ ID NO: 55 [SYWMD] or a sequence variant thereof;

[0241] The variable light chain CDR3 sequence comprises or consists of: SEQ ID NO: 26 [ALYMYTGSNNGRV] or a sequence variant thereof, or SEQ ID NO: 29 [LSYESSGYAV] or a sequence variant thereof, or SEQ ID NO: 60 [AQSTYYPYD] or a sequence variant thereof;

[0242] The variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 25 [NTVARHS] or a sequence variant thereof, or SEQ ID NO: 28 [GNSNRPS] or a sequence variant thereof, or SEQ ID NO: 59 [QVSNRAS] or a sequence variant thereof;

[0243] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 24 [GLSSGSVTRNNYPD] or a sequence variant thereof, or SEQ ID NO: 27 [QGGNFGSYYAS] or a sequence variant thereof, or SEQ ID NO: 58 [KASQSLVHTDGKTYLS] or a sequence variant thereof; and

[0244] Wherein the sequence variant comprises one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in said sequence.

[0245] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to human latent TGF-β1 is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein

[0246] The variable heavy chain CDR3 sequence comprises or consists of: SEQ ID NO: 11 [GGSIDLTYGMDY] or a sequence variant thereof, or SEQ ID NO: 14 [SRYGVVITTTYFGGVDY] or a sequence variant thereof;

[0247] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 10 [AIRWNGVTTYYAESMKG] or a sequence variant thereof, or SEQ ID NO: 13 [AINTGGGITYYTDSVKG] or a sequence variant thereof;

[0248] The variable heavy chain CDR1 sequence comprises or consists of: SEQ ID NO: 9 [DYTMN] or a sequence variant thereof, or SEQ ID NO: 12 [HYWMY] or a sequence variant thereof;

[0249] The variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 26 [ALYMYTGSNNGRV] or a sequence variant thereof, or SEQ ID NO: 29 [LSYESSGYAV] or a sequence variant thereof;

[0250] The variable light chain CDR2 sequence comprises or consists of: SEQ ID NO: 25 [NTVARHS] or a sequence variant thereof, or SEQ ID NO: 28 [GNSNRPS] or a sequence variant thereof;

[0251] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 24 [GLSSGSVTRNNYPD] or a sequence variant thereof, or SEQ ID NO: 27 [QGGNFGSYYAS or a sequence variant thereof; and

[0252] Wherein the sequence variant comprises one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in said sequence.

[0253] In certain embodiments, antibodies and antigen-binding fragments that bind to human latent TGF-β1 comprise or consist of a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein VH comprises or consists of an amino acid sequence of SEQ ID NO: 37, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and VL comprises or consists of an amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0254] In certain embodiments, antibodies and antigen-binding fragments that bind to human latent TGF-β1 comprise or consist of a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein VH comprises or consists of an amino acid sequence of SEQ ID NO: 39, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and VL comprises or consists of an amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0255] In certain embodiments, antibodies and antigen-binding fragments that bind to human latent TGF-β1 comprise or consist of a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein VH comprises or consists of an amino acid sequence of SEQ ID NO: 61, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and VL comprises or consists of an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0256] In certain embodiments, the antibodies and antigen-binding fragments that bind to human latent TGF-β are selected from antibodies and antigen-binding fragments comprising or consisting of a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the following:

[0257] (i) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO: 37, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto;

[0258] (ii) a VH comprising or consisting of the amino acid sequence of SEQ ID NO:39, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; and

[0259] (iii) a VH comprising, or consisting of, the amino acid sequence of SEQ ID NO:61, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, the amino acid sequence of SEQ ID NO:62, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0260] In certain embodiments, the antibodies and antigen-binding fragments that bind to human latent TGF-β are selected from antibodies and antigen-binding fragments comprising or consisting of a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the following:

[0261] (i) a VH comprising or consisting of an amino acid sequence of SEQ ID NO:37, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising or consisting of an amino acid sequence of SEQ ID NO:38, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; and

[0262] (ii) a VH comprising or consisting of an amino acid sequence of SEQ ID NO:39, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising or consisting of an amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0263] For embodiments in which the domains of an antibody or antigen-binding fragment are defined by a particular percent sequence identity to a reference sequence, the VH and / or VL domains may retain CDR sequences that are identical to those present in the reference sequence, such that variations exist only within the framework regions.

[0264] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to murine latent TGF-β is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein

[0265] The variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 17 [DRIRSSYEYDS] or a sequence variant thereof;

[0266] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 16 [HISAAGISRYYADAVKG] or a sequence variant thereof;

[0267] The variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 15 [NYGVH] or a sequence variant thereof;

[0268] The variable light chain CDR3 sequence comprises or consists of NO: 32[GCYDSSLSTGYV] or a sequence variant thereof;

[0269] The variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 31 [NANNRAS] or a sequence variant thereof;

[0270] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 30 [TGSSSNIGGGYYLS] or a sequence variant thereof; and

[0271] wherein the sequence variant comprises one, two or three amino acid changes (e.g., substitutions (e.g., conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in the sequence.

[0272] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to murine latent TGF-β1 is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein

[0273] The variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 20 [GLVSSGLNGMDY] or a sequence variant thereof;

[0274] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 19 [AITPGGTATYYANSVKG] or a sequence variant thereof;

[0275] The variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 18 [RSGMA] or a sequence variant thereof;

[0276] The variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 34 [GCYDDSLNTYV] or a sequence variant thereof;

[0277] The variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 33 [NTNNRAS] or a sequence variant thereof;

[0278] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 30 [TGSSSNIGGGYYLS] or a sequence variant thereof; and

[0279] wherein the sequence variant comprises one, two or three amino acid changes (e.g., substitutions (e.g., conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in the sequence.

[0280] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to murine latent TGF-β1 is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein

[0281] The variable heavy chain CDR3 sequence comprises or consists of SEQ ID NO: 23 [DLNSRSYKGMGD] or a sequence variant thereof;

[0282] The variable heavy chain CDR2 sequence comprises or consists of SEQ ID NO: 22 [GTSIGGGSTWYADSVKG] or a sequence variant thereof;

[0283] The variable heavy chain CDR1 sequence comprises or consists of SEQ ID NO: 21 [SYYMY] or a sequence variant thereof;

[0284] The variable light chain CDR3 sequence comprises or consists of SEQ ID NO: 36 [GCYDSSLSSVV] or a sequence variant thereof;

[0285] The variable light chain CDR2 sequence comprises or consists of SEQ ID NO: 31 [NANNRAS] or a sequence variant thereof or a sequence variant thereof;

[0286] The variable light chain CDR1 sequence comprises or consists of SEQ ID NO: 35 [TGSSSNIGGGYYLN] or a sequence variant thereof; and

[0287] Wherein the sequence variant comprises one, two or three amino acid changes (eg, substitutions (eg, conservative substitutions, humanizing substitutions, affinity variant substitutions), additions or deletions) in the sequence.

[0288] In certain embodiments, the antibodies and antigen-binding fragments that bind to human latent TGF-β1 are selected from antibodies and antigen-binding fragments comprising or consisting of a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the variable heavy chain domain (VH) and the variable light chain domain (VL) are selected from:

[0289] (i) a VH comprising, or consisting of, an amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence of SEQ ID NO:42, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto;

[0290] (ii) a VH comprising or consisting of an amino acid sequence of SEQ ID NO:43, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising or consisting of an amino acid sequence of SEQ ID NO:44, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto; and

[0291] (iii) a VH comprising or consisting of an amino acid sequence of SEQ ID NO:45, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising or consisting of an amino acid sequence of SEQ ID NO:46, or an amino acid sequence at least 90%, 95%, 97%, 98% or 99% identical thereto.

[0292] For embodiments in which a domain of an antibody or antigen-binding fragment is defined by a particular percent sequence identity to a reference sequence, the VH and / or VL domains may retain CDR sequences that are identical to those present in the reference sequence, such that variations exist only within the framework regions.

[0293] The present invention also provides antibodies or antigen-binding fragments thereof that bind to the same epitope as the TGF-β1 antibodies exemplified herein.

[0294] The exemplary TGF-β1 antibodies and antigen-binding fragments described herein are TGF-β1 activating antibodies, i.e., they bind to latent TGF-β1 and release mature TGF-β1 cytokines from LAP-mediated inhibition. The activation properties of the exemplary TGF-β1 antibodies and antigen-binding fragments can be measured by any of the methods described in Section B above. In addition, the exemplary TGF-β1 antibodies and antigen-binding fragments described herein can take the form of any antibody according to the embodiments described in Section B above.

[0295] In preferred embodiments, the exemplary TGF-β1 antibody molecules having the above-described CDR sequences exhibit a high degree of human homology, such as being humanized or germline variants of the antibodies or antigen-binding fragments thereof from which the CDR sequences are derived.

[0296] In non-limiting embodiments, exemplary TGF-β1 antibodies and antigen-binding fragments thereof having CDR, VH and / or VL sequences described herein may comprise a CH1 domain and / or a CL domain (from the heavy chain and light chain, respectively) whose amino acid sequences are completely or substantially human. For antibody molecules for human therapeutic use, the entire constant region of the antibody, or at least a portion thereof, typically has a completely or substantially human amino acid sequence. Thus, one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may be completely or substantially human in terms of their amino acid sequence.

[0297] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may all have completely or substantially human amino acid sequences. In the case of a constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97% or at least 99% with a human constant region. The term "human amino acid sequence" in this context refers to an amino acid sequence encoded by a human immunoglobulin gene, including germline, rearranged and somatically mutated genes. The present invention also contemplates polypeptides comprising a constant domain of a "human" sequence that has been altered by one or more amino acid additions, deletions or substitutions relative to a human sequence, except for those embodiments that explicitly require the presence of a "fully human" hinge region. Any exemplary Fc region modification described herein may be incorporated into a TGF-β1 antibody having the above-described CDR and / or VH / VL domain sequences. In certain embodiments, a TGF-β1 antibody having the above CDR and / or VH / VL domain sequences comprises a modified human IgG Fc domain comprising or consisting of amino acid substitutions H433K and N434F, wherein the Fc domain numbering conforms to EU numbering. In certain embodiments, a TGF-β1 antibody having the above CDR and / or VH / VL domain sequences comprises a modified human IgG Fc domain comprising or consisting of amino acid substitutions M252Y, S254T, T256E, H433K and N434F.

[0298] Unless otherwise specified in the application, the % sequence identity between two amino acid sequences can be determined by comparing the two sequences aligned in an optimal manner, wherein the amino acid sequence to be compared may contain additions or deletions relative to the reference sequence in order to achieve optimal alignment between the two sequences. The percent identity is calculated by determining the number of identical positions where the amino acid residues are identical between the two sequences, dividing the number of identical positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent identity between the two sequences. For example, the BLAST program "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250) can be used, which is available on the website http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, and the parameters used are the parameters given by default (in particular, the parameters "open gap penalty": 5, and "extension gap penalty": 2; the selected matrix is, for example, the matrix "BLOSUM 62" proposed by the program), and the percentage of identity between the two sequences to be compared is calculated directly by the program.

[0299] For embodiments in which a domain of an antibody or antigen-binding fragment is defined by a particular percent sequence identity to a reference sequence, the VH, VHH and / or VL domains may retain CDR sequences that are identical to those present in the reference sequence, such that variations exist only within the framework regions.

[0300] D. Nucleic acid encoding TGF-β1 activating antibody

[0301] The present invention also provides a polynucleotide molecule or nucleic acid encoding the TGF-β1 antibody or antigen-binding fragment of the present invention. Provided are polynucleotide molecules or nucleic acids encoding full-length antibodies, as well as polynucleotide molecules or nucleic acids encoding fragments or single chains of the antibodies, such as the VH, VHH and / or VL domains of the TGF-β1 antibodies described herein. Also provided are expression vectors containing the nucleotide sequence of the present invention, the nucleotide sequence being operably linked to a regulatory sequence that allows the antibody or its fragment to be expressed in a host cell or a cell-free expression system; and host cells or cell-free expression systems containing the expression vectors.

[0302] Polynucleotide molecules encoding antibodies or antigen-binding fragments of the present invention include, for example, recombinant DNA molecules. The terms "nucleic acid," "polynucleotide," or "polynucleotide molecule" used herein are interchangeable and refer to any single-stranded or double-stranded DNA or RNA molecule, or, if single-stranded, to a molecule of its complementary sequence. When discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule may be described herein in accordance with the normal convention of providing a sequence in the 5' to 3' direction. In some embodiments of the present invention, nucleic acids or polynucleotides are "isolated." The term, when applied to nucleic acid molecules, refers to nucleic acid molecules separated from sequences immediately adjacent to the naturally occurring genome of the organism from which it originates. For example, "isolated nucleic acid" may include DNA molecules inserted into a vector (e.g., a plasmid or viral vector), or DNA molecules integrated into the genomic DNA of a prokaryotic or eukaryotic cell or a non-human host organism. When applied to RNA, the term "isolated polynucleotide" refers primarily to RNA molecules encoded by isolated DNA molecules as defined above. Alternatively, the term may refer to RNA molecules purified / isolated from other nucleic acids associated with it in its natural state (i.e., in a cell or tissue). An isolated polynucleotide (DNA or RNA) may further refer to a molecule produced directly by a biological or synthetic process and separated from other components present during its production.

[0303] In order to recombinantly produce the antibodies of the present invention, recombinant polynucleotides encoding the antibodies or recombinant polynucleotides encoding different chains or domains can be prepared (using standard molecular biology techniques) and inserted into replicable vectors for expression in selected host cells or cell-free expression systems. Suitable host cells can be prokaryotes, yeasts or higher eukaryotic cells, particularly mammalian cells. Examples of useful mammalian host cell lines are: monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or subcloned 293 cells grown in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL 8287) or NS0 (HPA Culture Collection No. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2), and DSM's PERC-6 cell line. Expression vectors suitable for each of these host cells are also generally known in the art.

[0304] It should be noted that the term "host cell" generally refers to a cultured cell line. An intact human into which an expression vector encoding an antigen-binding polypeptide of the invention has been introduced is specifically excluded from the definition of "host cell".

[0305] E. Antibody Production

[0306] On the other hand, the present invention also provides a method for producing the antibody or antigen-binding fragment of the present invention, which comprises culturing a host cell (or a cell-free expression system) containing a polynucleotide or nucleic acid (e.g., an expression vector) encoding the antibody or antigen-binding fragment under conditions that allow expression of the antibody or fragment, and recovering the expressed antibody. This recombinant expression process can be used for large-scale antibody production, including the activating TGF-β1 antibody of the present invention, including monoclonal antibodies for human therapeutic use. Suitable vectors, cell lines, and production processes for large-scale production of recombinant antibodies suitable for in vivo therapeutic use are generally available in the art and are well known to those skilled in the art.

[0307] F. Pharmaceutical Compositions

[0308] The scope of the present invention includes pharmaceutical compositions containing one or a combination of the antibodies or antigen-binding fragments described herein, formulated together with one or more pharmaceutically acceptable carriers or excipients. Such compositions may include a combination of (i.e., two or more different) activating TGF-β1 antibodies. Techniques for formulating monoclonal antibodies for human therapeutic use are well known in the art and have been reviewed in, for example, Wang et al., Journal of Pharmaceutical Sciences 2007; 96: 1-26, the contents of which are incorporated herein in their entirety.

[0309] Pharmaceutically acceptable excipients that can be used to formulate the composition include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (e.g., sodium carboxymethyl cellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0310] In certain embodiments, the composition is formulated for administration to a subject via any suitable route of administration, including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal administration.

[0311] G. Treatment methods

[0312] As reported in the Examples herein, TGF-β1 activating antibodies according to the invention are able to improve the outcome of a mouse model of graft versus host disease (GvHD). Without being bound by theory, this effect is likely due to the role of TGF-β1 as an immunosuppressive cytokine in regulating immune responses.

[0313] As can be seen from the above, the activated TGF-β1 antibodies and antigen-binding fragments described herein can be used in treatment methods. Therefore, the present invention provides a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein, which is used as a medicament. The TGF-β1 antibodies and antigen-binding fragments used as medicaments are generally formulated as pharmaceutical compositions. Importantly, all embodiments described above related to the activated TGF-β1 antibodies and antigen-binding fragments are also applicable to the methods described herein.

[0314] Provided herein is a method for inhibiting a pathological immune response in a subject in need, wherein the method comprises administering to the subject a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein. Also provided is a method for preventing or treating an autoimmune disease in a subject in need, wherein the method comprises administering to the subject a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein. Also provided is a method for preventing or treating an alloimmune disease in a subject in need, wherein the method comprises administering to the subject a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein. The subject is typically a human subject. As used herein, the term "therapeutically effective amount" is intended to mean the amount or dose of a TGF-β1 antibody or antigen-binding fragment sufficient to produce a therapeutic effect, such as the amount or dose required to eliminate or at least alleviate symptoms associated with a disease or condition. The appropriate amount or dose may be determined by a physician as needed. For example, the dose may be adjusted according to factors such as the size or weight of the subject to be treated, the age of the subject to be treated, the general physical condition of the subject to be treated, the condition to be treated, and the route of administration.

[0315] The present invention also provides a TGF-β1 activating antibody or an antigen binding fragment thereof according to the present invention, which is used to inhibit a pathological immune response in a subject in need thereof. Also provided is a TGF-β1 activating antibody or an antigen binding fragment thereof according to the present invention, which is used to prevent or treat an autoimmune disease in a subject in need thereof. Also provided is a TGF-β1 activating antibody or an antigen binding fragment thereof according to the present invention, which is used to prevent or treat an alloimmune disease in a subject in need thereof. The subject is typically a human subject.

[0316] Autoimmune and alloimmune diseases or conditions to be prevented or treated according to the present invention include, but are not limited to, inflammatory bowel disease (IBD), multiple sclerosis (MS), graft versus host disease (GVHD), allogeneic transplant rejection, antibody-mediated allogeneic transplant rejection (AMR), allogeneic islet transplant rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), ANCA vasculitis, adrenal autoimmune disease, autoimmune encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis, and Orchitis, immune thrombocytopenia (ITP or idiopathic thrombocytopenic purpura or idiopathic thrombocytopenic purpura or immune-mediated thrombocytopenia), autoimmune urticaria, Behcet's disease, bullous pemphigoid (BP), cardiomyopathy, Castleman syndrome, celiac dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, COVID-19-mediated postural tachycardia syndrome (POTS), Crohn's disease, delayed graft function after renal transplantation, dilated cardiomyopathy, discoid lupus, acquired Epidermolysis bullosa, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome (GBS), Goodpasture syndrome, Hashimoto's thyroiditis, hemophilia A, hemolytic disease of the fetus and newborn (HDFN), idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, IgA neuropathy, IgM polyneuropathy, juvenile arthritis, Kawasaki disease, lichen planus, lichen sclerosus, lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, membranous neuropathy, membranous nephropathy, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, transplant type 1 diabetes, multifocal motor neuropathy (MMN), Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), myasthenia gravis (MG), generalized myasthenia gravis (gMG), ocular myasthenia gravis (OMG), myositis, neuromyelitis optica (NMO), paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris (PV), pemphigus foliaceus (PF), pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis (DM), necrotizing autoimmune myopathy (NAM), antisynthetase syndrome (ASyS), primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Raynaud's phenomenon, Reiter's syndrome,Rheumatoid arthritis (RA), sarcoidosis, scleroderma, Sjögren's syndrome, solid organ transplant rejection, stiff-man syndrome, Takayasu's arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, thyroid eye disease, ulcerative colitis, uveitis, warm autoimmune hemolytic anemia (wAIHA), dermatitis herpetiformis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegener's granulomatosis.

[0317] In addition to its important role as an immunosuppressive cytokine in regulating immune responses, TGF-β1 also plays an important role in cancer development, and strategies targeting TGF-β1 are being used in cancer therapy (Fonseca Teixeira et al., Front. Cell. Dev. Biol. 2020; 8: 605). In the context of cancer, TGF-β1 plays a complex dual role, with both tumor-promoting and tumor-suppressing activity. Especially in the early stages of cancer development, TGF-β1 is believed to play an important immunosuppressive role by inhibiting the proliferation of epithelial cells and precancerous cells. In the later stages of cancer progression, tumors can acquire resistance to TGF-β1-mediated cell inhibition.

[0318] In view of the key tumor suppressor function of TGF-β1, especially in the early stages of cancer development, provided herein is a method for preventing or treating a tumor disease in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein. Also provided is a TGF-β1 activating antibody or antigen-binding fragment thereof according to the present invention, which is used to prevent or treat a tumor disease in a subject in need thereof. In the context of preventing or treating a tumor disease, the TGF-β1 activating antibody or antigen-binding fragment described herein can activate latent TGF-β1 associated with the surface of tumor cells (e.g., cells of benign tumors) in the pre-malignant tumor stage.

[0319] As used herein, a method of "preventing" a disease or condition refers to preventing the onset of a disease, preventing the worsening of symptoms, preventing the progression of a disease or condition, or reducing the risk of a subject developing a disease or condition. As used herein, a method of "treating" a disease or condition refers to curing the disease or condition and / or alleviating or eliminating symptoms associated with the disease or condition, thereby alleviating the patient's suffering.

[0320] For clinical applications, in certain embodiments, the TGF-β1 antibody described elsewhere herein is administered to a subject at one or more doses of about 0.1 mg / kg body weight to about 20 mg / kg body weight. In certain embodiments, the TGF-β1 antibody is administered to a subject at a dose of about 0.1 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the TGF-β1 antibody is administered to a subject at a dose of about 0.5 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the TGF-β1 antibody is administered to a subject at a dose of about 1 mg / kg body weight to about 10 mg / kg body weight.

[0321] H. Kit

[0322] Any of the TGF-β1 activating antibodies or antigen-binding fragments described herein, or pharmaceutical compositions comprising the same, may be packaged as a kit and optionally include instructions for use.

[0323] In some embodiments, the kit is used to implement the treatment methods described herein. In some embodiments, the kit is used to prevent or treat alloimmune and / or autoimmune diseases or conditions.

[0324] I. Others

[0325] The present invention also provides an antibody or antigen-binding fragment according to the first or second aspect of the invention or a pharmaceutical composition according to the invention for use in the manufacture of a medicament.

[0326] Provided herein is a method of activating TGF-β1 in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein.

[0327] Also provided is a method of increasing TGF-β1 activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein. As used herein, the term "increasing TGF-β1 activity" means that the TGF-β1 activity is increased by at least 1.2 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times compared to the TGF-β1 activity in the absence of a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to the present invention. The activity of TGF-β1 can be assessed by any method known in the art.

[0328] Another object is to provide a method for down-regulating the immunity of a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to any aspect or embodiment described elsewhere herein. As used herein, the term "down-regulating immunity" refers to reducing the immunity of the subject by at least 1%, at least 1.5%, at least 2%, at least 2.5% compared to the immunity of the subject in the absence of a therapeutically effective amount of a TGF-β1 activating antibody or antigen-binding fragment according to the present invention. The immunity of the subject can be assessed by any method known in the art.

[0329] Compared with the TGF-β1 antibodies of the prior art, the antibodies of the present invention may present the following advantages:

[0330] - In some embodiments, the antibodies of the present invention exhibit the ability to activate TGF-β1 or increase the activity of TGF-β1;

[0331] - In some embodiments, the antibodies of the invention exhibit increased affinity for TGF-β1 compared to TGF-β1 antibodies of the prior art;

[0332] - In some embodiments, the antibodies of the invention exhibit increased avidity for TGF-β1 compared to TGF-β1 antibodies of the prior art. Example

[0333] The invention will be further understood with reference to the following non-limiting examples.

[0334] Example 1: 14 monoclonal antibodies that activate latent TGF-β1

[0335] A total of 134 monoclonal antibodies (mAbs) against human or mouse GARP, the GARP:(latent)TGF-β1 complex, or latent TGF-β1 were screened in a TGF-β reporter gene assay for their ability to activate TGF-β1. HEK-293T cells, which do not express GARP and secrete very low levels of soluble latent TGF-β1, were co-transfected with i) plasmids encoding GARP and latent TGF-β1 to express high levels of surface GARP:TGF-β1, and ii) a reporter plasmid (pCAGA-12) encoding firefly luciferase under the control of a TGF-β1-responsive promoter and a control plasmid (pRL-TK) encoding Renilla luciferase under the control of a constitutive promoter as a control for transfection efficacy. Transfected cells were incubated in the presence of mAbs (20 μg / ml) or with serial dilutions of recombinant human active TGF-β1 (rhTGF-β1) as a positive control. Firefly and Renilla luciferase activities were measured 24 hours later. The ratio of firefly to Renilla luminescence measured in the absence of rhTGF-β1 or mAb was used as a reference negative signal. Firefly / Renilla ratios above the mean + 2SD of negative signals were considered positive signals for TGF-β1 activity. In these assays, 14 of the 134 mAbs tested activated latent TGF-β1 (see Figure 1 A and 1B). 14 TGF-β1 activating mAbs were directed against either human latent TGF-β1 (6 of 24 mAbs directed against llama-derived anti-human TGF-β1 (named LHT-1 to LHT-24)) or mouse latent TGF-β1 (8 of 11 mAbs directed against llama-derived anti-mouse TGF-β1 (named LMT-1 to LMT-3 and LMT-5 to LMT-12)). All activating mAbs were not directed against GARP and did not cross-react with human and mouse latent TGF-β1 ( Figure 1 A). Figure 1 B shows the results obtained using TGF-β1 activating mAb and some non-activating mAbs in a reporter gene assay (upper panel: LMT mAb; lower panel: LHT mAb). They are representative of at least 3 independent experiments. Figure 1 The shaded bar in B highlights the TGF-β1 activating mAbs. Dilutions of recombinant active human TGF-β1 (rhTGF-β1) were used as controls to monitor the detection of TGF-β1 activity. Potential TGF-β1 activating mAbs triggered signals equivalent to 40 to 320 pg / ml of rhTGF-β1. Several serial dilutions of TGF-β1 activating mAbs were tested in the reporter gene assay to determine the EC50, which ranged from 0.2 to 2.1 nM ( Figure 1 C).

[0336] Example 2: Antibodies against mouse or human latent TGF-β1 are used as bivalent, monospecific full-length mAbs or F(ab')2 It activates TGF-β1 when used as a fragment, but not when used as a monovalent Fab fragment

[0337] We tested the ability of four TGF-β1-activating LMT antibodies (i.e., LMT-5, -7, -9, and -10) to activate latent TGF-β1 when used in various formats in the reporter gene assay described above ( Figure 2 A). The four LMT-activating antibodies activated TGF-β1 when used as bivalent full-length mAbs or F(ab')2 fragments, but none of them activated TGF-β1 when used as monovalent Fab fragments ( Figure 2 A) We conclude that monospecific antibodies require bivalent binding to latent TGF-β1 in order to mediate TGF-β1 activation.

[0338] Example 3: TGF-β1 activating antibodies can activate latent TGF-β1 regardless of whether it is presented by GARP on the cell surface Potential TGF-β1

[0339] TGF-β1 activating mAbs were identified by screening in HEK-293 reporter cells overexpressing the transmembrane protein GARP and latent TGF-β1 to maximize the expression and presentation of latent TGF-β1 on the cell surface. In fact, during the production and processing of latent TGF-β1 in cells, GARP binds to the LAP part of latent TGF-β1 via a disulfide bond, increases the production of latent TGF-β1 by acting as a molecular chaperone, and ultimately presents and tethers latent TGF-β1 on the cell surface (Gauthy et al. PLoS One 2013; 8(9): e76186; Liénart et al. Science 2018; 362(6417): 952-956). We tested whether TGF-β1 activating mAbs can also activate latent TGF-β1 in the absence of GARP by reporter gene assays, in which HEK-293 cells were transfected with plasmids encoding latent TGF-β1, with or without plasmids encoding GARP ( Figure 2 B). In the presence of GARP, all three tested TGF-β1-activating mAbs (i.e., LMT-7, -9, and -10) induced TGF-β1 activity well above the background activity in reporter cells ( Figure 2 In the absence of GARP, two of the three mAbs (i.e., LMT-7 and -10) also induced TGF-β1 activity above background, although at lower levels than in the presence of GARP ( Figure 2 B, middle panel). The reduced activity may be due to lower levels of latent TGF-β1 bound to the cell surface in the absence of GARP. In reporter cells that were not transfected to express latent TGF-β1, none of the three mAbs induced TGF-β1 activity ( Figure 2B, right). This suggests that the TGF-β1-activating mAb preparation was not contaminated with active TGF-β1 that might have co-purified with the mAb. We performed ELISA (data not shown) and immunoblotting ( Figure 2 C) Confirmation of the absence of contamination of the mAb preparation by active TGF-β1.

[0340] Example 4: TGF-β1 activates LMT and unique epitope bound by LHT mAb

[0341] TGF-β1 activating mAbs bind either human or mouse TGF-β1, but not both simultaneously (non-cross-reacting mAbs). We took advantage of this observation to map the epitopes bound by human or mouse TGF-β1 activating mAbs, respectively. Sequence alignment of human and mouse TGF-β1 proteins showed that most of the amino acid differences are located between residues 87 and 278, a region that is the LAP and is located outside of the highly conserved so-called “latency lasso” ( Figure 3 A). We constructed four plasmids encoding HA-tagged mouse-human-mouse chimeric TGF-β1. In each chimera, the amino acids from a fragment of mouse LAP were replaced by the amino acids found in the corresponding fragment of human LAP. The mouse LAP fragment replaced by the corresponding human LAP in the four plasmids contained residues 87-135, 136-200, 201-222, or 223-278, respectively ( Figure 3 B) We co-transfected plasmids encoding GARP and chimeric TGF-β1 into HEK-293T cells to express the chimeric form of latent TGF-β1 on the cell surface and analyzed the binding of TGF-β1-activating and non-activating mAbs to the transfected cells by flow cytometry.

[0342] As expected, all LMT mAbs bound to cells expressing mouse TGF-β1 but not to cells expressing human TGF-β1 ( Figure 3 B). The loss of binding of any given LMT mAb to a particular mouse-human-mouse chimera compared to mouse TGF-β1 allowed the identification of the region of mouse TGF-β1 that was required for binding by that particular mAb. We observed that five activating LMT mAbs (LMT-5, -6, -7, -10, and -11) required region 136-200 for binding ( Figure 3 B). We can refine this binding requirement more precisely to residues 158-163 within region 136-200 (data not shown). The other three activating LMT mAbs (LMT-2, -9, and -12) require region 201-222 for binding ( Figure 3B). In contrast, the non-activating LMT-1 mAb required both regions 136-200 and 201-222 for binding (LMT-1), while the non-activating LMT-8 did not require any of the 4 regions tested in the chimera ( Figure 3 B).

[0343] Also as expected, all LHT mAbs bound to cells expressing human TGF-β1 but not to cells expressing mouse TGF-β1 ( Figure 3 B). Here, the gain of binding of any given LHT mAb to a particular mouse-human-mouse chimera as compared to mouse TGF-β1 determines the region required for binding by that particular mAb. We observed that 2 activating LHT mAbs (LHT-18 and -22) required region 201-222 for binding (i.e., the same region required for activating LMT-2, -9, and -12). The non-activating LHT-8 mAb also required region 201-222 for binding, while the activating LHT-15 mAb required region 87-135.

[0344] In summary, we observed that three activating LMT mAbs and two activating LHT mAbs required region 201-222, but not other regions, highlighting a common binding requirement for activating mAbs directed against mouse or human TGF-β1, respectively. Figure 3 LHT-8 (shown in C) and LHT-11 (not shown) also require this region but not the other regions for binding.

[0345] To further identify the amino acids in region 201-222 required for activated mAb binding, we constructed a series of plasmids, each encoding HA-tagged mouse or human TGF-β1 in which a single amino acid was replaced with alanine (A). A series of 20 Ala mutant plasmids covered 20 positions in region 201-222 in mouse TGF-β1, and another series of 20 plasmids covered 20 positions in human TGF-β1 ( Figure 3 C) To perform “Ala scanning” analysis, we co-transfected HEK-293T cells with plasmids encoding mouse or human GARP and individual Ala mutants of mouse or human TGF-β1 and analyzed the binding of TGF-β1-activating and non-activating mAbs using flow cytometry ( Figure 3 C).

[0346] When L at position 208 (L208) or D at position 212 (D212) was mutated to A, the mouse TGF-β1 activating mAbs LMT-2 and LMT-12 lost their ability to bind to mouse TGF-β1. Therefore, activation of LMT-2 and LMT-12 requires L208 and D212 for binding to mouse TGF-β1, and no other single residues are required. No other LMT or LHT mAbs (whether activating or non-activating) that bind to region 201-222 require L208 and D212, nor do they require other amino acids for binding. For example, the non-activating LMT-1 mAb requires L208, G211, D212, and F217, while the non-activating LMT-8 mAb does not require any of the 20 single residues in region 201-222. The requirement for L208 / D212 was unique to the TGF-β1-activating mAbs LMT-2 and LMT-12 and was not observed for non-activating mAbs that bound human or mouse TGF-β1.

[0347] When the G at position 212 (G212) or the E at position 215 (E215) was mutated to A, the human TGF-β1 activating mAb LHT-22 lost the ability to bind to human TGF-β1. Therefore, LHT-22 requires G212 and E215 for binding to human TGF-β1, but no other single residues. No other LMT or LHT mAb (either activating or inactive) that binds to region 201-222 requires G212 and E215, nor does it require other amino acids for binding. The inactive LHT-8 mAb requires E213 and E215, while the inactive LHT-11 requires R205 and R210. The requirement for G212 / E215 is unique to the activating mAb LHT-22, and this requirement is not observed for the inactive mAbs that bind to human or mouse TGF-β1.

[0348] Together, these observations suggest that the binding requirement to L208 / D212 in murine TGF-β1 or G212 / E215 in human TGF-β1 is unique to TGF-β1-activating mAbs.

[0349] We also examined whether LMT-12 and LHT-22 lost their ability to activate single A mutants in 293T reporter cells ( Figure 3 D). As expected, the mAbs lost the ability to activate the mutants to which they did not bind ( Figure 3C: L208A and D212A for LMT-12; G212A and E215A for LHT-22). But in addition, LHT-22 also lost the ability to activate the five single A mutants (W207A, L208A, G211A, I214A, and F217A) to which it did bind, as well as WT TGF-β1. We therefore identified five additional TGF-β1 residues required for LHT-22 activity.

[0350] In summary, using chimeras and individual A mutants, we identified residues required for mAb binding and activity ( Figure 3 B, C, D). To activate hTGF-β1, LHT-22 requires residues W207, L208, G211, G212, I214, E215, and F217 in LAP. To activate mTGF-β1, LMT-12 requires residues L208 and D212 in LAP. The LHT-22 and LMT-12 epitopes are very similar to each other and are not shared with the non-activating mAbs. Interestingly, all TGF-β1 activating mAbs appear to bind to a loop close to the LAP dimerization interface ( Figure 2 D) This interface has been described as being critical for TGF-β1 latency by binding LAP monomers together (Shi et al.).

[0351] Example 5: Treatment of mice with TGF-β1 activated LMT-10 and LMT-12 increased the incidence of graft-versus-host disease in the model Lifetime

[0352] Graft-versus-host disease (GvHD) is a life-threatening side effect of allogeneic hematopoietic stem cell transplantation, which is caused by the potent immunoreactivity of donor-derived T cells against allogeneic recipient cells. Due to its potent immunomodulatory properties, TGF-β1 can reduce the activity of donor-derived T cells against allogeneic recipient antigens. Therefore, we tested the therapeutic efficacy of TGF-β1-activating mAb to treat GvHD in mice. We used a GvHD mouse model in which C57BL / 6H-2 b / b Splenocytes from mice were injected intraperitoneally (ip) into F1(C57BL / 6x DBA2)H-2 b / d Donor T cells to allogeneic H-2 in F1 recipients d MHC molecule reactivity leads to the development of GvHD, which can be scored by monitoring weight loss, hunched posture, loss of mobility, and ruffled hair, and may lead to death 10-21 days after donor cell transplantation. b / b One day before spleen cell transplantation, F1 H-2 b / d Recipients were administered TGF-β1 activating mAb or isotype control (400 μg / mouse intraperitoneally weekly) ( Figure 4 A).

[0353] Mice that received TGF-β1 activated LMT-10 or LMT-12 had significantly improved survival compared to mice that received an isotype control antibody ( Figure 4 B). Median survival increased from 16 days in mice receiving isotype control to 27 days in mice treated with LMT-10. More than 50% of mice treated with LMT-12 even showed long-term survival (>30 days), precluding the estimation of median survival ( Figure 4 B). GvHD scores of individual mice treated with LMT-10 or LMT-12 showed slower disease progression or reduced disease severity, or still achieved complete GvHD regression compared to isotype controls ( Figure 4 C).

[0354] Example 6: LMT-12 activates latent TGF-β1 from GARP-independent sources in GvHD

[0355] As in Example 4 and Figure 2 As described above, TGF-β1-activating mAb activated latent TGF-β1 regardless of whether latent TGF-β1 was presented by GARP on the cell surface. Nevertheless, when latent TGF-β1 was presented by GARP in HEK-293 cells, activation by TGF-β1-activating mAb was completely inhibited in the presence of blocking anti-GARP mAb. Figure 5 As shown in A, the blocking anti-human GARP mAb LHG-10 inhibited human TGF-β1 activation by LHT-4, -15, -18, -20, -22, and -25 (left panel), and the blocking anti-mouse GARP:latent TGF-β1 complex mAb 58A2 inhibited murine TGF-β1 activation by LMT-12 (right panel). This observation provided an opportunity to measure whether TGF-β1-activating mAbs could still exert therapeutic activity in mouse GvHD in the presence of blocking anti-mouse GARP antibodies, indicating that TGF-β1-activating mAbs can activate latent TGF-β1 independently of latent TGF-β1 presentation by GARP-expressing cells in vivo.

[0356] As mentioned above, we will b / b Spleen cell transplantation into F1 H-2 b / d Recipient mice were treated with TGF-β1 alone to activate LMT-12 or in combination with the blocking anti-GARP:latent TGF-β1 complex mAb 58A2 ( Figure 5B). As expected, 58A2 alone had no therapeutic effect, whereas LMT-12 alone exerted a significant therapeutic effect in GvHD, increasing median survival, slowing disease progression, and reducing disease severity compared to an isotype control antibody. Interestingly, LMT-12 exerted the same therapeutic activity when used alone or in combination with 58A2 ( Figure 5 B). Therefore, we conclude that TGF-β1-activating mAbs can exert therapeutic activity by activating latent TGF-β1 produced in vivo by cells that do not express GARP. TGF-β1 activation and therapeutic activity of the activating mAbs are independent of context and independent of the GARP:TGF-β1 complex.

[0357] Example 7: Binding activity of TGF-β1 antibody

[0358] The binding properties of the activated anti-human and anti-mouse latent TGFβ1 antibodies binding to region 201-222 were tested by surface plasmon resonance (Biacore 3000, GE Healthcare). The CM5 chip was immobilized with approximately 500 RU of human latent TGFβ1 and mouse latent TGFβ1 in sodium acetate buffer pH 4.5 (GE Healthcare, #BR100350) using amine coupling and depletion HBS-EP buffer. The flow rate was set to 30 μl / min, using the "kinject" injection mode, regenerative injection of 10 mM glycine pH 1.5. The binding rate (k a ), dissociation rate (k d ) and affinity (K D ) was determined based on six concentration measurements of each antibody: 100 nM, 50 nM, 25 nM, 12 nM, 6 nM, and 3 nM. All clones tested were in the hIgG1 backbone, except LHT-18 which had a ScFv-Fc hIgG1 backbone. All clones demonstrated binding to immobilized latent TGFβ1, with LHT-18 binding to human latent TGFβ1 at a K of D The K value of LHT-22 for binding to human latent TGFβ1 is 66 nM. D The K value of the clone binding to mouse latent TGFβ1 was 2320 nM. D The values ​​ranged from 63 nM to 687 nM (Table 4).

[0359] Table 4: Affinity determination by SPR

[0360]

[0361]

[0362] NB = No Binding

[0363] Example 8: Specificity determination assay

[0364] The specificity of LHT-22 and LHT-18 for latent TGF-β1 (compared to latent TGF-β2 and latent TGF-β3) was tested by ELISA experiments for human latent TGF-β and flow cytometry for mouse latent TGF-β, both of which were performed in a single experiment.

[0365] ELISA was performed to ensure that LHT-22 specifically binds to human latent TGF-β1, but not to human latent TGF-β2 and human latent TGF-β3. Briefly, 96-well Maxisorp plates were coated with human latent TGF-β1, TGF-β2, or TGF-β3. Three different concentrations of LHT-22hIgG1 were then incubated, and bound antibodies were detected with anti-hIgG-HRP antibody. Antibodies with OD values ​​<0.1 were defined as non-binding antibodies. A negative control sample (Motavizumab) was also included. All concentrations of LHT-22 tested (4, 1, and 0.25 μg / mL) showed binding to human latent TGF-β1, but not to human latent TGF-β2 and latent TGF-β3 (Table 5).

[0366] Flow cytometry was used to ensure that LMT-12 specifically binds to mouse latent TGF-β1, but not to mouse latent TGF-β2 and mouse latent TGF-β3. In short, 293T cells were transfected with plasmids encoding HA-tagged TGF-β1, β2 or β3 and plasmids encoding GARP. They were then stained with LMT-12mIgG1 or anti-HA mIgG1 antibodies at specified concentrations, and then stained with goat anti-mouse antibodies (GAM-FITC) coupled to FITC as secondary antibodies. By using flow cytometry, as measured with a viability dye (Bioscience, cat.65-0865-14), the histograms were gated on live cells. All tested concentrations of LMT-12 bind to mouse latent TGF-β1, but not to mouse latent TGF-β2 and latent TGF-β3 ( Figure 6 ).

[0367] Table 5: Specificity of LHT-22 to human latent TGFβ1 by ELISA (OD 450nm value)

[0368]

[0369]

[0370]

[0371]

[0372] The scope of the present invention is not limited to the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. In addition, all embodiments described herein are considered to have broad applicability and can be combined with any and all other consistent embodiments as desired.

[0373] Incorporated by Reference

[0374] In the foregoing description and the following examples, various publications are cited, each of which is herein incorporated by reference in its entirety.

Claims

1. An antibody or an antigen-binding fragment thereof that binds to latent TGF-β1, wherein the antibody or the antigen-binding fragment thereof activates TGF-β1.

2. The antibody or antigen-binding fragment of claim 1, which activates TGF-β1 by releasing mature TGF-β1 cytokine from latent associated peptide (LAP)-mediated inhibition.

3. The antibody or antigen-binding fragment according to claim 1 or claim 2, which binds to an epitope comprising one or more amino acids from a region consisting of amino acids 87-222 of human or mouse latent TGF-β1, wherein human latent TGF-β1 is represented by SEQ ID NO: 1 and mouse latent TGF-β1 is represented by SEQ ID NO:

5.

4. The antibody or antigen-binding fragment according to claim 1 or claim 2, which binds to an epitope comprising one or more amino acids from a region consisting of amino acids 87-135 of human or mouse latent TGF-β1, wherein human latent TGF-β1 is represented by SEQ ID NO: 1 and mouse latent TGF-β1 is represented by SEQ ID NO:

5.

5. The antibody or antigen-binding fragment according to claim 1 or claim 2, which binds to an epitope comprising one or more amino acids from a region consisting of amino acids 136-200 of human or mouse latent TGF-β1, wherein human latent TGF-β1 is represented by SEQ ID NO: 1 and mouse latent TGF-β1 is represented by SEQ ID NO:

5.

6. The antibody or antigen-binding fragment according to claim 1 or claim 2, which binds to an epitope comprising one or more amino acids from a region consisting of amino acids 201-222 of human or mouse latent TGF-β1, wherein human latent TGF-β1 is represented by SEQ ID NO: 1 and mouse latent TGF-β1 is represented by SEQ ID NO:

5.

7. The antibody or antigen-binding fragment according to claims 3-6, which binds to human latent TGF-β1.

8. The antibody or antigen-binding fragment according to claim 7, which binds to an epitope comprising amino acid residues E119, K123, S138, R141, S209, R210, G212, E213, I214 and / or E215 of human latent TGF-β1, wherein human latent TGF-β1 is represented by SEQ ID NO:

1.

9. The antibody or antigen-binding fragment of claims 3-6, which binds to murine latent TGF-β1.

10. The antibody or antigen-binding fragment of claim 9, which binds to an epitope comprising amino acid residues S138, R141, P145, E146, S175, L208, N209 and / or D212 of murine latent TGF-β1, wherein murine latent TGF-β1 is represented by SEQ ID NO:

5.

11. The antibody or antigen-binding fragment of claim 10, which binds to an epitope comprising amino acid residues S138, R141, P145, E146, S175, L208, N209 and / or D212 of murine latent TGF-β1, wherein the epitope does not comprise another amino acid residue from the region consisting of amino acids 135-222 of murine latent TGF-β1, wherein murine latent TGF-β1 is represented by SEQ ID NO:

5.

12. The antibody or antigen-binding fragment of any one of claims 1-11, which activates TGF-β1 when latent TGF-β1 is complexed with GARP.

13. The antibody or antigen-binding fragment of any one of claims 1-12, which activates TGF-β1 when latent TGF-β1 binds to the surface of one or more cell types selected from the group consisting of regulatory T cells (Tregs); megakaryocytes; platelets; B lymphocytes; endothelial cells; fibroblasts; mesenchymal cells; and hepatic stellate cells.

14. The antibody or antigen-binding fragment of any one of claims 1-11, which activates latent TGF-β1 associated with one or more cell types selected from the group consisting of: myeloid cells; lymphoid cells; hematopoietic cells; and cancer cells at a precancerous stage.

15. The antibody or antigen-binding fragment of any one of claims 1-14, wherein the antibody or antigen-binding fragment is bivalent.

16. The antibody according to claim 15, wherein the antibody is IgG, preferably IgG1.

17. The antibody or antigen-binding fragment according to claim 15 or claim 16, wherein the antibody or antigen-binding fragment comprises a CH1 domain, a hinge region, a CH2 domain and / or a CH3 domain of human IgG, preferably human IgG1.

18. The antigen-binding fragment of any one of claims 1-17, wherein the antigen-binding fragment is selected from: an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH or VHH); a single-chain antibody (scFv); a (scFv)2 fragment, a F(ab')2 fragment; a Fab fragment; a Fd fragment; a Fv fragment; a one-armed (monovalent) antibody; a diabody; a triabody; and a tetrabody.

19. The antibody or antigen-binding fragment of any one of claims 1-8 or 12-18, wherein the antibody or antigen-binding fragment comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise CDR sequences selected from the group consisting of: (i) a HCDR3 comprising or consisting of SEQ ID NO: 11; a HCDR2 comprising or consisting of SEQ ID NO: 10; a HCDR1 comprising or consisting of SEQ ID NO: 9; a LCDR3 comprising or consisting of SEQ ID NO: 26; a LCDR2 comprising or consisting of SEQ ID NO: 25; a LCDR1 comprising or consisting of SEQ ID NO: 24; (ii) a HCDR3 comprising or consisting of SEQ ID NO: 14; a HCDR2 comprising or consisting of SEQ ID NO: 13; a HCDR1 comprising or consisting of SEQ ID NO: 12; a LCDR3 comprising or consisting of SEQ ID NO: 29; a LCDR2 comprising or consisting of SEQ ID NO: 28; a LCDR1 comprising or consisting of SEQ ID NO: 27; and (iii) HCDR3 comprising or consisting of SEQ ID NO:57; HCDR2 comprising or consisting of SEQ ID NO:56; HCDR1 comprising or consisting of SEQ ID NO:55; LCDR3 comprising or consisting of SEQ ID NO:60; LCDR2 comprising or consisting of SEQ ID NO:59; LCDR1 comprising or consisting of SEQ ID NO:58; Or any CDR having an amino acid sequence that shares at least about 90% identity with SEQ ID NOs: 9-14, 24-29, 55-60.

20. The antibody or antigen-binding fragment of claim 19, wherein the antibody or antigen-binding fragment comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (i) a VH comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; (ii) a VH comprising the amino acid sequence of SEQ ID NO:39, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; and (iii) a VH comprising the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto.

21. The antibody or antigen-binding fragment of any one of claims 1-6 or 9-18, wherein the antibody or antigen-binding fragment comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise CDR sequences selected from the group consisting of: (i) a HCDR3 comprising or consisting of SEQ ID NO: 17; a HCDR2 comprising or consisting of SEQ ID NO: 16; a HCDR1 comprising or consisting of SEQ ID NO: 15; a LCDR3 comprising or consisting of SEQ ID NO: 32; a LCDR2 comprising or consisting of SEQ ID NO: 31; a LCDR1 comprising or consisting of SEQ ID NO: 30; (ii) a HCDR3 comprising or consisting of SEQ ID NO:20; a HCDR2 comprising or consisting of SEQ ID NO:19; a HCDR1 comprising or consisting of SEQ ID NO:18; a LCDR3 comprising or consisting of SEQ ID NO:34; a LCDR2 comprising or consisting of SEQ ID NO:33; a LCDR1 comprising or consisting of SEQ ID NO:30; and (iii) a HCDR3 comprising or consisting of SEQ ID NO:23; a HCDR2 comprising or consisting of SEQ ID NO:22; a HCDR1 comprising or consisting of SEQ ID NO:21; a LCDR3 comprising or consisting of SEQ ID NO:36; a LCDR2 comprising or consisting of SEQ ID NO:31; a LCDR1 comprising or consisting of SEQ ID NO:35; Or any CDR having an amino acid sequence that shares at least about 90% identity with SEQ ID NOs: 15-23, 30-36.

22. The antibody or antigen-binding fragment of claim 21, wherein the antibody or antigen-binding fragment comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (i) a VH comprising the amino acid sequence of SEQ ID NO:41, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:42, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; (ii) a VH comprising the amino acid sequence of SEQ ID NO:43, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; and (iii) a VH comprising the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:46, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto.

23. An antibody or antigen-binding fragment thereof that binds to latent TGF-β1, wherein the antibody or antigen-binding fragment comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise CDR sequences selected from the group consisting of: (i) a HCDR3 comprising or consisting of SEQ ID NO: 11; a HCDR2 comprising or consisting of SEQ ID NO: 10; a HCDR1 comprising or consisting of SEQ ID NO: 9; a LCDR3 comprising or consisting of SEQ ID NO: 26; a LCDR2 comprising or consisting of SEQ ID NO: 25; a LCDR1 comprising or consisting of SEQ ID NO: 24; (ii) a HCDR3 comprising or consisting of SEQ ID NO: 14; a HCDR2 comprising or consisting of SEQ ID NO: 13; a HCDR1 comprising or consisting of SEQ ID NO: 12; a LCDR3 comprising or consisting of SEQ ID NO: 29; a LCDR2 comprising or consisting of SEQ ID NO: 28; a LCDR1 comprising or consisting of SEQ ID NO: 27; and (iii) HCDR3 comprising or consisting of SEQ ID NO:57; HCDR2 comprising or consisting of SEQ ID NO:56; HCDR1 comprising or consisting of SEQ ID NO:55; LCDR3 comprising or consisting of SEQ ID NO:60; LCDR2 comprising or consisting of SEQ ID NO:59; LCDR1 comprising or consisting of SEQ ID NO:58; Or any CDR having an amino acid sequence that shares at least about 90% identity with SEQ ID NOs: 9-14, 24-29, 55-60.

24. The antibody or antigen-binding fragment thereof according to claim 23, wherein the antibody or antigen-binding fragment comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (i) a VH comprising the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; (ii) a VH comprising the amino acid sequence of SEQ ID NO:39, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; and (iii) a VH comprising the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto.

25. An antibody or antigen-binding fragment thereof that binds to latent TGF-β1, wherein the antibody or antigen-binding fragment comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise CDR sequences selected from the group consisting of: (i) a HCDR3 comprising or consisting of SEQ ID NO: 17; a HCDR2 comprising or consisting of SEQ ID NO: 16; a HCDR1 comprising or consisting of SEQ ID NO: 15; a LCDR3 comprising or consisting of SEQ ID NO: 32; a LCDR2 comprising or consisting of SEQ ID NO: 31; a LCDR1 comprising or consisting of SEQ ID NO: 30; (ii) a HCDR3 comprising or consisting of SEQ ID NO:20; a HCDR2 comprising or consisting of SEQ ID NO:19; a HCDR1 comprising or consisting of SEQ ID NO:18; a LCDR3 comprising or consisting of SEQ ID NO:34; a LCDR2 comprising or consisting of SEQ ID NO:33; a LCDR1 comprising or consisting of SEQ ID NO:30; and (iii) a HCDR3 comprising or consisting of SEQ ID NO:23; a HCDR2 comprising or consisting of SEQ ID NO:22; a HCDR1 comprising or consisting of SEQ ID NO:21; a LCDR3 comprising or consisting of SEQ ID NO:36; a LCDR2 comprising or consisting of SEQ ID NO:31; a LCDR1 comprising or consisting of SEQ ID NO:35; Or any CDR having an amino acid sequence that shares at least about 90% identity with SEQ ID NOs: 15-23, 30-36.

26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the antibody or antigen-binding fragment comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (i) a VH comprising the amino acid sequence of SEQ ID NO:41, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:42, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; (ii) a VH comprising the amino acid sequence of SEQ ID NO:43, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto; and (iii) a VH comprising the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO:46, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto.

27. An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody of any one of claims 23-26.

28. An isolated nucleic acid encoding the antibody or antigen-binding fragment or VH, VHH or VL domain thereof of any one of claims 1-27.

29. An expression vector comprising the nucleic acid of claim 28 operably linked to regulatory sequences that allow expression of the antibody, antigen binding fragment, VH domain, VHH domain or VL domain in a host cell or a cell-free expression system.

30. A host cell or cell-free expression system comprising the expression vector of claim 29.

31. A method for producing a recombinant antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell or cell-free expression system of claim 30 under conditions that allow expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

32. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-27 and at least one pharmaceutically acceptable carrier or excipient.

33. The antibody or antigen-binding fragment according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 34 for use as a medicament.

34. A method of inhibiting a pathological immune response in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-27.

35. A method for preventing or treating an autoimmune disease and / or alloimmune disease in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to any one of claims 1-27, or the pharmaceutical composition according to claim 32.

36. The method of claim 35, wherein the disease is selected from the group consisting of: inflammatory bowel disease (IBD), multiple sclerosis (MS), graft versus host disease (GVHD), allogeneic transplant rejection, antibody-mediated allogeneic transplant rejection (AMR), allogeneic islet transplant rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune Addison's disease, Alzheimer's disease, anti-neutrophil cytoplasmic autoantibodies (ANCA), ANCA vasculitis, adrenal autoimmune disease, autoimmune encephalitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, immune platelet thrombocytopenia (ITP or idiopathic thrombocytopenic purpura or idiopathic thrombocytopenic purpura or immune-mediated thrombocytopenia), autoimmune urticaria, Behcet's disease, bullous pemphigoid (BP), cardiomyopathy, Castleman syndrome, celiac dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, COVID-19-mediated postural tachycardia syndrome (POTS), Crohn's disease, delayed graft function after renal transplantation, dilated cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita, original mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome (GBS), Goodpasture syndrome, Hashimoto's thyroiditis, hemophilia A, hemolytic disease of the fetus and newborn (HDFN), idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, IgA neuropathy, IgM polyneuropathy, juvenile arthritis, Kawasaki disease, lichen planus, lichen sclerosus, lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, membranous neuropathy, membranous nephropathy, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, transplant type 1 diabetes, multifocal motor neuropathy (MMN), myelin oligodendrocyte glycoprotein antibodies myasthenia gravis (MG), generalized myasthenia gravis (gMG), ocular myasthenia gravis (OMG), myositis, neuromyelitis optica (NMO), paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris (PV), pemphigus foliaceus (PF), pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis (DM), necrotizing autoimmune myopathy (NAM), antisynthetase syndrome (ASyS), primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis (RA), sarcoidosis,Scleroderma, Sjögren's syndrome, solid organ transplant rejection, stiff-man syndrome, Takayasu's arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, thyroid eye disease, ulcerative colitis, uveitis, warm autoimmune hemolytic anemia (wAIHA), dermatitis herpetiformis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegener's granulomatosis.

37. A method for preventing or treating a tumor disease in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to any one of claims 1-27, or the pharmaceutical composition according to claim 32.

38. The antibody or antigen-binding fragment according to any one of claims 1-27 or the pharmaceutical composition according to claim 32, for use in treating an autoimmune disease and / or alloimmune disease in a subject in need thereof.

39. The antibody or antigen-binding fragment according to any one of claims 1-27 or the pharmaceutical composition according to claim 32, for use in treating a tumor disease in a subject in need thereof.

40. A kit comprising the antibody or antigen-binding fragment of any one of claims 1-27 or the pharmaceutical composition of claim 32 and optionally instructions for use.

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